Quantity, distribution and phenotype of newly generated cells in the intact spinal cord of adult macaque monkeys

Abstract

The existence of proliferating cells in the intact spinal cord, their distribution and phenotype, are well studied in rodents. A limited number of studies also address the proliferation after spinal cord injury, in non-human primates. However, a detailed description of the quantity, distribution and phenotype of proliferating cells at different anatomical levels of the intact adult non-human primate spinal cord is lacking at present. In the present study, we analyzed normal spinal cord tissues from adult macaque monkeys (Macaca fuscata), infused with Bromo-2'-deoxyuridine (BrdU), and euthanized at 2h, 2 weeks, 5 weeks and 10 weeks after BrdU. We found a significantly higher density of BrdU + cells in the gray matter of cervical segments as compared to thoracic or lumbar segments, and a significantly higher density of proliferating cells in the posterior as compared to the anterior horn of the gray matter. BrdU + cells exhibited phenotype of microglia or endothelial cells (∼50%) or astroglial and oligodendroglial cells (∼40%), including glial progenitor phenotypes marked by the transcription factors Sox9 and Sox10. BrdU + cells also co-expressed other transcription factors known for their involvement in embryonic development, including Emx2, Sox1, Sox2, Ngn1, Olig1, Olig2, Olig3. In the central canal, BrdU + cells were located along the dorso-ventral axis and co-labeled for the markers Vimentin and Nestin. These results reveal the extent of cellular plasticity in the spinal cord of non-human primates under normal conditions.

Keywords: Central canal; Proliferation; Spinal cord; Transcription factor.

Fig. 1

Experimental paradigms. A. Time-frame of animal experiments and BrdU injection protocols used in the study. Group I is short term group; Groups II, III, IV could be combined in long term group. B. Schematic presentation of a horizontal section through the spinal cord. The regions of interest are annotated.

Fig. 2

Density of BrdU + cells, in cervical (A,D,G,J,M) or lumbal (B,E,H,K,N) segments. The images correspond to representative sections through the spinal cord of the short term survival animals (2h after BrdU). The spinal cord was divided in five regions of interest: anterior horn (AH), posterior horn (PH), anterior column (AC), lateral column (LC), posterior column (PC). Inset in A: a BrdU + doublet. Inset in E: BrdU + cell nuclei with a round morphology. Inset in G: BrdU + cell nuclei with an elongated morphology. Scale bar, 1 mm (N). Statistical evaluation of the density of BrdU + cells at different anatomical levels in the gray (C,F) or white (I,L,O) matter is presented by bar plots. *, P < 0.05.

Fig. 3

Reduction of BrdU + cells in long-term survival after BrdU infusion. Representative spinal cord micrographs taken from animals surviving 2h (A,C,E,G,I) or 5 weeks (B,D,F,H,J) after BrdU. The spinal cord is divided in five regions of interest: anterior horn (AH), posterior horn (PH), anterior column (AC), lateral column (LC), posterior column (PC). Single BrdU + nuclei are depicted by arrows while BrdU + doublets - by arrowheads. Scale bar, 1 mm (N).

Fig. 4

Statistical evaluation of BrdU + cell density and BrdU + cell “doublets” at various time points after BrdU. A. Number of BrdU + cells per section. *, P < 0.05. B. Proportion of BrdU + cells located in “doublets” at 2h, 2 weeks (wk), 5 weeks or 10 weeks following the infusion with BrdU. C. Double-labeling for BrdU (red) and Ki67 (green) in gray matter of the spinal cord at 2h survival after BrdU. Scale bar, 40 μm.

Fig. 5

Phenotype of BrdU + cells in the short-term survival group (2h after BrdU). A-E. Double-staining for BrdU (red) and different cell markers (green). Double-positive cells are depicted by arrows. F. Schematic map of the spinal cord indicating the position of the images from panels A–E. Scale bar, 10 μm (E).

Fig. 6

Phenotype of BrdU + cells in a long-term survival group (5 weeks after BrdU). A-E. Double-staining for BrdU (red) and different cell markers (green). Double-positive cells are depicted by arrows. F. Schematic map of the spinal cord indicating the position of the images from panels A–E. Scale bar, 20 μm (E).

Fig. 7

Proportions of proliferating cells expressing cell specific markers at different survival time points after BrdU: A- 2 h (2h), B, C, D- 2–10 weeks (2w/5w/10w).

Fig. 8

Proliferating cells express embryonic transcription factors in adult monkey spinal cord. Double-staining for BrdU (red) and different transcriptions (green) in the spinal cord of the short-term survival group (2h after BrdU). Double-positive cells are depicted by arrows. Scale bar, 20 μm (H).

Fig. 9

Location and phenotype of BrdU + cells in the central canal of the macaque spinal cord. A-C. BrdU immunolabeling of monkey sections at 2h (A) and 5 weeks (B) following BrdU infusion. The BrdU + cells are at the ventral or the dorsal poles of the canal (arrows). The images are aligned according the map in panel D. Insets demonstrate BrdU + cells within the ependymal layer. C. Statistical evaluation of the BrdU + cell density in the central canal after different survival times following BrdU application. *, P < 0.05. E. Immunostaining for Vimentin (green) and BrdU (red) demonstrates the dorso-ventral arrangement of the Vimentin + cells (E1; arrows). Double-labeled cells in E2-E5 are depicted by arrows. In panel E5, Vimentin + basal processes are depicted by arrowheads. F. Immunostaning for PSA-NCAM demonstrates numerous positive cells in the ependymal layer of the canal with the strongest signal at the dorsal pole (arrows). G. Dual immunolabeling for PSA-NCAM (green) and GFAP (red) demonstrates the lack of GFAP staining in the ependymal cells and a dense meshwork of fibers surrounding the canal (arrows). H. Anti-vWF (red) immunolabeling counterstained by DAPI shows the anatomical relation of blood vessels (arrows) to the central canal. I. Nestin/Vimentin double-staining revealing double-positive cells at the ventral and dorsal poles of the central canal (arrows). A basal process directed toward a blood vessel (bv) is depicted by an arrowhead. Asterisk, lumen of the central canal. Scale bars, 50 μm (A, B, E1, F1, G, H); 20 μm (F2, I3); 10 μm (E3).