The in vivo time course for elimination of adrenalectomy-induced apoptotic profiles from the granule cell layer of the rat hippocampus

Abstract

Although apoptotic cellular degeneration has been reported to be extremely rapid with the use of in vitro models, the time needed to clear apoptotic neurons in the in vivo brain is unknown. In this study we used a simple morphological approach to solve this problem. Four days after adrenalectomy (ADX), all of the operated rats morphologically displayed hippocampal granule cell apoptosis that was prevented completely by corticosterone replacement immediately after ADX. Therefore, we intravenously injected the rats with corticosterone 4 d after ADX and subsequently maintained them on corticosterone replacement in saline drinking water. This corticosterone replacement could protect healthy granule cells promptly and continuously against hormone-deficient apoptosis, because the normal glucocorticoid receptor immunoreactivity within the granule cell nuclei, which disappeared after ADX, was identified 1 hr after corticosterone replacement was started, and this effect persisted for several days. However, this corticosterone treatment could not prevent the irreversible apoptosis of the already degenerated granule cells at various stages of the same progressive apoptotic process. Then we successively traced the disappearance of apoptotic granule cells throughout the hippocampus at different time points by Nissl and silver staining. Given that the apoptotic cells at the earliest stage of the degenerating process when the ADX rats received corticosterone injection were the last to disappear, the period from corticosterone injection until the disappearance of the last degenerating debris of apoptotic cells was taken to represent the time course for elimination of apoptotic neurons in vivo. We discovered that the elimination of apoptotic granule cells took 72 hr.

Fig. 1.

Silver-stained sections reveal degenerating cells within the suprapyramidal blade of the granule cell layer 4 d after ADX. Numerous silver-impregnated granule cells are seen after ADX. Different ADX rats exhibited varied numbers of the silver-stained granule cells (arrows in A,B). Scale bar, 200 μm (for A,B).

Fig. 2.

Nissl-stained sections show different morphological profiles of apoptotic granule cells 4 d after ADX. These apoptotic profiles are the clumping of chromatin within the nucleus of the normal-sized granule cell (the first type,arrow in A), condensation of the degenerating nucleus and cytoplasm into a darkly stained ball (the second type, arrows in B), gradual disassembly of the pyknotic granule cells into fragments (the third type, arrows from C–E), and the gradual disappearance of the degenerating debris (the fourth type, small arrows in E, F). Scale bars: in C, 15 μm; in F, 50 μm (applies to A, B,D–F).

Fig. 3.

Apoptotic cells with different morphological profiles 4 d after ADX are present within the granule cell layer at the same section. B–D are higher magnification views of the same sites labeled by small capital lettersb, c, andd in A. The apoptotic cells at the first (arrows in B), second and third (arrows in C, D), and fourth types (small arrow in D) are distributed in the same section. Most apoptotic granule cells exhibited the morphological profiles at the second and third types. Scale bars: in A, 200 μm; in D, 70 μm (forB–D).

Fig. 4.

Photomicrographs illustrate the silver-stained granule cells (arrows) after corticosterone replacement in rats 4 d after ADX. A–D show the silver-impregnated cells within the granule cell layer at 1, 12, 24, and 48 hr after corticosterone treatment, respectively. The disappearance of silver-impregnated granule cells was obvious in the inner portion of the granule cell layer. Scale bar, 150 μm.

Fig. 5.

Nissl-stained sections show the morphological changes of the apoptotic granule cells at different times after corticosterone replacement in the rats 4 d after ADX. At 1 (A) and 4 hr (B) after corticosterone replacement, different morphological profiles of apoptotic cells were identified. Large and medium-sized arrowsin A and B indicate the clumping of chromatin within the nucleus and pyknosis of apoptotic cells, respectively. The main morphological profile of apoptotic cells at 12 hr was pyknosis of degenerating cells (medium-sized arrows in C) and disassembly of pyknotic cells (open arrows in C). Double small arrows indicate the degenerating debris. At 24 hr later, the breaking up of pyknotic cells into fragments became the main morphological feature (open arrows in D). At 48 hr, degenerating debris with different sizes (double small arrows in E) still were observed. The apoptotic cells with clumping of chromatin within the nucleus disappeared from the granule cell layer at 12, 24, and 48 hr after corticosterone replacement. Scale bar, 80 μm.

Fig. 6.

Photomicrographs illustrating silver-stained granule cells and GR–IR within the hippocampus. In the sham-operated rat no silver-stained granule cells were seen (open arrows in A), and the normal GR–IR is present within the granule cell layer (arrows inB). At 4 d post-ADX, numerous silver-stained cells (arrows in C) and the complete disappearance of GR–IR were present in the hippocampus (open arrows in D indicate the granule cell layer). The reappearance of normal GR–IR in the granule cell layer (arrows in F) was identified clearly 1 hr after corticosterone replacement, although many silver-stained cells (arrows in E) still were distributed within the suprapyramidal blade of the granule cell layer. Scale bars: in E, 200 μm (for A,C, E); in F, 250 μm (forB, D, F).