Experimental Models to Study the Neuroprotection of Acidic Postconditioning Against Cerebral Ischemia

Summary

Acidic postconditioning protects against cerebral ischemia. Here we present two models to execute APC. They are achieved respectively by transferring corticostriatal slices to acidic buffer after oxygen-glucose deprivation in vitro and by inhaling 20% CO₂ after middle cerebral artery occlusion in vivo.

Abstract

Stroke is one of the leading causes of mortality and disability worldwide, with limited therapeutic approaches. As an endogenous strategy for neuroprotection, postconditioning treatments have proven to be promising therapies against cerebral ischemia. However, complicated procedures and potential safety issues limit their clinical application. To overcome these disadvantages, we have developed acidic postconditioning (APC) as a therapy for experimental focal cerebral ischemia. APC refers to the mild acidosis treatment by inhaling CO₂ during reperfusion following ischemia. Here we present two models to execute APC in vitro and in vivo, respectively. The oxygen-glucose deprivation (OGD) treatment of mice and the corticostriatal occlusion and middle cerebral artery occlusion (MCAO) of mice were employed to mimic cerebral ischemia. APC can be simply achieved by transferring brain slices to acidic buffer bubbled with 20% CO₂, or by mice inhaling 20% CO₂. APC showed significant protective effects against cerebral ischemia, as reflected by tissue viability and brain infarct volume.

Introduction

Stroke is one of the leading causes of mortality and disability worldwide. Great efforts have been made to find effective treatments for stroke in the last decades, however, the achievement is quite unsatisfactory. Postconditioning is a process manipulated by subtoxic stresses following an ischemic episode. Postconditioning, including ischemic, hypoxic, low-glucose and remote ischemic postconditioning, trigger endogenous adaptive mechanisms, and have been proven to be promising therapies against cerebral ischemia^1,2,3,4. However, ischemic po....

Protocol

All experiments were approved by and conducted in accordance with the ethical guidelines of the Zhejiang University Animal Experimentation Committee and were in complete compliance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals. Efforts were made to minimize any pain or discomfort, and the minimum number of animals was used.

1. OGD of Corticostriatal Slices

1. Solution preparation:
   1. Prepare 1,000 mL r-ACSF (124 mmol/L, NaCl, 5 mmol/L KCl, 1.25 mmol/L KH₂PO₄, 2 mmol/L MgSO₄, 26 mmol/L NaHCO₃, 2 mmol/L CaCl₂, 10....

Results

In the corticostriatal slice model described above, corticostriatal slice viability was quantified by TTC assay at 1 h after reperfusion. TTC conversion was calculated by normalizing the absorption at 490 nm to the control slice. According to TTC conversion, APC protected against OGD-induced reperfusion injury in an onset time and duration-dependent manner. In detail, both 1 and 3 min of acidosis treatment significantly improved viability at 5 min after 15 min OGD, whereas 5 min did not (.......

Discussion

Here we present two experimental models to study the neuroprotection of APC against cerebral ischemia. In brain slices, APC is achieved by incubating mice corticostriatal slices in acidic buffer bubbled with 20% CO₂ after reperfusion onset, while in the MCAO model, APC is achieved by inhaling 20% CO₂ to mice after reperfusion. Both models reflect the neuroprotection of APC against cerebral ischemia. The protection was comparable with that achieved by ischemic postconditioning but with a wider time w.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Sodium chloride	Sigma	S5886
Potassium chloride	Sigma	P5405
Potassium phosphate monobasic	Sigma	P9791
Magnesium sulfate	Sigma	M2643
Sodium bicarbonate	Sigma	S5761
Calcium chloride dihydrate	Sigma	C5080
D-(+)-Glucose	Sigma	G7021
Vibratome	Leica	VT1000 S
2,3,5-triphenyltetrazolium hydrochloride	Sigma	T8877
Absolute Ethanol	Aladdin Industrial Corporation	E111993
Dimethyl sulfoxide	Sigma	D8418
Laser Doppler Flowmetry	Moor Instruments Ltd	Model Moor VMS-LDF2
Diethyl ether anhydrous	Sinopharm Chemical Reagent Corporation	80059618
Trichloroacetaldehycle hydrate	Sinopharm Chemical Reagent Corporation	30037517
10% Formalin	Aladdin Industrial Corporation	F111936
24-well plates	Jet Biofil	TCP-010-024