An Alternative Approach to Study Primary Events in Neurodegeneration Using Ex Vivo Rat Brain Slices

Summary

We present a method which can provide further insights into the early events underlying neurodegeneration and based on the established ex-vivo brain technique, combining the advantages of in vivo and in vitro experiments. Moreover, it represents a unique opportunity for direct comparison of treated and untreated group in the same anatomical plane.

Abstract

Despite numerous studies that attempt to develop reliable animal models which reflecting the primary processes underlying neurodegeneration, very few have been widely accepted. Here, we propose a new procedure adapted from the well-known ex vivo brain slice technique, which offers a closer in vivo-like scenario than in vitro preparations, for investigating the early events triggering cell degeneration, as observed in Alzheimer's disease (AD). This variation consists of simple and easily reproducible steps, which enable preservation of the anatomical cytoarchitecture of the selected brain region and its local functionality in a physiological milieu. Different anatomical areas can be obtained from the same brain, providing the opportunity to perform multiple experiments with the treatments in question in a site-, dose-, and time-dependent manner. Potential limitations which could affect the outcomes related to this methodology are related to the conservation of the tissue, i.e., the maintenance of its anatomical integrity during the slicing and incubation steps and the section thickness, which can influence the biochemical and immunohistochemical analysis. This approach can be employed for different purposes, such as exploring molecular mechanisms involved in physiological or pathological conditions, drug screening, or dose-response assays. Finally, this protocol could also reduce the number of animals employed in behavioral studies. The application reported here has been recently described and tested for the first time on ex vivo rat brain slices containing the basal forebrain (BF), which is one of the cerebral regions primarily affected in AD. Specifically, it has been demonstrated that the administration of a toxic peptide derived from the C-terminus of acetylcholinesterase (AChE) could prompt an AD-like profile, triggering, along the antero-posterior axis of the BF, a differential expression of proteins altered in AD, such as the alpha7 nicotinic receptor (α7-nAChR), phosphorylated Tau (p-Tau), and amyloid beta (Aβ).

Introduction

AD is a chronic pathology characterized by gradual neurodegenerative impairment affecting different brain areas, such as the entorhinal cortex (EC), BF, hippocampus (HC), and olfactory bulb (OB)^1,2,3,4,5. The late stages of AD development lead to a progressive cognitive decline, making this disease the most common form of dementia, approximately accounting for 70% of all cases⁶. Despite extensive attempts to understand the initial stages causing AD, there is not currently a defined e....

Protocol

All animal studies have been performed under approved protocols.

NOTE: In this section, the sequence of the main phases performed during the experimental procedure and the suggested time interval is provided (Figure 1). Moreover, a step-by-step description of the protocol is supplemented by an illustrative panel, showing critical actions ranging from brain removal to tissue homogenization after the incubation period (Figure 2). The details regarding the materials and instructions to build the apparatus and the subsequent phase for the WB analysis are previously described

Results

The protocol presented here indicates that the administration of a toxic peptide, T30, modulates in a site-dependent manner the expression of α7-nAChR, p-Tau, and Aβ in BF-containing sections (Figure 3A). The nicotinic receptor shows a significant increase in the rostral-treated hemislice compared to its control counterpart (slice 1, p = 0.0310) (Figure 3B), while the intermediate slice does not reveal any change between.......

Discussion

The principal aspect of this protocol, based on the well-established ex vivo brain technique, allows to synchronously test two specular hemislices, obtained from the same anatomical plane, monitoring their response after the application of a specific condition (control or treated); this therefore offers an experimental paradigm as tightly controlled as possible. The possibility of evaluating in a time-, dose-, and site-specific manner different neurochemicals related to neuronal impairment, as seen dur.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Sodium chloride (NaCl)	Sigma-Aldrich, Germany	S7653	Reagent for aCSF preparation
Potassium chloride (KCl)	Sigma-Aldrich, Germany	P9333	Reagent for aCSF preparation
Sodium bicarbonate (NaHCO3)	Sigma-Aldrich, Germany	S5761	Reagent for aCSF preparation
Magnesium sulphate heptahydrate (MgSO4 (7H2O))	Sigma-Aldrich, Germany	63138	Reagent for aCSF preparation
Potassium phosphate monobasic (KH2PO4)	Sigma-Aldrich, Germany	P5655	Reagent for aCSF preparation
Hepes salt	Sigma-Aldrich, Germany	H7006	Reagent for aCSF preparation
Hepes acid	Sigma-Aldrich, Germany	H3375	Reagent for aCSF preparation
Glucose	Sigma-Aldrich, Germany	G7528	Reagent for aCSF preparation
Calcium chloride dehydrate	Sigma-Aldrich, Germany	223506	Reagent for aCSF preparation
T30 peptide	Genosphere Biotechnologies, France		AChE-derived peptide tested
Surgical dissecting kit	World Precision Instruments, USA	Item #: MOUSEKIT	Brain removal step
Surgical blades	Swann-Morton, UK	BS 2982	Brain removal step
Filter paper	Fisher Scientific, USA	11566873	Brain preparation for slicing
Glue			Brain preparation for slicing
Vibratome	Leica, Germany	VT1000 S	Slicing
Brushes			Tissue handling
Oxygen canister			Sectioning and incubation phase
1x Phosphate buffer saline (PBS)	Fisher Scientific, USA	BP2438-4	Homogenization step
Phosphatase inhibitors	Fisher Scientific, USA	1284-1650	Homogenization step
Protease inhibitors	Roche complete PIC, USA	4693116001	Homogenization step
Pestles	Starlab, UK	I1415-5390	Homogenization step
Microcentrifuge
Pierce 660 nm Protein Assay	Thermo Scientific, USA	22660	Protein concentration