FLOW CYTOMETRY IN PRECLINICAL TOXICOLOGY/SAFETY ASSESSMENT - Flow Cytometry in Drug Discovery and Development - page 135

was likely due to the absence of other cell types that are necessary for outgrowth in
culture. Monocytes and T cells have both been implicated as necessary for endothelial
maturation and/or vascularization [29, 30].

6.6.4 Drug Toxicity Experiment

All experiments up to this point were performed in-house using freshly prepared lysed
whole blood samples from just a few rats at a time. But the in-life portion of definitive
studies, including dosing with tool compounds, were to be performed at remote
locations. Each experiment consisted of four (vehicle control, low-, mid-, and high-)
dose groups with six rats per group. This required that samples be fixed and/or frozen
to survive transport and short-term storage (only a few rat samples could be sorted per
day). In initial experiments, paraformaldehyde (PFA) fixation proved incompatible
with mRNA analysis, so an alternative preservation method was sought that provided
satisfactory flow cytometry results and sufficient amount of recoverable mRNA. In
addition, all fixatives and buffers weremadewithDEPC-treatedwater. All vessels that
came into contact with samples were rinsed with DEPC-treated water before use. The
cell sorter was decontaminated with bleach and rinsed with DEPC-treated water
before each sort. RNA integrity/yield was compared among sorted and unsorted
leukocytes isolated from fresh lysed whole blood and preservation methods under
consideration included PFA (4% and 0.2%, repeated with DEPC-treated water
formulation), RNAlater , Cyto-Chex , “RNA-friendly” fixative (formalin/EtOH/

AmOH), formaldehyde/MetOH, 90% EtOH, 90% MeOH, EtOH/acetone, and fresh-
frozen in RPMI-10% DMSO. Further attempts to improve integrity included adding
RNAsecure to PFA, formulating with chloride/citrate buffer instead of PBS, and

trial of different commercially available RNA isolation and extraction kits (two each).
In the end, only fresh-frozen samples gave both satisfactory flow cytometry and RNA
integrity results. Samples could be stored up to 7 months without appreciable impact
on immunophenotyping results. There was a slight reduction in RNA integrity/yield
after four weeks.
For definitive studies, global gene expression profiling of CD36 þ /Lin cells by
microarray was performed. The goals of this effort were to identify potential protein
targets useful for more robust immunophenotypic characterization and to provide
information on vascular injury pathogenesis in rats dosed with known vascular
toxicants. Doses were chosen based on previous experience and/or literature review.
One such study is outlined as follows.

6.6.5 Methods

6.6.5.1 Dosing/Sample Collections Male Sprague-Dawley rats (350-500 g; 6 per
dose group) were given vehicle (sterile water), 1, 3, or 10mg/kg/day serotonin (5HT)
subcutaneously (2mL/kg) for 4 days and blood was collected terminally
approximately 24 h after the last dose. Postnecropsy pathologic examination of
mesenteric rolls with lesion scoring and statistical analysis confirmed induction
of dose-dependent mesenteric arterial vascular injury using this study design.