Proliferation and differentiation of progenitor cells throughout the intact adult rat spinal cord

Abstract

The existence of multipotent progenitor populations in the adult forebrain has been widely studied. To extend this knowledge to the adult spinal cord we have examined the proliferation, distribution, and phenotypic fate of dividing cells in the adult rat spinal cord. Bromodeoxyuridine (BrdU) was used to label dividing cells in 13- to 14-week-old, intact Fischer rats. Single daily injections of BrdU were administered over a 12 d period. Animals were killed either 1 d or 4 weeks after the last injection of BrdU. We observed frequent cell division throughout the adult rodent spinal cord, particularly in white matter tracts (5-7% of all nuclei). The majority of BrdU-labeled cells colocalized with markers of immature glial cells. At 4 weeks, 10% of dividing cells expressed mature astrocyte and oligodendroglial markers. These data predict that 0.75% of all astrocytes and 0.82% of all oligodendrocytes are derived from a dividing population over a 4 week period. To determine the migratory nature of dividing cells, a single BrdU injection was given to animals that were killed 1 hr after the injection. In these tissues, the distribution and incidence of BrdU labeling matched those of the 4 week post injection (pi) groups, suggesting that proliferating cells divide in situ rather than migrate from the ependymal zone. These data suggest a higher level of cellular plasticity for the intact spinal cord than has previously been observed and that glial progenitors exist in the outer circumference of the spinal cord that can give rise to both astrocytes and oligodendrocytes.

Fig. 1.

Quantitative method for counting BrdU-positive nuclei in the intact spinal cord. Coronal sections (40 μm) from C7, T8, and L2 were selected for quantitation and immunohistochemically labeled for BrdU (A). Electronic templates that divided the spinal cord into concentric annuli (B) and radial sectors (C) were overlaid onto stained sections. With the aid of an image analysis system, the perimeters of the templates were delineated by electronically circumscribing the outer circumference of each tissue section (D). The limit was drawn just beneath the pial surface. All BrdU-positive nuclei were counted, and the area of each region was calculated after the regions were delineated. BrdU-labeled cells at the ependymal layer of the central canal were counted independently. BrdU distribution and cell migration was determined in a group of animals, where a single injection of BrdU was given followed by killing at 1 hr or 1 day after injection (E, F). Three consecutive 40 μm sections from a representative animal were traced at the level of T8.Dots indicate the presence of a BrdU-labeled nucleus. Each section and corresponding nuclei are represented as a separate color. At 1 hr pi most BrdU-labeled cells are found as single nuclear profiles, and nuclei are distributed in the medial and outer circumference of the spinal cord (E). At 1 day pi, the number of BrdU-labeled nuclei increases, and many of the nuclear profiles are located in clusters of two (arrows) and four (arrowhead) nuclei, indicating cell division.

Fig. 2.

Immunohistochemical staining for BrdU in the adult cervical spinal cord. Photomicrographs are taken from 1 d after the last of 12 daily BrdU injections. BrdU-labeled nuclei were most commonly found in the outer white matter where they typically exhibited small, ellipsoid nuclei that were associated with radial elements (A, arrows indicate radial elements, 200×). A less common but distinct nuclear morphology was that of a rounded, large nucleus not associated with radial elements of the spinal cord (B, 200×). Dense BrdU labeling was also noted in the gray matter, particularly in the superficial dorsal horn (C, 100×). The substantia gelatinosa (delineated bylines) was the only gray matter region where clusters of BrdU-labeled nuclei were noted (arrowheads). BrdU labeling was rarely noted in the ependymal layer of the central canal, but occasional clusters of these cells could be detected (D, 400×). BrdU-labeled pial cells were also noted (E, arrows, 400×). The template maps the anatomical origin of photomicrographs A–E.

Fig. 3.

Quantitation of BrdU-labeled nuclei using an annulus template. BrdU-positive nuclei were counted using a template that divides the spinal cord into five concentric annuli consisting of 200 μm steps beginning at the central canal. The number of BrdU-positive nuclei is expressed as a percentage of the total number of DAPI-labeled nuclei for each region. All comparisons are corrected for surface area. The index of BrdU increases from the medial to the outer annuli, indicating a gradient of cell division. In addition, only a small decrease in BrdU density is found between the 1 d and 4 week pi time period. This finding indicates that labeled cells persist for at least 4 weeks. (*p < 0.05 when compared to the 0–200 μm annulus, †p < 0.05 when compared to the 600–800 μm annulus.)

Fig. 4.

Quantitation of BrdU-labeled nuclei using a sector template. BrdU-positive nuclei were counted using a template that divides the spinal cord into radial sectors. Dorsal, lateral, and ventral sectors from C7 are compared. The number of BrdU-positive nuclei is expressed as a percentage of the total number of DAPI-labeled nuclei for each region. Comparisons are corrected for surface area. No statistical differences were noted among sectors. This finding indicates that cell division cannot be modeled by dorsal to ventral gradients. Non-pooled sectors were also compared, but no statistical differences were found (data not shown).

Fig. 5.

Comparison of the incidence of BrdU-positive nuclei at all three spinal segments. The gradient of BrdU-positive nuclei from the medial to outer annuli described for the cervical cord is also present at thoracic and lumbar levels. The number of BrdU-positive nuclei is expressed as a percentage of the total number of DAPI-labeled nuclei for each region, and comparisons are corrected for surface area. Note that the concentration of BrdU-labeled cells is initially higher in the outer annulus of cervical and lumbar versus that of the thoracic cord (†p < 0.05 when compared to the same region and time of the thoracic spinal cord). Importantly, cells persist at all levels with only a slight decrease in BrdU number between the 1 d and 4 week time points (*p < 0.01 when compared to the medial annulus of the same spinal level).

Fig. 6.

NG2 colocalization with BrdU in the adult spinal cord. NG2 immunoreactivity was used to classify BrdU-positive cells as glial progenitor cells. NG2 immunoreactivity was found throughout the spinal cord and labeled cells exhibited unipolar, bipolar, and multipolar cell morphologies in both gray and white matter. NG2 colocalized with BrdU more commonly than any other immunohistochemical marker. Throughout the spinal cord BrdU/NG2 colocalized cells were found that did not colabel with the glial marker APC (A, arrowhead, 100×). Many BrdU/NG2-colabeled cells were located near the pial layer with processes extending into this region (A, arrows). These cells were not found near the central canal. The predominant morphology was that of a bipolar cell (B, top, 400×). At 4 weeks, a small population had complex, multipolar processes (B, bottom, 400×). These cells did not colocalize with the mature glial marker APC. At 1 hr after a single pulse of BrdU, most BrdU-labeled cells colocalized with NG2 (C, 630×). Single confocal sections of each marker are presented for both NG2-immunoreactive cells.

Fig. 7.

BrdU/S100β colocalization in the adult spinal cord. Confocal microscopy was used to determine the incidence of BrdU/S100β colocalization. A confocal z-series allows examination of nuclei through their entire z-axis in 1 μm steps (A, 400×). In this series a BrdU-positive nucleus (red only) is associated with S100β immunoreactivity (green only). These markers consistently colocalize (red and greenmerge) throughout the series (arrowheads). S100β astrocytes (arrowhead) often exhibited a radial morphology with long central to lateral processes (B,arrows, 200×). Occasionally BrdU-positive astrocytes had processes that contained a lumen associated with microvascular elements (C, arrows, 800×).

Fig. 8.

APC, GFAP, and RIP colocalization with BrdU in the adult spinal cord. APC and GFAP immunoreactivity were used to classify BrdU-positive cells into immature oligodendrocytes or astrocytes. APC immunoreactivity was found throughout the spinal cord, especially in the white matter where astrocytes and oligodendrocytes were found in radially oriented chains (A, arrows, 200×). Astrocytes were characterized by a small somal size and colocalization with GFAP (A, arrowhead;B, 800×). Oligodendrocytes were also detected with this method. Oligodendrocytes contained large, rounded cell bodies that did not colocalize with GFAP (C, 8000×). Separate color channels are presented at the bottom of B andC. Mature oligodendrocytes were identified by colocalization of BrdU and RIP immunoreactivity (D, arrowhead, 400×). Confocal microscopy was used to determine if RIP-positive cell bodies contained BrdU-positive nuclei (E, 800×). Separate confocal channels are presented to the right.

Fig. 9.

Quantitation of BrdU-labeled cells in the adult spinal cord. One hundred BrdU-positive cells from at least three sections from spinal levels C7, T8, and L2 were examined with a confocal microscope. Only nuclei that could be localized to one of the phenotypic markers throughout the z plane were considered positive. Medial measurements are taken from the medial annulus (0–600 μm from the central canal), and outer measurements are taken from the outer annulus (>600 μm from the central canal). Cells were counted as oligodendrocytes (A) if they were immunoreactive for RIP/BrdU or APC/GFAP−/BrdU. At all spinal levels the number of oligodendrocytes was low 1 d after the last BrdU injection, but significantly increased to 5% at 4 weeks. APC/GFAP+/BrdU-labeled astrocytes followed a similar progression (B) and significantly increased at the 4 week time point. S100β/BrdU-immunoreactive astrocytes (B) represented a unique population in that their numbers did not significantly increase over time. Note that 1 d after the last injection, the number of S100β/BrdU-immunoreactive astrocytes in the outer annulus is ∼6% and remains stable at 4 weeks (C). The most abundant population of BrdU-labeled cells were immunoreactive for NG2/BrdU. This population of glial progenitors was ∼50–60% of the total BrdU-labeled cells at 1 d. Although there was a trend toward decreased NG2/BrdU colabeling at 4 weeks, this did not reach statistical significance. A small portion of BrdU labeling was associated with microglial or microvascular markers (D). OX-42/BrdU-immunoreactive microglia and RECA/BrdU-immunoreactive endothelial cells accounted for <2% of the total BrdU, and these populations decreased significantly at 4 weeks. (*p < 0.01 when compared to the 1 d time point of the same phenotype).

Fig. 10.

Models of stem cell proliferation and migration in the intact adult spinal cord. Three models illustrate how dividing stem cells may give rise to progenitors that migrate and proliferate. Model I corresponds to early postnatal gliogenesis. The current data suggest that in the adult, dividing cells are located primarily in the outer circumference of the spinal cord, and therefore Models II and III more likely reflect adult gliogenesis in the intact spinal cord.