Awake Rat Model of Ischemic Stroke with Neuropsychiatric Complications During a Seven-Day Observation

Summary

The new model of focal ischemic stroke in awake rats is proposed. This was achieved by injecting an air embolus into the internal carotid artery of rats. The development of focal ischemic stroke was confirmed by brain staining with triphenyltetrazolium chloride, functional tests, and assessment of physiological parameters.

Abstract

The translational model of experimental stroke in rats induced by a gas embolism was created to clarify the features of stroke's development and to evaluate the neuroprotective effect of novel remedies and proven drugs. The moderately time and labor-consuming protocol allows for observing the dynamics of rapid neuron death in awake animals, providing a unique opportunity to assess the acute phase of stroke, including delirium, as well as to monitor autonomic disturbances such as changes in blood pressure, body temperature, and respiratory minute volume. Atmospheric air (100 µL) was administered by infusion through a vascular catheter placed retrogradely into the common carotid artery via the external carotid artery, using an infusion pump. Twenty-four hours after embolization, the animals exhibit stereotypical behavioral, motor deficits, and histopathological differences. By the seventh day of the experiment, a significant improvement in neurological status was observed, and the results in behavioral tests approached those of the control values. At the same time, a transition from neutrophilic to macrophage infiltration is observed in the ischemic lesion. The proposed model can be utilized in preclinical studies, as it reproduces the neuropsychiatric complications of the post-stroke period, along with the corresponding physiological, functional, and histopathological changes.

Introduction

The objective of this research was to develop a model of focal cerebral ischemic stroke in awake rats and to achieve the closest approximation of this model to the clinical aspects of ischemic stroke in humans. As demonstrated in the research by Söyland et al., 62.6% of patients exhibited significant symptoms of ischemic stroke in an awake state, while only 19% experienced a wake-up stroke¹. Consequently, the occurrence of an awake state during cerebral artery embolization in animals is of the greatest interest. The classical models employed for the study of focal cerebral ischemic stroke include the following: transcranial occlusion, endo....

Protocol

The study was conducted in accordance with the standard operating procedures of the laboratory and was approved by the Bioethical Commission of the Institutional Animal Care and Use Committee of the Branch of the Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry of the Russian Academy of Sciences (Protocol No. 1016/25, dated January 17, 2025). In this study, 28 male Sprague-Dawley (SD) rats, aged 10-12 weeks, were used, with an average body weight of 301 g and a microbiological status of Specific Pathogen Free (SPF). The reagents and the equipment used are listed in the Table of Materials.

1. Animal preparation<....

Results

Survival and overall condition
Immediately after administration of the air bubble, all rats showed a disturbed state and a pronounced tremor for several seconds, followed by cessation of all motor activity and loss of consciousness for 1-2 min. Some animals lost support on their paws for 10-15 min after regaining consciousness.

The survival rate of animals 3 h after CAE was 100%, but after 24 h the survival rate decreased to 80%, and on the 3^rd day it was 60%. A.......

Discussion

MCAO is a common method of modeling focal ischemic stroke in rats, which is achieved by thrombus formation, local spasm of the middle cerebral artery, and temporary occlusion by filament². The aim of such models is (1) to achieve focal apoptosis of neural brain tissue¹⁶ with standardized lesion volume and area¹⁷, (2) to monitor the neurological deficit of animals for 2-7 days¹⁸.

The selection of the.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
3-component syringe with a 2 mL needle	LEIKO	523222	23G
Aladdin Single infusion pump	World Precision Instruments	AL-1000
Ciprofloxacin	Pharmavet	3897744	5% solution of ciprofloxacin
Computerized system PowerLab 8/35	ADInstruments	3508-1090
Dezin	Apteka.ru		0.5% chlorhexidine bigluconate in 70% ethanol
Digital thermometer	B.Well Swiss AG	LOT 0413
Electronic dynamometer Grip Strength Meter	BioMed Easy Technologies	007231024415MC01
ELISA Kit for S100 Calcium Binding Protein B (S100B)	CLOUD-CLONE CORP.(CCC)	SEA567Ra	48T
Fishing line			Diameter 0.9 mm
Gas anesthesia system	UGO BASILE	21100
GraphPad statistical software	Prism		v.10
Grooved Needle Director	Fisher Scientific	Catalog no. 11-00	12.7 cm
Heparin	Endopharm. Moscow Endocrine Plant	30122
ImageJ	National Institutes of Health and the Laboratory for Optical and Computational Instrumentation
Insulin syringe	SFM Hospital Products	160303	0.5 mL, U-100, 30G
Intravenous catheter	KD Medical		G24
Isoflurane (Isoflurin)	Vetpharma		100 mL, 1000 mg/kg
Lab animal immobilizer for laboratory animals	Open Science	AE1001-R1
Light microscope	ZEISS		Axio Scope.A1
Light microscopy slides	Thermo Scientific	15442933
Medical grade polyethylene micro tubing	Scientific Commodities Inc.	BB31695-PE/4	Inner diameter 0.76 mm
Medical sterile gauze wipes	New life		45x29 CM
Micro tube	SARSEDT AG & Co. KG	S044378	1.5 mL
Micropipette Eppendorf research plus	Eppendorf		200 µL
Microscope slides	Fisher Scientific	11562203
Microsurgical forceps (curved)	Kazan Medical Instruments Plant	F0106
Microsurgical forceps (straight)	RWD Life Science	F11029-11
Microtome blades	Thermo Scientific	3052835	MX35 Premier+
Multiconditioning System	TSE Systems	131202-10
Neutral buffered formalin	hem	1072024	pH 6,8-7,2
Non-absorbable Surgical Suture Lavsan	Lintex	2022-01
Ophthalmic scissors for iridectomy	CM Instrumente	AJ-410-05
Oxygen concentrator	Armed	YYT32700960	7F-3L
Phosphate buffer	AMRESCO	2120C462
Piptte tips	Servicebio		200 µL
Rats	obtained from the Laboratory Animal Nursery “Pushchino” (www.spf-animals.ru), Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry of the Russian Academy of Sciences.
Refrigerated Lab Multifunctional Centrifuge	Eppendorf	5804 R
Rota-Rod Rotamex-5	COLUMBUS INSTRUMENTS	210426
Scissors METZENBAUM	Medstandart-Instrument	MT-H-256
Surgical suture material	REPROMED	114/0042011124	Kaproag 4/0
Surgical table	VetKormTorg	VETBOT STL-1, TD00472
Telazol	Zoetis	672913	50 mg/mL tiletamine and 50 mg/mL zolazepam in 5 mL
Triphenyltetrazolium chloride	Dia-M	TTC1809
Xylazine	NITA-FARM	151	2% xylazine hydrochloride in 50mL