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. 2015 Jan 1;24(1):51-66.
doi: 10.1089/scd.2014.0135.

Systemic injection of CD34(+)-enriched human cord blood cells modulates poststroke neural and glial response in a sex-dependent manner in CD1 mice

Shilpa D Kadam  1 , HuiGen Chen, Geoffrey J Markowitz, Saba Raja, Shanu George, Tatayana Verina, Elisabeth Shotwell, Brett Loechelt, Michael V Johnston, Naynesh Kamani, Ali Fatemi, Anne M Comi
Affiliations

Systemic injection of CD34(+)-enriched human cord blood cells modulates poststroke neural and glial response in a sex-dependent manner in CD1 mice

Shilpa D Kadam et al. Stem Cells Dev. .

Erratum in

  • Correction.
    [No authors listed] [No authors listed] Stem Cells Dev. 2015 Apr 1;24(7):916. doi: 10.1089/scd.2014.0135.cxn. Stem Cells Dev. 2015. PMID: 25785760 Free PMC article. No abstract available.

Abstract

Stroke in the developing brain is an important cause of neurological morbidity. We determined the impact of human cord blood-derived CD34(+)-enriched mononuclear cells (CBSC) intraperitoneally injected 48 h after an ischemic stroke at postnatal day 12 by evaluating poststroke neurogenic niche proliferation, glial response, and recovery in CD1 mice. Percent brain atrophy was quantified from Nissl-stained sections. Density of BrdU, Iba-1, and GFAP staining were quantified in the dentate gyrus and the subventricular zone (SVZ). Immunohistochemistry for human nuclear antibody, human mitochondrial antibody, and human CD34(+) cells was done on injured and uninjured brains from CBSC- and vehicle-treated mice. Developmental neurobehavioral milestones were evaluated pre- and post-treatment. No significant differences in stroke severity were noted between CBSC and vehicle-treated injured animals. With a 1×10(5) CBSC dose, there was a significant increase in subgranular zone (SGZ) proliferation in the CBSC-versus vehicle-treated stroke-injured male mice. SVZ glial fibrillary acidic protein (GFAP) expression was increased contralaterally in injured females treated with CBSC but suppressed in injured males. Significant negative correlations between severity of the stroke-injury and spleen weights, and between spleen weights and SGZ proliferation, and a positive correlation between GFAP expression and severity of brain injury were noted in the vehicle-treated injured mice but not in the CBSC-treated mice. GFAP expression and SVZ proliferation were positively correlated. In conclusion, neurogenic niche proliferation and glial brain responses to CBSC after neonatal stroke may involve interactions with the spleen and are sex dependent.

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Figures

<b>FIG. 1.</b>
FIG. 1.
Morphometric quantification of immunohistochemistry. (A–C) Quantification of glial fibrillary acidic protein (GFAP) expression in the granule cell layer and hilus of the dentate gyrus. Color images of GFAP immunolabeling (A) were converted to black and white (B) and then inverted in Adobe Photoshop. The dentate gyrus (DG) was subdivided into the granule cell layer (GCL) and the hilar regions, and immunopositive area was quantified (C) using MCID software. (D–F) Quantification of BrdU and GFAP expression in the subventricular zone (SVZ) proper, white matter dorsal to SVZ, and the striatum below the SVZ. The regions of interest were outlined using MCID software on DAPI-stained sections images taken at 10× and using the parameters indicated in (D). A line separating the striatum from white matter was drawn in linked images from GFAP-stained sections (E) and GFAP immunopositive proportional target area quantified in the three regions of interest. Then BrdU proportional target area was quantified in linked images from BrdU-stained sections (F). (G–I) Quantification of density of Iba-1-labeled cells in the SVZ. The SVZ was outlined using Axiovision software on DAPI-stained sections images taken at 10× and (G) linked to Iba-1-stained images (H, I) in which the number of Iba-1-labeled cells (I, arrow) in the SVZ were counted.
<b>FIG. 2.</b>
FIG. 2.
Developmental milestones following P12 ligation in CD1 mice. (A) Poststroke weight gain in ligation-injured CBSC- and vehicle-treated mice compared to naïve controls. Stroke-injured mice showed an age-dependent lag in weight-gain following stroke that remained significant at P21 (P<0.05) among both injured CBSC- (*) and vehicle-treated mice (‡). This lag did not show improvement in the CBSC-treated group of injured-mice when compared to the vehicle-treated group of injured mice. (B) Open-field: stroke-injured mice showed a delayed progress in open-field activity compared with naïve controls (P<0.05); however, by P21 they were not significantly different from controls; ligation-uninjured animals (n=8; three vehicle-treated and five CBSC-treated) were not, however, significantly different from ligation-injured animals (data not shown). CBSC-treated injured mice did not show significant improvements in open-field activity compared to the vehicle-treated group in the 7 day follow-up after treatment (ie, P14- P21). (C) Geotaxis: milestones for negative geotaxis scores were not significantly different in ligation-injured mice compared to naïve controls irrespective of treatment. (D) Forelimb grasp milestones were not significantly affected in the ligation-injured mice irrespective of treatment.
<b>FIG. 3.</b>
FIG. 3.
Severity of infarct injury following P12 ligations quantified at P21 (7 days following treatment with CBSC or vehicle at P14; A and B). No significant differences were noted in either hemispheric (left) or hippocampal atrophy (right) between CBSC-treated and vehicle-treated groups either when all animals were analyzed together (A, B) or when analyzed by animal sex (C, D).
<b>FIG. 4.</b>
FIG. 4.
Density of BrdU-positive cells in the subgranular zone (SGZ) after treatment with vehicle versus CBSC (A–C). A nonsignificant (P=0.065) increase was noted in density on the contralateral side of injured animals (A-right). However, when broken down by animal sex (B) this increase was significant and seen only in males ipsilaterally, P<0.05 (B-left). (C) Rostro-caudal analyses by hippocampal section along the AP axis revealed a significant sex difference by Poisson regression (see text, page 6, column 2) in CBSC-treated versus vehicle-treated injured littermates. The upregulation of SGZ proliferation was driven by CBSC-treated males within the data set. (*P<0.05).
<b>FIG. 5.</b>
FIG. 5.
BrdU expression in the SVZ and surrounding regions of vehicle-treated versus CBSC-treated males (left) and female (right) injured mice (A–D). A nonsignificant increase (P=0.085) was noted in BrdU expression in the contralateral SVZ region in injured females treated with CBSC (A). This trend was mirrored by similar average increases in the contralateral SVZ proper (B) and white matter above the SVZ (C) of injured females treated with CBSC that did not reach significance.
<b>FIG. 6.</b>
FIG. 6.
GFAP expression quantified in the granule cell layer and in the hilus of the dentate gyrus (A) and in the subventricular zone (b) of all injured and uninjured mice. GFAP expression was significantly increased ipsilaterally (A, B) in both neurogenic niches (GCL #P<0.05; Hilus ###P<0.0005; SVZ ##P<0.002) and was decreased contralaterally in the SVZ (B) 9 days after injury (###P<0.001).
<b>FIG. 7.</b>
FIG. 7.
Quantification of GFAP expression in the SVZ and surrounding regions of vehicle-versus CBSC-treated males (left) and female (right) injured mice (A–D). There was a trend for a decrease in GFAP expression in the SVZ region ipsilaterally in CBSC-treated males (P=0.07; A-left), while in females GFAP expression in the SVZ region was significantly increased (P<0.005; A-right). There were also trends for increased GFAP expression in the SVZ itself (P=0.08; B-right) and in the white matter above the SVZ (P=0.06; C-right) in injured females treated with CBSC. (*P<0.05).
<b>FIG. 8.</b>
FIG. 8.
Density of Iba-1 expression in the SGZ and SVZ of injured versus uninjured mice. Expression of Iba-1 was significantly increased in the SGZ hilus of injured mice after carotid ligation at P12 (###P<0.000005 ipsilaterally and ##P<0.005 contralaterally; (A). Average expression of Iba-1 in the ipsilateral SVZ was increased but not significantly (B).
<b>FIG. 9.</b>
FIG. 9.
Confocal images (60×) of fixed coronal brain sections from ipsilateral stroke-injured hemispheres (ie, infarct borders) of ligated mice treated with 1×105 CBSC-dosage of CD34+ enriched fraction of human CB cells or vehicle (Plasma Lyte A) stained with mouse-specific anti-CD68 and human mitochondrial (HuMit, MAB 1273) markers. Nonspecific labeling with HuMit antibody was noted in the injured hemispheres of both CBSC and vehicle-treated animals.
<b>FIG. 10.</b>
FIG. 10.
Apotome images (60×) of fixed coronal brain sections from ipsilateral stroke-injured hemispheres of ligated mice treated with 1×105 CBSC-dosage of CD34+-enriched fraction of human CB cells or vehicle (Plasma Lyte A) stained with human nuclear antibody (A and E, green), human CD34+ (B and F, red), and Dapi (arrow C and G, blue); merged images (D and H) demonstrate that double-labeling is imaging the same cell. Nonspecific labeling with antibodies was noted in the injured hemispheres of both CBSC (A–D) and vehicle-treated (E–H) animals.
See this image and copyright information in PMC

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