Neuroprotective and Antiapoptotic Activity of Lineage-Negative Bone Marrow Cells after Intravitreal Injection in a Mouse Model of Acute Retinal Injury

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) (/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. .

(a)

(b)

(c)

(d)

(e)

(f)

(g)

(h)

(i)

(j)

(k)