Effects of 2-mercaptoethanol on survival and differentiation of fetal ...

Neuroscience Letters, 163 (1993) 159 162 ~ 1993 Elsevier Ireland Ltd. All rights reserved 0304-3940/93/$ 06.00

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Effects of 2-mercaptoethanol on survival and differentiation of fetal mouse brain neurons cultured in vitro K a z u h i r o Ishiia'*, M a s a h i r o K a t a y a m a a, K o t a r o H o r i b, Junji Y o d o i b, T o h r u N a k a n i s h i c "Department 0[' Cell Biology and hDepartment of Clinical Immunology, Institute fi)r Virus Research, Kyoto University, Sakyou-Ku, Kyoto 606, Japan 'Department of Biochemistry, Okayama University Dental School, Sikata-Cho, Okayama 700, Japan (Received 11 June 1993; Revised version received 2 August 1993; Accepted 18 August 1993)

Key words: 2-Mercaptoethanol: Striatum; Cerebral cortex; Differentiation; Conditioned medium Effects of"2-mercaptoethanol on primary cultures of fetal mouse brain neurons have been investigated. The addition of 2-mercaptoethanol to the culture medium increased 6- or 200-fold the survival rates of embryonic day-16 murine striatum neurons and day-18 cerebral cortical neurons cultured in serum-free medium, respectively, and also induced neurite outgrowth, particularly being prominent in cortical neurons. Moreover, this drug enhanced trophic activities of the conditioned medium of VR-2g or BIM cells. These findings indicate that 2-mercaptoethanol can support the viability and differentiation of fetal mouse brain neurons.

Primary cultures of mammalian fetal brain neurons have been available for studies on the differentiation and maturation of neurons [2, 8] and for bioassays of neurotrophic factors [11, 20]. In such primary cultures, one prominent feature is that there occurs drastic neuronal cell death [21]. This is probably due to two reasons: first, neurons are subject to the programmed cell death [7, 17], and second, fetal brain tissue cells are exposed to critical environmental conditions such as treatment with trypsin for cell dissociation and different environmental conditions from those in vivo, including low cell density, absence of glial cell feeders, and serum-free medium [11, 19]. Therefore, to attain stable cultures of brain neurons under such conditions, improvement of the culture medium is required. In the course of studies on the neurotrophic activity of phosphoglucose isomerase [15] dissolved in a solution containing 2-mercaptoethanol (2-ME), we found that 2ME supported the survival of primary cultured neurons (unpublished). Thus, we have investigated in more details such effects of 2-ME. In this report, we demonstrate that 2-ME exerted the enhancing effects on the viability and differentiation of fetal brain neurons cultured in a serum-free medium and also in the conditioned medium of VR-2g [11] and BIM cells [9]. For the primary culture, embryonic day- 16 (E 16) murine striatum or embryonic day-18 (El8) cerebral cortex *Corresponding author. Fax: (81).(75) 761-5626.

neurons were used, because neuronal cells derived from these ages of fetal brains showed better responses to the conditioned medium (used as sources of neurotrophic factors) as tested in the preliminary experiments. As shown in Fig. 1, 2-mercaptoethanol (2-ME) added in a serum-free medium exhibited the prominent effects on the survival of both El6 striatum and El8 cerebral cortical neurons. Three points should be noted. Firs't, in the cultures of striatum neurons in a serum-free medium, the number of viable neurons drastically decreased on culture day 4, while in those in the 2-ME medium, it was not so, indicating that 2-ME can protect neurons from cell death. Second, at the optimal concentration of 2-ME (10 to 50/IM), the survival rates of cells were approximately 6-fold or 200-fold higher than those in control cultures for striatum or cortical neurons, respectively (Fig. 1). Third, when primary cultured cells were treated with M T T for determination of the viability as described in the legend of Fig. 1, cells were more densely stained in the presence of 2-ME than in the absence of 2-ME (data not shown), suggesting that the metabolic levels of cells, i.e. the mitochondrial activity to cleave M T T [1], were enhanced by 2-ME. 2-ME had also the ability to induce neurite outgrowth in striatum and cerebral cortical neurons, particularly being prominent in cortical neurons (Fig. 2). When cultured in serum-free medium alone, most of viable cortical cells had no or very short neurites (Fig. 2g), but when 2-ME was added in the serum-free medium, most of the

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Fig. 1. The dose effects of 2-mercaptoethanol on the viability of fetal brain neurons. E 16 striatum (left) and E 18 cerebral cortical neurons (right) were cultured for 4 days in a serum-free medium ( ), the conditioned medium of VR-2g cells (-.-) or BIM cells (---) with 0 to 500/.tM o f 2-mercaptoethanol. In the inset in the right panel, the survival rates in the presence of 0 to 5 # M of 2-mercaptoethanol are expressed in a logarithm. To compute the survival rate, cells were stained with MTT according to the method of Mosmann [1, 16]. Briefly, cells were incubated in 3-(4,5-dimethyl-2-thiazolyl)2,5-diphenyl tetrazolium bromide (MTT) (Sigma) (0.5 mg/ml) at 37°C for 90 min, and fixed in 10% formalin solution for 20 min. Viable cells will be stained dark blue because of the cleavage of the dye by the mitochondriat enzymes. Under the microscope (× 200 in the magnification), viable cells were counted in three optical fields per well in duplicate. The inoculation density corresponds to 200 cells per optical field. For cell dissociation, the fetal mouse brain tissue was cut into pieces, washed twice in PBS, and incubated at 37°C for 5 min in PBS for E 16 striatum or in 0.01% trypsin solution (Difco)/0.05% DNase-I for El8 cerebral cortex. Then, the tissue pieces were dissociated into single cells by trituration. Cells were washed twice in serum-free medium and inoculated in 96-well plates coated with poly-D-lysine (Sigma) at l04 cells per well. The serum-free medium was prepared according to the slight modification of the method of Bottenstein and Sato [3], that is, Dulbecco-Vogt's modification of Eagle M E M was supplemented with non-essential amino acids such as alanine (3 mg/l), asparagine-H20 (5 mg/l), aspartic acid (4.4 mg/1), glutamic acid (5 mg/l) and proline (11.5 mg/l) (called DMEM-N), transferrin (10 #g/ml), insulin (5 #g/ml), progesterone (5 nM), selenium salt (25 nM), putrescine (25 # M ) and bovine serum albumin (40 #g/ml). The conditioned media o f VR-2g [11] and BIM cells [10] were prepared to be used as sources of neurotrophic factors. Briefly, cells were inoculated at 1.6 × 106 cells per 100-mm dish in D M E M - N containing 10% fetal calf serum, and 2 days later, the medium was replaced by the serum-free medium. After 1 day, the culture fluids were collected, centrifuged at 3000 rpm for 10 min, and the supernatants were used.

viable cortical neurons extended long neurites (Fig. 2h) where 50% of the viable cortical cells had longer neurites than 67 /.tm (Table I). In contrast, striatum neurons formed only short neurites even when cultured in the medium containing 2-ME (Fig. 2d and Table I). Another interesting effect of 2-ME is that this drug enhanced the trophic activities of the conditioned medium (Fig. 1). First, the survival rates of both striatum and cortical neurons in the conditioned medium were increased several times by the addition of 2-ME (Fig. 1). Second, the ability of the conditioned medium to induce neurite outgrowth was drastically enhanced by the addition of 2-ME, particularly being prominent in striatum neurons (Table I). The cooperation of 2-ME and the conditioned medium in respect of the trophic activity was in a synergistic manner for striatum neurons and in an additive one for cortical neurons (Table I). In the present investigation, it has been clearly demon-

TABLE I THE P E R C E N T A G E OF N E U R O N S HAVING L O N G NEURITES IN VARIOUS C U L T U R E M E D I A El6 murine striatum and El8 cortical neurons were cultured for 3 days in various media, fixed in formalin, and photographed. The numbers of neurons bearing longer neurites than 67 # m were counted on the pictures, and the percentages of those to total viable neurons were computed. Values are mean + S.D. *Conditioned medium. #2-Mercaptoethanol ( 10 #M). Medium

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Fig. 2. The differentiation of E 16 fetal murine striatum and E 18 cerebral cortical neurons cultured for 3 days in various media, a f: striatum neurons; g,h: cortical neurons, a,g: serum-free medium: d,h: 2-mercaptoethnol (I 0/aM); b,e/c,f: the conditioned medium of VR-2g or BIM cells in the absence (b,c) or presence (e,f) of 2-mercaptoethanol (10/aM). Note that the cell bodies of neurons look white by halation, probably because of the membrane differentiation (star-marked in f.h). Bar = 50/am.

strated that 2-mercaptoethanol had the trophic activity by itself at least for cerebral cortical neurons and also the ability to enhance the trophic activity of the conditioned medium. The functional roles of 2-ME in the primary culture of neurons is not known at present. However, it has been well known that lymphocyte proliferation and activation are controlled by the thiol compounds such as 2-mercaptoethanol and glutathione (for review, refs 14 and 18). In this case, the intracellular uptake ofcystine in the culture medium is accelerated through its reduction by 2-ME [12], and then, the synthesis of glutathione and IL-2 are enhanced [5]. It is highly possible that similar mechanisms of 2-ME may function in primary cultures

of neurons. Moreover, it is possible that 2-ME is involved in regulatory mechanisms to release cells from the oxidation stress, since fetal neurons cultured in vitro which are known to be extremely sensitive to oxidation stress leading to cell death [4, 6, 13] were rescued from cell death by 2-ME as described here. These findings suggest two possibilities: first, the culture system described here will be available for development of bioassay systems to detect unknown neurotrophic factors of weak activities. Second, the studies of functional mechanisms of 2-ME will be useful for analysis of neurological disease such as Parkinson's disease and Alzheimer's disease which result from neuronal cell abnormality.

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