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 experimental indication elucidating them. In addition, the most popular theory - the "amyloid hypothesis" - is increasingly questioned since it does not provide a complete profile in explaining the AD pathobiology, nor a pharmaceutical target that has proved effective^7,8,9.

An alternative theory which is receiving increasing attention suggests that the initial mechanisms occurring during neurodegeneration are related to a neuronal cluster primarily susceptible in AD^{3,10,11,12,13,14}. This heterogeneous cellular hub encompassed within the BF, midbrain, and brainstem, projects to multiple regions, such as the EC, HC, and OB^15,16. Despite its diversity in neuronal morphology and neurotransmitter synthesis, this core of cells shares a common feature in expressing AChE, which can also have a non-enzymatic function^17,18. This non-classical role as a novel signaling molecule mediates calcium (Ca²⁺) flow into neurons which can undergo trophic or toxic events in relation to Ca²⁺ dose, availability, and neuronal age^17,18,19.

During neurodegeneration, the observed cellular loss might be therefore associated to this non-enzymatic function^17,18,20, which is attributable to a 30mer peptide (T30) cleaved from the AChE C-terminus²⁰. In line with previous results, carried on cell culture and optical imaging^18,21 preparations, we demonstrated, through a novel approach based on ex vivo rat brain slices containing BF structures, that T30 induced an AD-like profile²². Specifically, this new methodology offers a more physiological scenario than cell culture since it maintains many of the characteristics of an intact tissue, ranging from anatomical to circuitry preservation, albeit for a time window of hours. We applied this protocol to explore the events taking place during the early phases of neurodegeneration, monitoring the acute response upon T30 application.

Despite the large body of literature on using brain slices to investigate molecular pathways implied in neuronal damage or neurogenesis^23,24, this protocol provides for the first time a more immediate and sensitive read out compared to the common use of organotypic slices. However, as is the case for organotypic brain sections, this acute slice procedure can also be adopted for several purposes, such as the evaluation of neuroprotective or neurotoxic molecules, discovery of primary molecular changes occurring in a specific process, immunohistochemical analysis, and pharmacological assays for central nervous system related pathologies.

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 described²².

1. Surgical Tools, Vibratome Settings and Treatment Preparation

NOTE: Execute the following steps before starting the experimental procedure:

1. Prepare two different artificial cerebrospinal fluids (aCSFs), one used for the brain slicing and the other for the incubation step as previously described²⁹. Briefly, the working concentrations (in mmol) of the two aCSFs are for the "slicing" aCSF: 120 NaCl, 5 KCl, 20 NaHCO₃, 2.4 CaCl₂, 2 MgSO₄, 1.2 KH₂PO₄, and 10 glucose; 6.7 HEPES salt and 3.3 HEPES acid; pH: 7.1. While for the "recording" aCSF: 124 NaCl, 3.7 KCl, 26 NaHCO₃, 2 CaCl₂, 1.3 MgSO₄, 1.3 KH₂PO₄, and 10 glucose; pH: 7.1.
2. Mix well the solutions and then put on ice and oxygenate the slicing aCSF in order to have it cold while removing the brain and the subsequent sectioning. Keep at room temperature (RT) and supply oxygen to the recording aCSF.
3. Place the instruments under the dissection hood for the decapitation and brain removal, i.e., guillotine, surgical dissection kit (scissors, forceps, blades), and spatula.
4. Set up the vibratome parameters by selecting the slice thickness (300 µm in this preparation) and the frequency at 7 and the speed at 6. Insert the vibratome chamber in its appropriate space and surround it with ice.
5. Open the carbogen (95% O_2, 5% CO₂) valve to oxygenate the "slicing" (aCSF) during the brain sectioning. Select the minimum flow rate, around 2 mL/min.
6. Take two 15-mL tubes to prepare the conditions to test. Add in one tube 10 mL of the "recording" aCSF alone (control group) and in the other, 10 mL of the "recording" aCSF enriched with T30 at a concentration of 2 µM (treated group). Vortex both tubes and keep them on ice until their use in the setup preparation phase (section 3).

2. Anesthesia, Brain Extraction and Slicing

NOTE: In this study, 9 wild type P14 male Wistar rats were used. Brain extraction and subsequent slicing constitute a critical part of the protocol since they should be quickly performed (within 10 min) in order to prevent or at least slow down tissue degeneration processes.

1. Add 1.5 mL of 100% w/w of isoflurane on a cotton layer into the induction chamber, place the animal inside, and close the lid. Wait until the animal is deeply anesthetized, confirming the absence of the pedal reflex, and then decapitate the animal using the guillotine.
2. Keep the head of the animal within a pot containing ice-cold slicing aCSF during the brain removal step (Figure 2A - C).
3. Incise the skin over the skull with a surgical blade, then cut the skull using scissors following the midline, from the posterior part and proceed anteriorly until reaching the bone above the OBs (Figure 2A).
4. Pull laterally the two sides of the skull using a pair of forceps in order to have access to the brain (Figure 2B). Insert a spatula ventrally to the brain, gently scoop it out, and keep it hydrated in ice-cold "slicing" aCSF for approximately 5 min (Figure 2C).
5. Dispose the brain on filter paper and cut out the cerebellum using a blade (Figure 2D). Glue the brain vertically on the vibratome disc and insert it inside the sectioning chamber, fill it with ice-cold "slicing" aCSF, and provide oxygen (Figure 2E).
6. Adjust the cutting interval, and proceed with the brain sectioning (Figure 2F). Collect the sections containing the region of interest, such as the medial septum (MS), diagonal band of Broca (DBB), and substantia innominata (SI), as previously described²². From top to the bottom, the three slices contain the rostral (R), intermediate (I), and caudal (C) portion of the BF (Figure 2G).
7. Divide along the midline each section with small scissors, obtaining two specular hemislices (Figure 2H), used individually as the control and treated condition at the same anatomical plane.
8. Keep the hemisections in the vibratome chamber for 5 - 10 min, then transfer them with a glass pipette into a bubbling pot containing recording aCSF. Keep at RT for approximately 30 min to recover.
9. Fill, in the meantime, three glass vials with 3 mL of recording aCSF alone (previously prepared in step 1.6) for the control group and three vials with 3 mL of aCSF enriched with T30 (previously prepared in step 1.6) for the treated group.

3. Setup Preparation

1. Transfer the hemisections into their corresponding glass vials (Figure 2I) in order to have three consecutive hemislices (including the rostral, intermediate, and caudal BF region) for the control group (green frame, Figure 2J) and their matching counterparts for the treated group (red frame, Figure 2J).
2. Transfer the brain tissue with two different brushes, one for the control hemislices and another for the treated counterparts, in order to prevent any contamination between conditions.
3. Seal each glass vial with the corresponding lid, presenting the oxygenating needle (21G) (Figure 2J). Connect the main tube of the apparatus to the carbogen source (Figure 2K). Deliver oxygen to the hemislices throughout the incubation period with a minimal flow rate of 2 mL/min in order to avoid any movement of the brain tissue and possible mechanical damage during the experiment. Start the 5 h incubation. Execute the steps 3.1 - 3.3 within 5 min.
   NOTE: Check frequently (every 15 - 20 min) whether the oxygen is supplied into all the vials and that its flow is not moving the tissue from the bottom.

4. Sample Homogenization

1. Stop the oxygen flow after the incubation and remove all the lids from the vials. Transfer the hemislices, using the proper brushes, into 1.5 mL tubes containing 250 µL of lysis buffer, maintaining the tubes on ice (Figure 2L). To make the lysis buffer, mix PBS 1x with phosphatase and protease inhibitors cocktails both diluted at 1:100, and homogenize the tissue with different pestles to prevent contamination among the samples. Complete these actions within 10 - 15 min.
2. Centrifuge the brain lysate at 1,000 x g for 5 min at 4 °C. Transfer the supernatant into new tubes and keep the samples at -80 °C.

5. Reading of the Protein Concentration and Western Blot Analysis

1. Determine the protein concentration of each sample and subsequently prepare the aliquots for Western blot analysis as previously described²².
2. Perform statistical analysis to evaluate the effects of the treatment on protein expression.

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