НЕЙРОХИМИЯ, 2014, том 31, № 1, с. 42-46


УДК 577


Department of Neurology, Sichuan Academy of Medical Science and Sichuan Provincial People's Hospital,

Chengdu, P. R. China

Abstract—Chronic treatment of rats with D-galactose (D-gal) can mimic brain aging process. In the present study, we investigated whether and how astrocytes undergo morphological alterations due to long-term D-gal exposure. After 8 weeks of daily subcutaneous injection of D-gal, total glial cells (marked by Holzer's crystal violet staining) and astrocytes (marked by anti-glial fibrillary acidic protein immunohistochemical labeling) of the medulla of the motor area were comparatively analyzed in control and D-gal-treated rats. We found a significant increase in the quantity of both total glial cells and astrocytes, with a higher ratio of astrocytes to total glial cells in the experimental group than the control; astrocytic body size, process number and area, and glial fibrillary acidic protein immunoreactive intensity were also increased in D-gal-treated animals. Our present study provides direct evidence that chronic administration of D-gal enhances brain astrocytic activities, which may exert compensatory functions on D-gal-induced deteriorative neurons.

Keywords: astrocyte, glial cell, cerebral medulla, D-galactose, rat.

DOI: 10.7868/S1027813314010142


While normal levels of intracorporeal D-galactose (D-gal) are converted into glucose, an excess of D-gal can cause oxidative damage to various tissues by reacting with the free amines of amino acids and forming advanced glycation end-products [1—4]. Abundant evidence from mice, rats, and Drosophila suggests that chronic administration of D-gal can induce acceleration of senescence, mimicking animal and cell aging models [2, 4—6]. The D-gal-induced brain aging is largely reflected in morphological and/or functional damages to cerebral neurons, which includes neuronal mitochondrial dysfunction [4], neurotransmitter imbalances [6], P-amyloid accumulation [7], nerve fiber degeneration [8], neuronal apoptosis [4, 5], and many related behavioral deficits [5, 8, 9].

Astrocytes are the most abundant and widely investigated glial cells in the central nervous system. They perform multiple functions, including physically supporting neurons, maintaining the internal environment, forming the blood-brain barrier that provides nutrients to neurons, repairing and scarring traumatized tissue, propagating intercellular Ca2+ waves over long distances in response to stimulation, and releasing gliotransmitters [10, 11]. Interestingly, astrocytes become more active in adverse physiological or early

♦Corresponding author, address: No 32 Western Section 2, Yi-huan Road, Chengdu 610072, P. R. China, e-mail: liangzg2013@gmail.com.

pathological conditions, showing stronger expression of glial fibrillary acidic protein (GFAP) and increases in their quantity, size, and the process development [12-14].

However, whether and how the astrocytes undergo morphological alterations due to chronic D-gal insults remains largely unclear. Thus, in the present study, we sought to identify changes in the quantity, somatic size, process number and area, and astrocyte to total glial cell ratio in the cerebral medulla between control and chronic D-gal-administered rats. With these findings, we aimed to expand our understanding of the influence of D-gal on astrocytic activities, which may play compensatory roles in D-gal-induced degeneration in the aging brain.


Animal and Drug Treatment

Male Sprague-Dawley (SD) rats (2 months old, 230-250 g, n = 20) were used in this study The animals were randomly assigned to the following two groups of ten: (1) saline-treated group (0.9% saline, 1 mL/kg body weight, subcutaneous injection once daily for 8 consecutive weeks); (2) D-gal-treated group (D-gal; Sigma, USA; 100 mg/kg dissolved in 0.9% saline; a total volume of 1 mL/kg; the injection protocol was similar to the control group). Rats were raised in a temperature-controlled (22-24°C) and light/dark-cycled (12 h/12 h) house with food and wa-

ter ad libitum. All animal experiments were conducted in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals.

Tissue Preparation

Rats were anesthetized with intraperitoneal injection of 40 mg/kg sodium pentobarbital. Animals were perfused through the heart with 0.9% saline, followed immediately with 200 mL fixative solution containing 4% paraformaldehyde in 0.1 M phosphate-buffered saline (PBS, pH 7.4). The skull was opened and the left primary motor area was dissected out, and then trimmed to 2 cm x 1 cm x 1 cm for further fixation in the above fixative (4°C, 24 h).

The tissue blocks were dehydrated in ethanol, cleared in xylene, and embedded in paraffin. Consecutive coronal sections of 6 ^m were cut before mounting on microscope slides coated with 3-aminopropyl-triethoxysilane (APES; Sigma, USA; 1: 50 in acetone) for Holzer's crystal violet staining and anti-GFAP im-munostaining.

Holzer's Crystal Violet Staining

In each series, 5 sections were taken at intervals of 120 ^m apart for Holzer crystal violet staining, as previously described [15]. Sections were treated in freshly prepared 0.5% phosphomolybdic acid in alcohol (37°C, 3 min). After draining and washing, the sections were immersed in a solution of 80% chloroform and 20% alcohol until the sections became translucent. The sections were transferred to a staining rack containing the same chloroform—ethanol solution plus 5% crystal violet (Sigma, USA; 37°C, 30 s). After treatment with a solution of 10% potassium bromide in distilled water (37°C, 1 min) to differentiate the background, the glial cells were clearly labeled in deep violet.

Anti-GFAP Immunohistochemical Staining

The adjacent sections were used for anti-GFAP immunohistochemical staining. Sections were depar-affinized in xylene, and hydrated through a graded series of ethanol to water. The sections were treated with 3% hydrogen peroxide (37°C, 5 min) to inhibit endogenous peroxidase activity. After washing in PBS (3 x 5 min at 37°C, the same washing procedure was performed between each of the following steps), the sections were incubated with 10% goat serum (37°C, 5 min) to suppress nonspecific staining. Subsequently, sections were incubated with anti-GFAP monoclonal antibody (Sigma, USA; 1 : 500; 4°C, 24 h), followed by incubation with biotinylated anti-mouse IgG antibody (Sigma, USA; 1 : 800; 37°C, 10 min). Sections were treated with preformed avidin—biotin—peroxidase complexes (ABC; Sigma, USA; 37°C, 30 min), and finally incubated in 0.05% 3,3-diaminobenzidine (DAB)/0.01% hydrogen

peroxidase (Sigma, USA; 37°C, 10 min) in PBS for identification of astrocytes. The remaining adjacent sections were processed for the immunoreactive controls, that is, the anti-GFAP antibody was replaced by PBS.


Density Calculation of the Total Glial Cells and the Astrocytes

The glial cells (from Holzer's crystal violet stained slides) and astrocytes (from the anti-GPAP immunohistochemical stained slides) were counted using a calibrator (50 ^m x 50 ^m) under a microscope (Motic China Group Co. Ltd, China) at 400x. Five areas were randomly selected from the subcortical medulla in each section, and the cell density (cells/mm2) was calculated. The ratio of astrocytes to total glial cells was calculated as the mean density of astrocytes divided by the mean density of total glial cells.

Astrocyte Somatic Diameter Calculation

The somatic diameter of the astrocytes was directly measured (5 random cells in each section) from the cells with a clear border and distinct nucleus (vacancy in the somatic center), using an eyepiece micrometer at 1000x. The diameter of the astrocyte soma was roughly estimated as: d = (a x b)1/2 (a and b were the longitudinal and transversal diameters of an astrocyte, respectively).

The Optical Absorption Calculation

In order to evaluate the GFAP-immunoreactive intensity, we compared their immunostaining intensity with the background. Under high magnification (400x), the gray scale images were captured using a CCD camera while holding all image-gathering parameters constant. Five background intensity values were averaged as the background intensity in each section, and 5 astrocytes in each section were selected to determine their somatic immunoreactive density by using software BI-2000 (Taimeng Sci & Tec Co. Ltd, China). The background intensity was then measured relative to the GFAP-immunoreactive intensity of each cell to provide an individual optical intensity value.

Calculation of Astrocytic Process Number and Area

In each section, astrocytes meeting the following criteria were selected for measurement: (1) the entire processes were completely and distinctly stained in the sections; (2) the cell was distinct from neighboring cells. For each section, the first 5 cells satisfying the requirements were imaged by a Motic microscope with imaging software at 400x. The area of the processes was measured by circumscribing a polygon around the end points of the process and then calculating the area

HEHPOXHMHH TOM 31 № 1 2014

Astrocyte parameters in the cerebral medulla of control and D-gal-treated rats

Control rat (n = 10) D-gal-treated rat (n = 10)

Number of the total glial cell/mm2 579.96 ± 23.83 690.74 ± 12.12**

Number of the astrocytes/mm2 289.40 ± 8.69 395.79 ± 14.04**

Ratio of astrocytes to the total glial cells 48.92 ± 1.44% 56.29 ± 1.33%**

Diameter of astrocytes (p.m) 7.45 ± 0.13 8.32 ± 0.17**

Number of astrocytic processes 6.25 ± 0.29 7.35 ± 0.35*

Area of astrocytic processes (p.m2) 660.47 ± 20.42 796.53 ± 26.85**

Optical absorption of anti-GFAP immunoreaction 42.72 ± 1.39% 48.10

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