) PC12 cells died when they were differentiated with h NGF and subsequently deprived of serum and h NGF.When infected with a lentiviral vector encoding Bcl-x L, PC12 cells differentiated with h NGF survived for an additional 3 weeks ( transduction of adult rat septal neurons expressing neuronal markers (Neu N; red), cholinergic markers (Ch AT; blue), and the h NGF or Bcl-x L transgene (h NGF/Bcl-x L; green).The images obtained from each individual staining and from the merged images are shown.
Coronal sections (50 μm) through the medial septum stained for Ch AT immunoreactivity 6 weeks after h NGF, Bcl-x L, or GFP vector injection or saline control injection and 3 weeks after unilateral aspirative lesion of the fimbria fornix, ipsilateral to the injection.
The medial septum was defined as the area lying above a line between the midportion of the anterior commissure, beneath the corpus callosum, and laterally limited by the ventricles.
Ch AT-positive cells displaying a round cell body and at least one process were counted for the lesioned and the unlesioned site of the septum.
Percentage of ipsilateral/contralateral septal neurons quantified after intraseptal vector injections and lesion of the fimbria fornix.
The percentage of Ch AT-immunoreactive cells (mean ± SEM) are shown for h NGF-, Bcl-x L-, and GFP-expressing vector and saline control.
h NGF and Bcl-x L viral vector-injected animals presented significantly higher numbers of surviving cholinergic septal neurons compared with GFP viral vector or saline-injected control animals (∗, model for the role of h NGF and Bcl-x L in neuronal survival.
Lentiviral transfer and expression of h NGF and Bcl-x L by lentiviral vectors is obtained in target and adjacent glial and neuronal cells.
Bcl-x L stably expressed is localized within the target cell in mitochondrial and nuclear membranes; however, it is not secreted.
Only infected cholinergic neurons can survive axotomy-induced cell death and trophic factor withdrawal by interfering with the cell death cascade by overexpression of Bcl-x L.
Surrounding cells do not contribute to cell survival and, therefore, cell saving is less than that obtained with overexpression of h NGF.
h NGF is expressed by the target neuron and adjacent glial and neuronal cells as a secreted protein; the autocrine and paracrine effects on the target cell allow for a higher number of cells saved by h NGF.