Neuroprotective and antiapoptotic activity of lineage-negative bone marrow cells after intravitreal injection in a mouse model of acute retinal injury

Abstract

We investigated effects of bone marrow-derived, lineage-negative cell (Lin(-)BMC) transplantation in acute retinal injury. Lin(-)BMCs were intravitreally injected into murine eyes at 24 h after NaIO3-induced injury. Morphology, function, and expression of apoptosis-related genes, including brain-derived neurotrophic factor (BDNF) and its receptor, were assessed in retinas at 7 days, 28 days, and 3 months after transplantation. Moreover, global gene expression at day 7 was analyzed by RNA arrays. We observed that Lin(-)BMCs integrated into outer retinal layers improving morphological retinal structure and induced molecular changes such as downregulation of proapoptotic caspase-3 gene, a decrease in BAX/BCL-2 gene ratio, and significant elevation of BDNF expression. Furthermore, transplanted Lin(-)BMCs differentiated locally into cells with a macrophage-like phenotype. Finally, Lin(-)BMCs treatment was associated with generation of two distinct transcriptomic patterns. The first relates to downregulated genes associated with regulation of neuron cell death and apoptosis, response to oxidative stress/hypoxia and external stimuli, and negative regulation of cell proliferation. The second relates to upregulated genes associated with neurological system processes and sensory perception. Collectively, our data demonstrate that transplanted Lin(-)BMCs exert neuroprotective function against acute retinal injury and this effect may be associated with their antiapoptotic properties and ability to express neurotrophic factors.

Figure 1

Quantification of the hematopoietic and mesenchymal origin of a BM-derived Lin⁻ cell population by flow cytometry. Lin⁻BMC distribution based on FSC (Forward Scatter) and SSC (Side Scatter) parameters that describe their size and granularity, respectively (a). The cells enclosed in region P1 were further subdivided into hematopoietic (region P2) or nonhematopoietic (region P3) cell populations based on their CD45 antigen expression (b). CD45⁻ BMCs were additionally analyzed based on their CD29 and CD105 antigen expression to determine their mesenchymal origin (c). Similarly, CD45⁺ BMCs were analyzed according to their CD34 antigen expression to exclude mature hematopoietic cells and to determine the number of hematopoietic stem/progenitor cells (d). Expression of mRNA for BDNF in isolated Lin⁻BMCs and BM-derived mononuclear cells (e). Immunocytofluorescence image of Lin⁻BMCs depicts the expression of BDNF protein (red). Nuclei are visualized with DAPI staining (blue) (f). Scale bar = 20 μm. ^* P < 0.05.

Figure 2

In vivo monitoring of injected cell in damaged retinal tissue. A representative SD-OCT image of an injured retina on the 7th day after Lin⁻BMC transplantation (a). The green lines on the OCT images (left side) indicate the boundaries of tissue depth displayed in an en face two-dimensional fundus projection (right side). The depth-dependent fundus image at this time point shows injected cells in the vitreous. The cells were uniformly distributed and were homogenous in appearance. The SD-OCT analyses on the 28th day (b) and the 3rd month (c) showed the absence of the injected Lin⁻BMCs in the vitreous cavity.

Figure 3

Longitudinal follow-up of the fate of Lin⁻BMCs at different time points post intravitreal transplantation. Immunofluorescence analysis of retinas after intravitreal GFP⁺Lin⁻BMCs delivery showed transplanted cells (green) on the 7th day (a, b) and at 3 months after injection (c). Quantitative analysis of the total number of incorporated Lin⁻BMCs per eye section (n = 5/time point) (d). Coreactivity of GFP (green) and Isolectin-B4 (red) by fluorescent lectin staining showed that transplanted GFP⁺Lin⁻BMCs locally differentiated into Isolectin-B4-positive macrophages (inserts) in Lin⁻BMC-injected eyes on the 7th day after transplantation (e) and at 3 months after injection (f). Scale bar = 20 μm. ^* P < 0.05 for day 7 versus other time points.

Figure 4

Molecular analyses of potential in vivo effects of Lin⁻BMCs on injured retinas at different time points post injury. BDNF mRNA (a) and BDNF protein (b-upper panel) expression time course analyses show a significant increase in Lin⁻BMC-treated eyes compared to PBS-injected eyes on the 7th day after transplantation. Immunoblotting for the BDNF, pMAPK, tMAPK, PCNA, and active caspase-3 of retinal lysates collected from control, uninjured animals (Control), PBS injected and Lin⁻BMC-treated mice on d7, d28, and m3 after injury (b). Double-stained retinal sections (anti-BDNF and anti-GFP) were used to visualize the coexpression of BDNF and GFP cell markers in transplanted Lin⁻BMCs (insert) (c). Quantitative PCR analysis for relative quantification of TRK-B mRNA displayed a markedly increased level on the 7th day after transplantation (d). Representative immunofluorescence for Trk-B expression following Lin⁻BMC transplantation revealed that its expression is mostly restricted to RPE/photoreceptor junction (e). The western blot analysis and densitometry for relative protein quantification of the active, phosphorylated form of p44/42 MAPK (Erk1/2) revealed its markedly increased expression on d7 and d28 in the Lin⁻BMC-transplanted retinas (f). The western blot analysis and densitometry for relative PCNA quantification demonstrated a strong overexpression of the protein in the retinas collected from the Lin⁻BMC-treated mice on d28 and m3 after injury (g). Mean values ± SDs are presented in the diagrams. Double-stained sections (anti-GFP and anti-PCNA) were used to visualize the location of proliferating Lin⁻BMCs (insert) (h). Quantitative PCR analysis of BAX/BCL-2 ratio following Lin⁻BMC transplantation (i) (n = 5/time point). There were significant reductions in the BAX/BCL-2 ratio on d7 after Lin⁻SPC injection. The immunofluorescent localization of active caspase-3 demonstrated that its expression was detected selectively in the PBS-injected, control retinas at the site of injury (j, k). The scale bar = 20 μm. ^* P < 0.05.

Figure 5

Effects of Lin⁻BMC transplantation on retinal morphology and the b-wave ERG amplitudes over time. The scotopic (a), mixed rod-cone (b), and photopic (c) response, as well as oscillatory potentials (d), were analyzed repeatedly on the same individual mice during the time of the experiment. The results are presented as the mean ± SD (n = 5). Representative in vivo SD-OCT images of the retina on the 7th and 28th day and at the 3rd month after injury showing a considerable improvement in the architecture of the outer and inner photoreceptor segments of the right eyes treated with Lin⁻BMCs (e). Quantitative comparative analysis of the total retinal thickness of the Lin⁻BMC-treated retinas compared with the control eyes (f). ^* P < 0.05 for the right eyes treated with Lin⁻BMCs versus the left eyes injected with PBS (n = 5).

Figure 6

Global changes in gene expression in murine retinas on the 7th day after NaIO₃-injury. The heat-map represents expression levels of genes representing particular gene ontologies (GOs) with large gene expression changes (fold change > 4) in Lin⁻BMC-treated eyes compared to PBS-injected control eyes. For a given GO, an average value was computed and subtracted from each observation. Each column comprises a set of horizontal lines, with each line representing a particular GO. Gene expression levels comprising a particular GO are indicated on a color scale, with yellow corresponding to the highest level of expression and blue corresponding to the lowest level; the range of expression rate from the analyzed genes is shown below the graph. The GO terms, listed on the left side of the graph (y-direction), were selected if the genes annotated with the corresponding GO terms were considered statistically significantly overrepresented.