Fahim Ebrahimi

Publications (3)6.23 Total impact

• Article: Time dependent neuroprotection of mycophenolate mofetil: effects on temporal dynamics in glial proliferation, apoptosis, and scar formation.

  Fahim Ebrahimi, Marco Koch, Philipp Pieroh, Chalid Ghadban, Constance Hobusch, Ingo Bechmann, Faramarz Dehghani
  [show abstract] [hide abstract]
  ABSTRACT: Immunosuppressants such as mycophenolate mofetil (MMF) have the capacity to inhibit microglial and astrocytic activation and to reduce the extent of cell death after neuronal injury. This study was designed to determine the effective neuroprotective time frame in which MMF elicits its beneficial effects, by analyzing glial cell proliferation, migration, and apoptosis. Using organotypic hippocampal slice cultures (OHSCs), temporal dynamics of proliferation and apoptosis after N-methyl-D-aspartate (NMDA)-mediated excitotoxicity were analyzed by quantitative morphometry of Ki-67 or cleaved caspase-3 immunoreactive glial cells. Treatment on NMDA-lesioned OHSCs with mycophenolate mofetil (MMF)100 μg/mL was started at different time points after injury or performed within specific time frames, and the numbers of propidium iodide (PI)+ degenerating neurons and isolectin (I)B(4)(+) microglial cells were determined. Pre-treatment with guanosine 100 μmol/l was performed to counteract MMF-induced effects. The effects of MMF on reactive astrocytic scar formation were investigated in the scratch-wound model of astrocyte monolayers. Excitotoxic lesion induction led to significant increases in glial proliferation rates between 12 and 36 hours after injury and to increased levels of apoptotic cells between 24 and 72 hours after injury. MMF treatment significantly reduced glial proliferation rates without affecting apoptosis. Continuous MMF treatment potently reduced the extent of neuronal cell demise when started within the first 12 hours after injury. A crucial time-frame of significant neuroprotection was identified between 12 and 36 hours after injury. Pre-treatment with the neuroprotective nucleoside guanosine reversed MMF-induced antiproliferative effects on glial cells. In the scratch-wound model, gap closure was reached within 48 hours in controls, and was potently inhibited by MMF. Our data indicate that immunosuppression by MMF significantly attenuates the extent of neuronal cell death when administered within a crucial time frame after injury. Moreover, long-lasting immunosuppression, as required after solid-organ transplantation, does not seem to be necessary. Targeting inosine 5-monophosphate dehydrogenase, the rate-limiting enzyme of purine synthesis, is an effective strategy to modulate the temporal dynamics of proliferation and migration of microglia and astrocytes, and thus to reduce the extent of secondary neuronal damage and scar formation.
  Journal of Neuroinflammation 05/2012; 9:89. · 3.83 Impact Factor
• Article: Propidium iodide staining: a new application in fluorescence microscopy for analysis of cytoarchitecture in adult and developing rodent brain.

  Marcus Hezel, Fahim Ebrahimi, Marco Koch, Faramarz Dehghani
  [show abstract] [hide abstract]
  ABSTRACT: Immunohistochemical visualization of antigens in specimen has evolved to an indispensable technique in biomedical research for investigations of cell morphology and pathology both in bright field and fluorescence microscopy. While there are couple of staining methods that reveal entire cytoarchitecture in bright field microscopy such as Nissl or hemalaun-eosin, there are still limitations in visualizations of cytoarchitecture in fluorescence microscopy. The present study reports a simple staining method that provides the required illustration of cell allocations and cellular composition in fluorescence microscopy in adult and in developing rodent central nervous system using the fluorophore propidium iodide (PI, 5μg/mL). PI is a well-accepted marker for degenerating cells when applied prior to fixation (pre-fixation PI staining). Here, PI was added to the sections after the fixation (post-fixation PI staining). This revised labeling procedure led to similar cytoarchitectural staining patterns in fluorescence microscopy as observed with hemalaun in bright field microscopy. This finding was proven in organotypic hippocampal slice cultures (OHSC) and brain sections obtained from different postnatal developmental stages. Excitotoxically lesioned OHSC subjected to pre-fixation PI staining merely showed brightly labeled condensed nuclei of degenerating neurons. In contrast, post-fixation PI staining additionally revealed extensive labeling of neuronal cell bodies and glial cells within the OHSC, thus allowing visualization of stratification of neuronal layers and cell morphology. Furthermore, post-fixation PI staining was combined with NeuN, calbindin, calretinin, glial fibrillary acidic protein or Griffonia simplicifolia isolectin B4 (IB(4)) in post natal (p1 and p9) and adult rats. In early post-natal brain sections almost all mentioned cellular markers led to an incomplete staining of the native cell organization and resulted in an inaccurate estimation of cell morphology when compared to adult brains. In contrast, post-fixation PI staining allowed investigation of the whole cytoarchitecture independent of the developmental stage. Taken together, post-fixation PI staining provides a detailed insight in the morphology of both developing and adult brain tissues in fluorescence microscopy.
  Micron 04/2012; 43(10):1031-8. · 1.53 Impact Factor
• Article: Analyses of neuronal damage in excitotoxically lesioned organotypic hippocampal slice cultures.

  Fahim Ebrahimi, Marcus Hezel, Marco Koch, Chalid Ghadban, Horst-Werner Korf, Faramarz Dehghani
  [show abstract] [hide abstract]
  ABSTRACT: Organotypic hippocampal slice cultures (OHSCs) are widely used to study the mechanisms of neurodegeneration and neuroprotection. However, there are still controversies about the most appropriate method for quantification of neuronal damage. The response to excitotoxic lesions can be determined by propidium iodide (PI) staining, which labels nuclei of degenerating cells. Semiquantitative measurements of PI staining are based on (1) recording of the propidium iodide (PI) fluorescence intensity or (2) counting of PI positive neuronal nuclei. Here, we investigated OHSCs lesioned by the application of increasing NMDA concentrations (10microM, 50microM and 500microM) at 6 days in vitro (div) for 4h or left untreated, respectively. After 9 div, PI staining was performed and the staining determined in the dentate gyrus and cornu ammonis (CA1) by measurement of PI-fluorescence intensity or by counting PI(+)-nuclei with a confocal laser scanning microscope. The fluorescence intensity of lesioned OHSCs did not show a NMDA concentration dependent difference. In contrast, confocal laser scanning microscopy revealed a significant and dose-dependent increase in the number of PI(+)-nuclei. Linear regression analysis showed a high correlation between NMDA concentration and the number of PI(+)-nuclei. A high correlation was also found between the mean number of PI(+)-nuclei determined in every third optical section and that determined in a single mid-stag optical section. The results show that proper analysis of neuronal damage requires counting of PI(+)-nuclei by confocal laser scanning microscopy.
  Annals of anatomy = Anatomischer Anzeiger: official organ of the Anatomische Gesellschaft 08/2010; 192(4):199-204. · 0.88 Impact Factor