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

Optimization of synthesis method of imidazole N1-derivatives with potential biological activity

We wstępie przedstawiono charakterystykę fizyko-chemiczną heterocyklicznego układu imidazolu oraz opisano jego występowanie w cząsteczkach biologicznie czynnych. Następnie przedstawiono wpływ wprowadzenia układu imidazolu na parametry farmakologiczne związków biologicznie czynnych, ze szczególnym omówieniem wpływu na oddziaływania ligand-receptor. W dalszej części pracy zostały opisane wybrane leki zawierające w swojej strukturze imidazol, między innymi tyreostatyki, leki przeciwbakteryjne, agoniści receptorów α-adrenergicznych, leki przeciwgrzybicze, sartany i leki przeciwnowotworowe. Następnie zostały przedstawione nowe związki zawierające imidazol, o udowodnionej aktywności biologicznej w wybranych kierunkach terapeutycznych (działanie przeciwcukrzycowe, przeciwgrzybicze, terapia choroby Alzheimera) lub będące w fazie badań klinicznych. Badania własne stanowią opis przeprowadzonych doświadczeń mających na celu opracowanie metod syntezy różnie podstawionych w położeniu N1 pochodnych imidazolu. Reakcje były prowadzone etapowo. W pierwszym etapie opracowano i zoptymalizowano metodę syntezy pochodnych imidazolu, 2-metyloimidazolu i 2-metylo-3-bromo-imidazolu, które w położeniu N1 posiadają ugrupowanie estrowe. Związki były pochodnymi dwóch estrów octanu etylu oraz maślanu etylu. W dalszych etapach podjęto próby hydrolizy kwaśnej oraz zasadowej otrzymanych estrów, w celu uzyskania odpowiednich wyjściowych kwasów do reakcji utworzenia wiązania amidowego. W równoległej syntezie otrzymane estry traktowano jako wyjściowy substrat do otrzymania amidów z różnie podstawionymi pochodnymi benzyloaminy. Część doświadczalna zawiera opis wszystkich zsyntetyzowanych związków oraz zastosowanych metod fizykochemicznych. Rozdział ten jest uzupełniony o szczegółową analizę widm 1H NMR i LC/MS.The introduction presents the physico-chemical characteristics of the heterocyclic imidazole system and its occurance in biologically active molecules. It also describes the effect of introducing the imidazole system on the pharmacological parameters of compounds. It focuses particulary on ligand-receptor interactions. The following part shows selected drugs containing imidazole in their structure, including antithyroid drugs, antibacterial agents, α-adrenergic agonists, antifungal agents, sartans and anticancer drugs. This section shows also new compounds with proven biological activity in selected therapeutic directions (antidiabetic, antifungal, Alzheimer's disease) or compounds being in the phase of clinical trials.Own research describes experiments to develop methods for the synthesis of variously N1-substitued imidazole derivatives. Reactions were carried out in stages. In the first stage, the method of synthesizing derivatives of 2-methylimidazole and 2-methyl-3-bromo-imidazole which has ester moiety in the N1 position, was developed and optimized. The compounds were derived from two esters: ethyl acetate and ethyl butyrate. In order to obtain appropriate starting acids for the formation of the amide bond, acid and basic hydrolysis experiments were carried out. In simultaneous synthesis the obtained esters were treated as starting substrates to get amides with variously substitued benzylamine derivatives.The experimental part contains a description of all synthesized compounds and applied physicochemical methods. This chapter also includes a detailed analysis of 1H NMR and LC/MS spectra

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

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

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

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

Activity of metalloproteinases in the adult gerbil hippocampus after global ischemia.

<p>Animals were subjected to 5 min of global forebrain ischemia followed by reperfusion. Confocal photomicrographs showing <i>in situ</i> zymography in DG and CA1 from a control animal (A, E) and from ischemic animals sacrificed at 7 (B, F), 14 (C, G) and 28 days (D, H) after ischemia. Note the increase of fluorescence signal in the DG and in <i>strata oriens</i> and <i>stratum radiatum</i> with simultaneous decrease in pyramidal cell layer of the CA1 area across the time of reperfusion. All images subjected to direct comparisons were captured at the same exposure and digital gain settings. Photomicrographs are representative of observations made from six animals per time point. Abbreviations: s.o – <i>stratum oriens</i>; s.r. – <i>stratum radiatum</i>.</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

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

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