Protocol for Three-dimensional Confocal Morphometric Analysis of Astrocytes

Summary

Astrocytes in the CNS change their functional and structural properties in response to harmful stimuli. This report presents a protocol for assessment of three-dimensional astrocyte morphology in diseased conditions or after therapeutic interventions.

Abstract

As glial cells in the brain, astrocytes have diverse functional roles in the central nervous system. In the presence of harmful stimuli, astrocytes modify their functional and structural properties, a condition called reactive astrogliosis. Here, a protocol for assessment of the morphological properties of astrocytes is presented. This protocol includes quantification of 12 different parameters i.e. the surface area and volume of the tissue covered by an astrocyte (astrocyte territory), the entire astrocyte including branches, cell body, and nucleus, as well as total length and number of branches, the intensity of fluorescence immunoreactivity of antibodies used for astrocyte detection, and astrocyte density (number/1,000 µm²). For this purpose three-dimensional (3D) confocal microscopic images were created, and 3D image analysis software such as Volocity 6.3 was used for measurements. Rat brain tissue exposed to amyloid beta_1-40 (Aβ_1-40) with or without a therapeutic intervention was used to present the method. This protocol can also be used for 3D morphometric analysis of other cells from either in vivo or in vitro conditions.

Introduction

In healthy central nervous system (CNS), astrocytes play an important role in the regulation of blood flow, energy metabolism, synaptic function and plasticity, and extracellular ion and neurotransmitter homeostasis^1-3. In addition, astrocytes respond to different harmful stimuli and abnormal conditions such as trauma, infection, ischemia or neurodegeneration via reactive astrogliosis which is characterized by hypertrophy, proliferation and functional remodeling of astrocytes^4,5.

Reactive astrogliosis can engineer the inflammatory response and repair process in the tissue and, therefore, can affect the clinical outcome....

Protocol

This study was carried out in accordance with the policies set forth in the Guide for the Care and Use of Laboratory Animals (NIH) approved by Ethic Committee of Iran University of Medical Sciences (Tehran, Iran).

1. Animals, Surgery and Specimen Preparations

NOTE: Prepare brain tissue for 3D confocal microscopic analysis.

1. Divide animals randomly into two groups: Aβ_1-40‒injection (n = 8), and Aβ_1-40‒injection with genistein treatment (n = 8); administer genistein (10 mg/kg diluted in a vehicle such as polyethoxylated castor oil) by gavage 1 hr before surgery....

Results

This section presents some examples of the qualitative and quantitative observations produced by 3D morphometric analysis. For complete results of all 12 parameters mentioned earlier, please see our previous publication¹⁰.

Qualitative Observations

Astrocytes exhibited thin or thick branches that were usually long in Aβ_1-40 injected rats (Figure 1). A few small stellate-shaped astrocytes with short branches were observed in the .......

Discussion

In the current protocol, we employed 3D confocal morphometry to evaluate 12 different parameters that were associated with astrocyte morphology. For this purpose, hippocampal tissue of rats with Aβ_1-40‒induced astrogliosis, with or without genistein pretreatment as an anti-inflammatory agent were used. By using 3D images and morphometric software, we were able to show the effect of genistein on astrogliosis i.e. the morphology of astrocytes.

Changes in.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Amyloid beta 1-40	Sigma Aldrich	79793	Keep in -70 °C
Genistein	Sigma Aldrich	446-72-0	keep in -20 °C
polycolonal rabbit antibodies against glial fibrillary acidic protein	DAKO	Z0334
alkaline phosphate-conjugated swine anti-rabbit IgG antibodies	DAKO
Liquid Permanent Chromogen	DAKO	K0640
Liquid permanent Red Substrate Buffer	DAKO	K0640
Cremophor EL	Sigma Aldrich	27963	Polyethoxylated castor oil - Step 1.1
LSM 700 Confocal Laser Scanning Microscopy	Carl Zeiss
Volocity 6.3	Perkin Elmer Inc.,
Image Analysis 2000	Tekno Optic
Streotaxic apparatus	Stoelting
Graph pad Prism 5	Graph pad software Inc.