Postnatal changes in Bax-immunoreactivity and apoptosis of the rat trigeminal primary neurons

Neuroscience Letters 258 (1998) 97–100

Postnatal changes in Bax-immunoreactivity and apoptosis of the rat trigeminal primary neurons Tomosada Sugimoto*, Chun Xiao, Hiroyuki Ichikawa Department of Oral Anatomy II, Okayama University Dental School, 2-5-1 Shikata-cho, Okayama, 700-8525, Japan Received 10 October 1998; received in revised form 23 October 1998; accepted 23 October 1998

Abstract The developmental changes of Bax protein-like immunoreactivity (Bax-ir) and naturally occurring cell death were investigated in the trigeminal primary neurons of rats, in the first 2 postnatal weeks. At 1 day postpartum, the trigeminal primary neurons exhibited intense cytoplasmic Bax-ir. A densitometric analysis indicated .95% of cells exhibited an ir-density (the staining intensity as compared to the background level) higher than 1.5. By 2 weeks the ir-density significantly decreased with .95% lower than 1.5, that was similar to the adult level. A nick-end labeling method revealed DNA fragmentation in apoptotic trigeminal primary neurons in newborn rats. During the first 11 days the percentage of apoptotic cells was highly correlated to the postnatal days with a regression line y = −0.083x + 0.877 (r = 0.976). A possible role of Bax in neonatal rat primary neurons is discussed.  1998 Elsevier Science Ireland Ltd. All rights reserved

Keywords: Bax; Apoptosis; Primary sensory neurons; Trigeminal ganglion; Newborn rat

A protein Bax heterodimerizes with Bcl-2 and counteracts Bcl-2’s protection against apoptosis [7]. In addition, Bax appears to have a proapoptotic activity by itself [1]. We recently found that sensory primary neurons of rats contained Bax protein-like immunoreactivity (Bax-ir) [8]. In adult rats, active turnover of primary neurons has not been reported, and the rate of cell death, if any, appears to be very low [4]. However, primary neurons newborn rats are highly susceptible to injury-induced apoptosis [9]. Moreover, a small proportion of the neonatal rat trigeminal ganglion (TG) neurons underwent DNA fragmentation without intentional stimulation [9]. In this study, we investigate postnatal development of Bax-ir and naturally occurring cell death in the rat TG neurons. Sixteen Sprague–Dawley rats of either sex were killed at 1 day (18 ± 6 h, n = 4), 1 week (n = 4), 2 weeks (n = 4) and 8 weeks (adult, n = 4), postpartum. They were deeply anesthetized by ether inhalation, and perfused through the left ventricle with saline followed by 4% formaldehyde in 0.1 M phosphate buffer (pH 7.3). The TGs were cryopro* Corresponding author. Tel.: +81 86 2356635; fax: +81 86 2356639; e-mail: [email protected]

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tected in 20% sucrose in 0.02 M phosphate-buffered saline (PBS, pH 7.3), and stored for up to 4 days at −80°C. Horizontal 10-mm thick cryosections from each of the four developmental stages were mounted on the same gelatincoated slides, air-dried and stained for Bax-ir, simultaneously. Similarly prepared sections were also processed for nick-end labeling of DNA fragments (TUNEL). 10 additional rats (two for each day) were killed at 2, 3, 5, 9 and 11 days, and the TGs processed only for TUNEL. Bax-ir was labeled by an avidin-biotin-HRP complex (ABC) method [8]. In brief, the sections were incubated with rabbit anti-Bax IgG (10 ng/ml, P-19, Santa Cruz Biotechnology, CA) for 4 overnights at 4°C followed by biotinylated goat anti-rabbit IgG and ABC complex (Vectastain ABC kit, Vector Laboratories, CA). The label was visualized by a nickel ammonium sulfate-intensified diaminobenzidine (DAB) histochemistry. The sections were dehydrated and cover-slipped with Entellan (Merck, FRG). The specificity of the antibody has been described [8]. TUNEL was performed according to Gold et al. [6] with a slight modification. Endogenous peroxidase activity was quenched by incubating with 0.3% H2O2 in 80% methanol for 60 min at RT. After thorough rinsing with Tris–HCl-

 1998 Elsevier Science Ireland Ltd. All rights reserved

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buffered saline (TBS, pH 7.4), the sections were incubated with a tailing buffer (25 mM Tris–HCl buffer, 1 mM CoCl2, 200 mM sodium cacodylate, 0.025% bovine serum albumin, pH 6.6) containing 0.1 mM dATP, 0.01 mM biotin-16dUTP and 250 units/ml terminal deoxinucleotidyl transfer-

ase from calf thymus (Boehringer-Mannheim, FRG) for 60 min at 37°C. The enzymatic reaction was stopped by rinsing with 0.05% Tween 20 in PBS. For negative control, either the enzyme or biotin-16-dUTP was omitted from the system. The sections were further reacted with ABC complex,

T. Sugimoto et al. / Neuroscience Letters 258 (1998) 97–100

and the label was visualized by the DAB histochemistry. The sections were counter-stained with cresyl violet. For electron microscopy, two rats were killed at 1 day. They were perfused with 1% glutaraldehyde, 1% formaldehyde and 0.2 mM CaCl2 in 0.12 M phosphate buffer (pH 7.3). One-hundred-micrometer thick vibratome sections of the TG were processed for TUNEL as described above, except that quenching of the endogenous peroxidase was totally omitted and that plain PBS (without Tween 20) was used for the stop bath. After the DAB reaction, the sections were osmicated, dehydrated and embedded in an epoxy resin. Serial thin sections were lightly contrasted with lead citrate. Granular Bax-ir was laid over the perikaryal cytoplasm but not the nucleus of TG neurons (Fig. 1A–D). Axons showed weak immunoreactivity. This basic staining pattern was unchanged but the intensity greatly decreased with time. The microscopic image of the maxillary division of TG was taken by computer and a densitometric analysis was performed using the NIH Image program. The cytoplasmic optical density of all the neuronal cell bodies including the nuclear profile was measured in the field, and divided by the background density. The quotient was recorded for each cell and will be referred to as the (Bax-) ir-density, hereafter. The average ir-density was calculated for each rat (n, 95– 110). At 1 day, the neurons were uniformly small (diameter ,20 mm) (Fig. 1A). The ir-density ranged from 1.38 to 1.89 with more than 95% of cells falling in a range higher than 1.5 (Fig. 1E). By 2 weeks the largest cells reached about 30 mm, and the ir-density decreased in the range 1.02–1.64 with more than 95% lower than 1.5 (Fig. 1C,E). Most neurons at 2 weeks, therefore, had a lower irdensity than at 1 day. The larger the cell bodies, the lower the ir-density was (Fig. 1C). The cell size and the ir-density at 1 week were in between (Fig. 1B,E). After 2 weeks, the change in ir-density was small but the cell body continued to enlarge (Fig. 1D,E). In the adult, the largest cells exceeded 60 mm. The difference in average ir-density between developmental stages was highly significant (P , 10−4, AN OVA) except between 2 and 8 weeks (Fig. 1F).

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TUNEL revealed DNA fragmentation during the first postnatal week (Fig. 2A,B). In lightly stained profiles, dark shriveled nuclei could be barely distinguished from relatively pale cytoplasm. In most labeled profiles, however, the intense DAB reaction products obscured the subcellular detail (Fig. 2B). In these, the neuronal identity was solely derived from their shape and location; they were round, of a size comparable to small to medium neurons, and located in the cluster of Nissl-stained neurons. Electron microscopy revealed that the labeled neurons had undergone morphological changes similar to the apoptosis, induced by neonatal capsaicin treatment [9]. On light microscopic examination of the plastic embedded tissue, the labeled structures varied 8–15 mm in diameter. Smaller ones turned out to be neurons in which the DNA fragmentation signal was confined to the nucleoplasm (Fig. 2C). In such neurons the membranous organelles appeared healthy except that the nuclear contour was somewhat ruffled with irregular indentations. Coarse granular label resembling the euchromatin were dispersed throughout the nucleoplasm. In addition, a few large masses of concentrated signal could be also seen in the nucleoplasm. In larger structures, the signal had spread all over the cytoplasm (Fig. 2D,E). Many of these neurons appeared to have undergone cytoplasmic condensation. Cytoplasmic organelles mainly consisting of mitochondria were aggregated in a part of the cytoplasm, while the rest of the cytoplasmic area was filled with tightly packed electron dense granules (Fig. 2D,E). The nuclear envelope had disappeared but a few large amorphous areas of various electron densities, that have been demonstrated in the capsaicin-induced apoptotic nucleus [9], were seen among organelles (Fig. 2D,F). Despite the above, the outer membrane of the cell appeared to have maintained the continuity because the extracellular discharge of organelles or DNA fragmentation signal was not noted. In view of the above, the initial event of the cytopathology was intranuclear DNA fragmentation, that was followed by the extranuclear spread of the DNA fragments and cytoplasmic condensation. The membrane deterioration has not been observed in the TUNEL-positive structures, so far. Therefore, we consider the TUNEL-

Fig. 1. Bax protein-like immunoreactivity in TG neurons at 1 day (A), 1 week (B), 2 weeks (C) and 8 weeks postpartum (D). At 2 and 8 weeks, the smaller cells exhibit the higher ir-density (arrows in C and D). Scale bar, 50 mm in (D) applies to the rest of micrographs. (E) Percentage distribution diagrams of cells plotted against the Bax-ir density. At 1 day >95% of cells exhibit a density higher than 1.5, while >95% lower than 1.5 at 2 weeks or in the adult (sample sizes for 1 day, 1 week, 2 weeks and adult are 412, 410, 420 and 432 cells form four rats each). (F) Mean ± SD of average ir-density (95–119 cells from each rat, n = 4) at each developmental stage. *The difference from the rest of all the developmental stages was statistically significant (P , 10−4, ANOVA). Fig. 2. Light (A,B) and electron micrographs (C–F) of TUNEL-positive TG neurons at 1 day (A,C–F) and 1 week postpartum (B). (A) The TUNELpositive cells (arrows) are similar in size to the healthy neurons surrounding them. Scale bar, 100 mm._(B) The subcellular detail is obscured by the intense DAB reaction. Scale bar, 10 mm. (C) This neuron exhibits the DNA fragmentation signal only in the nucleus (n). (Inset) Serial section shows an apparently normal Golgi apparatus (arrow) and mitochondria with well-preserved cristae (arrowheads). Scale bars, 1 mm. (D,E) An example of neuron whose nuclear envelope has been disrupted. A large aggregation of cytoplasmic organelles occupies most cytoplasmic area in one section (D), while in the other the cytoplasm is largely filled with tightly packed granular density (asterisk in E). This cell contains two large amorphous lipid-like particles (arrows in D). Scale bars, 2 mm. (F) Another example of neuron containing a large amorphous particle (p). Despite the disruption of nuclear envelope, mitochondria have well-preserved cristae (arrows). The serial section analysis revealed that the cell in (F) was unequivocally labeled at the tissue surface. Scale bar, 1 mm. (G) The rate of apoptosis (%) plotted against the postnatal interval (days).

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positive neurons observed in this study to be apoptotic. For quantification, three cryosections were randomly chosen for each rat. Total cell count for each section (each rat) varied in the range 1096–2006 (3585–5892). The percentage of apoptotic cells was calculated and recorded for each rat. The mean ± SD of the percentage of apoptotic cells at 1 day was 0.83 ± 0.03 (n = 4). The apoptotic cells decreased to 0.26 ± 0.01% at 1 week (n = 4). The percentage of apoptotic cells (y) was highly correlated to the postnatal interval (x days) in the period 1–11 days postpartum (y = −0.083x + 0.877, r = 0.976, Fig. 2G). Apoptotic cells were extremely rare at 2 weeks or absolutely none at 8 weeks. Progenitors of the rat primary neurons complete proliferation during the embryonic life [3,10]. Thus produced neurons send axonal branches to the peripheral and central targets, and maintain them throughout the rest of the life. However, about 16% of rat dorsal root ganglion cells are lost in a few days after birth [3]. This study showed that a small but readily recognizable proportion of rat TG neurons underwent apoptosis during the first postnatal week. Elimination by apoptosis is a rapid event and apoptotic cells in tissues remain recognizable only for 1–9 h [2,5]. It is, therefore, likely that apoptosis detected in a small fraction of cells at one time culminates to a substantial cell loss with time. The proportion r (%) of labeled cells plotted against the postpartum interval t (hours) well fit a line r = f(t) = −0.0035t + 0.877 [24 ≤ t ≤ 244, r = 0.976] (Fig. 2G). The total cell count at t hours postpartum [T(t)] consists of labeled [N + = T(t) × (f(t)/100] and unlabeled cells [N− = T(t) × ((1 - f(t)/100)]. If apoptosis of a given cell could be detected for a period of S (hours), the N+ must have been added to the detectable population within the last S hour-period and will be lost within the next S hour-period. The total cell count at (t + S) [T(t + S)] would be equal to T(t) × ((1 − f(t)/100), and consist of labeled [T(t + S) × (f(t + S)/100] and unlabeled cells [T(t + S) × (1 − f (t + S)/100)]. By repeating the calculation we can estimate the proportion of cell loss during the first 11 postnatal days. Using the duration of detectability (4–9 h) by Arends and Wyllie [2], the expected cell loss was calculated to be 12– 24%. The figure fairly well coincides with the 16% cell loss as obtained by an actual cell counting method [3]. A high level of Bax-ir during the first postnatal week suggests transient overexpression of bax in the developing rat TG neurons. Concomitantly, the TG neurons exhibited a high apoptotic. Toward the end of the second postnatal week, Bax-ir rapidly decreased to the adult level, and the apoptotic activity subsided. Although the exact anti- and

pro-apoptotic mechanisms of the Bcl-2 family members are unclear, it is widely accepted that the ratio of Bcl-2/ Bax is a major determinant of a cell’s susceptibility to apoptosis [7]. For example, overexpression of bcl-2 protected cultured sympathetic neurons that were deprived of nerve growth factor (NGF) [1]. Contrarily, overexpression of bax counteracted Bcl-2’s protection and promoted apoptosis [1,7]. Based on correlative observations on dorsal root ganglion cell count and peripheral innervation, Coggeshall et al. [3] suggested that postnatal loss of primary neurons may reflect competition for peripherally synthesized trophic factors such as NGF. This study indicated such neuronal selection is conducted by apoptosis. The high level of Bax-ir may contribute to natural selection of primary neurons during the first postnatal week of rats. It may also explain the primary neurons’ susceptibility to apoptotic stimuli, such as capsaicin and axotomy, in newborn rats [9]. [1] Antonsson, B., Conti, F., Ciavatta, A.M., Montessuit, S., Lewis, S., Martinou, I., Bernasconi, L., Bernard, A., Mermod, J.-J., Mazzei, G., Maundrell, K., Gambale, F., Sadoul, R. and Martinou, J.-C., Inhibition of Bax channel-forming activity by Bcl-2, Science, 277 (1997) 370–372. [2] Arends, M.J. and Wyllie, A.H., Apoptosis: mechanisms and roles in pathology, Int. Rev. Exp. Pathol., 32 (1991) 223–254. [3] Coggeshall, R.E., Pover, C.M. and Fitzgerald, M., Dorsal root ganglion cell death and surviving cell numbers in relation to the development of sensory innervation in the rat hindlimb, Dev. Brain Res., 82 (1994) 193–212. [4] Devor, M., Govrin-Lippman, R., Frank, I. and Raber, P., Proliferation of primary sensory neurons in adult rat dorsal root ganglion and the kinetics or retrograde cell loss after sciatic section, Somatosens. Res., 3 (1985) 139–167. [5] Gavrieli, Y., Sherman, Y. and Ben-Sasson, S.A., Identification of programmed cell death in situ via specific labeling of nuclear DNA fragmentation, J. Cell Biol., 119 (1992) 493–501. [6] Gold, R., Schmied, M., Giegerich, G., Breitschopf, H., Hartung, H.P., Toyka, K.V. and Lassmann, H., Differentiation between cellular apoptosis and necrosis by the combined use of in situ tailing and nick translation techniques, Lab. Invest., 71 (1994) 219–225. [7] Oltvai, Z.N., Milliman, C.L. and Korsmeyer, S.J., Bcl-2 heterodimerizes in vivo with a conserved homolog. Bax, that accelerates programmed cell death, Cell, 74 (1993) 609–619. [8] Sugimoto, T., Xiao, C., He, Y.-F. and Ichikawa, H., Bax proteinlike immunoreactivity in primary sensory and hypothalamic neurons of adult rats, Neurosci. Lett., 215 (1996) 37–40. [9] Sugimoto, T., Xiao, C. and Ichikawa, H., Neonatal primary neuronal death induced by capsaicin and axotomy involves an apoptotic mechanism, Brain Res., in press. [10] White, F.A., Chiaia, N.L., Macdonald, G.J. and Rhoades, R.W., Birth dates and survival after axotomy of neurochemically defined subsets of trigeminal ganglion cells, J. Comp. Neurol., 325 (1995) 308–320.