The Potential Role of Metalloproteinases in Neurogenesis in the Gerbil Hippocampus Following Global Forebrain Ischemia

BACKGROUND: Matrix metalloproteinases (MMPs) have recently been considered to be involved in the neurogenic response of adult neural stem/progenitor cells. However, there is a lack of information showing direct association between the activation of MMPs and the development of neuronal progenitor cells involving proliferation and/or further differentiation in vulnerable (Cornus Ammoni-CA1) and resistant (dentate gyrus-DG) to ischemic injury areas of the brain hippocampus. PRINCIPAL FINDINGS: We showed that dynamics of MMPs activation in the dentate gyrus correlated closely with the rate of proliferation and differentiation of progenitor cells into mature neurons. In contrast, in the damaged CA1 pyramidal cells layer, despite the fact that some proliferating cells exhibited antigen specific characteristic of newborn neuronal cells, these did not attain maturity. This coincides with the low, near control-level, activity of MMPs. The above results are supported by our in vitro study showing that MMP inhibitors interfered with both the proliferation and differentiation of the human neural stem cell line derived from umbilical cord blood (HUCB-NSCs) toward the neuronal lineage. CONCLUSION: Taken together, the spatial and temporal profiles of MMPs activity suggest that these proteinases could be an important component in neurogenesis-associated processes in post-ischemic brain hippocampus

Neural identity of newly divided cells in the CA1 area after global ischemia.

<p>Animals were subjected to 5 min of global forebrain ischemia followed by reperfusion. BrdU was administered 24 h prior to sacrifice. Brain sections from a control animal (A, D) and from an animal 6 days (B, E ) and 28 days (C, F) after ischemia were stained for BrdU immunoreactivity (green) and neuron-specific NF-200 (A, B, C, G) or the astrocyte-specific GFAP (D, E, F, H) markers (red). C, H represent magnification (z-stacks) of the picture C and E. Six days after ischemia the higher than in control number of BrdU+ cells are seen within the damaged pyramidal cell. Intensive BrdU labeling was observed in <i>strata oriens</i> and <i>radiatum</i>. Depending on the CA1 area BrdU-positive cells expressed distinct antigens – NF-200 exclusively in pyramidal cell layer and GFAP in <i>strata oriens</i> and <i>radiatum</i> as well as in pyramidal cell layer. Photomicrographs are representative of observations made from six animals per time point. Scale bar 10 µm. Abbreviations: p.l. – pyramidal layer; s.o – <i>stratum oriens</i>; s.r.- <i>stratum radiatum</i>.</p

Effect of SB-3CT (10 µM) on the Ki67 and Tuj1 positive cells in HUCB-NSs culture.

<p>Equivalent numbers of HUCB-NSCs were plated on fibronectin-coated coverslips and grown 8 days in serum-free medium with or without SB-3CT (10 µM). Cells were stained for Ki67 (red) and Tuj1 (green). The cell nuclei counterstained with Hoechst (blue). Note the decreased number of immunolabeled cells in the presence of SB-3CT. Scale bar 20 µm.</p

Newly-divided cells in the DG of adult gerbil hippocampus after global ischemia.

<p>Animals were subjected to 5 min of global forebrain ischemia followed by reperfusion. BrdU was administered 24 h prior to sacrifice. Brain sections were double-labeled with anti BrdU antibody (green) and anti-NF-200 (red) (A, B, C, G) or anti-GFAP (red) (D, E, F, H). Confocal photomicrographs show immunohistochemical reaction in control DG (A, D), 9 days after ischemia (B, E), and 28 days after ischemia (C, F). G, H represent magnification (z-stacks) of the picture C and F. Photomicrographs are representative of observations made from six animals per time point. Scale bar 10 µm. Abbreviations: s.g.z – subgranular zone, g.z. –granular zone.</p

Time- course of cell proliferation in the adult gerbil hippocampus after global ischemia.

<p>Animals were subjected to 5 min of global forebrain ischemia followed by 6, 9, 11, 14, 21 or 28 days of reperfusion. BrdU was administered 24 h prior to sacrifice and brains were processed for BrdU immunohistochemistry. Graphs show the number of BrdU-labeled nuclei in the neurogenic SGZ of the DG (A) and in CA1 (B) in a sham operated control animal (C) and at different times after ischemia. The number of proliferating, BrdU-positive cells, increases markedly at 9–11 days of reperfusion in SGZ of the DG and at 6 days of reperfusion in CA1. Values represent the means ± SEM of six animals per time point. One-way ANOVA and Bonferroni test: **p<0.01 and ***p<0.001 indicate a statistically significant difference vs control value.</p

Effect of SB-3CT, GM6001 and doxycycline on the growth and proliferation of HUCB-NSCs.

<p>Equivalent numbers of HUCB-NSCs were plated on fibronectin-coated coverslips and grown 8 days in serum-free medium with or without SB-3CT (10 µM), GM6001 (25 µM) or doxycycline (60 µM). A) Graph shows an average number of surviving cells after 4, and 8 days in culture. The addition of SB-3CT, GM6001 or doxycycline to the incubation medium decreased the number of surviving cells. The results (mean values +/− SEM) represent five independent experiments. One-way ANOVA and Bonferroni test: **p<.01; ***p<.001, between treatments (inhibitors vs control value). B) Graph shows the rate of HUCB-NSCs proliferation expressed as a % of control of Ki67- immunopositive cells. The present of SB-3CT, GM6001 or doxycycline in the culture reduced markedly the number of proliferating cells. The results (mean values +/− SEM) represent five independent experiments. One-way ANOVA and Bonferroni test: ***p<.001, between treatments (inhibitors vs control value).</p

Neurogenesis in the adult gerbil hippocampus after global ischemia.

<p>Animals were subjected to 5 min of global forebrain ischemia followed by reperfusion. BrdU was administered twice daily 6–9 days after ischemia. Brain sections from a control animal (A, D, G), and from an animal 14 days (B, E, H) or 28 days (C, F, I, K, L) after ischemia were stained for BrdU immunoreactivity (green), for specific neuronal markers (NF-200 and NeuN -red) and astrocytic marker (GFAP-red) in the DG (A, B, C) and the CA1 (D–I). J, K, L represent magnification (z –stacks) of the picture C, F, I, respectively. Note co-localization of BrdU with mature NeuN-positive neurons in SGZ, SG and hilus at 14 days after ischemia (B) and increased number of BrdU/NeuN positive cells in SG at 28 days after ischemia (C, J). Photomicrographs are representative of observations made from six animals per time point. Scale bar 10 µm. Abbreviations: s.g.z - subgranular zone, g.z.- granular zone, h - hilus, p.l.- pyramidal layer.</p

Distribution of metalloproteinases in the CA1 after global ischemia.

<p>Confocal photomicrographs following co-staining of <i>in situ</i> zymography (green) with neuronal markers: NeuN (A, B, C, J) (red) NF200 (D, E, F, K) (red) or the astrocyte marker GFAP (red) (G, H, I, L) in control (A, D, G) and at 7 (B, E, H) or 28 days (C, F, I, J, K, L) after ischemia. J, K, L represent magnification (z-stacks) of the picture C, F, I respectively. Note the association of MMPs activity with reactive astrocytes GFAP positive in the <i>stratum oriens</i> and <i>stratum radiatum</i>, probably because of the glial reactivity. Photomicrographs are representative of observations made from six animals per time point. Scale bar 10 µm. Abbreviations: p.l.- pyramidal layer; s.o – <i>stratum oriens</i>; s.r. – <i>stratum radiatum</i>.</p

Effects of GM6001 and doxycycline on the differentiation of HUCB-NSCs.

<p>Equivalent numbers of HUCB-NSCs were plated on fibronectin-coated coverslips and grown 8 days in serum-free medium with or without SB-3CT (10 µM), GM6001 (25 µM) or doxycycline (60 µM). The graph represents the percentages of immunolabeled cells relative to the total cell population that are present in the culture. Note the decrease of the relative number of cells presenting neuronal antigen and simultaneous increase of the relative amount of glial cells in the presence of SB-3CT, GM6001 and doxycycline. The results (mean values +/− SEM) represent five independent experiments. One-way ANOVA and Bonferroni test: **p<.01, ***p<.001, between treatments (inhibitors vs control value).</p

Distribution of metalloproteinases in the DG after global ischemia.

<p>Animals were subjected to 5 min of global forebrain ischemia followed by reperfusion. Confocal photomicrographs following co-staining of <i>in situ</i> zymography (green) with neuronal markers: NF200 (D, E, F, K) (red) and NeuN (A, B, C, J) (red) or the astrocyte marker GFAP (G, H, I, L) (red) in control ( A, D, G) and at 7 (B, E, H) or 28 days (C, F, I, J, K, L) after ischemia. J, K, L represent magnification (z-stacks)of the picture C,F,I, respectively. Note that MMPs activity was principally associated with neurons. Photomicrographs are representative of observations made from six animals per time point. Scale bar 10 µm Abbreviations: g.z – granular zone; s.g.z- subgranular zone.</p