Adult male Fischer 344 rats (Charles River Breeding Lab, Wilmington, MA) weighing between 200 and 240 g were used in all experiments. All animal procedures were performed in AAALAC accredited facilities, following institutionally approved protocols, in accordance with published recommendations for the proper care and use of laboratory animals. Food and water were provided ad libitum and consumption rates monitored daily. A total of 33 rats were randomly assigned to Radiation Treatment groups receiving WBI at prescribed doses of 0 (Control, n = 9), 10 Gy (n = 16), and 15 Gy (n = 8). Each of these Radiation Treatment groups was subdivided into drug treatment groups receiving either ramipril (RAM) or vehicle only (RAD), administered according to the same schedule. Ramipril therapy was initiated 24 hours post-WBI, and maintained continuously until sacrifice at 12 weeks post-WBI.
Rats were irradiated using a dedicated self-shielded 5000Ci 137Cs irradiator (Mark I, Model 68, J.L. Shepherd and Associates, San Fernando, CA), with a primary collimator used to create a 2 cm × 30 cm rectangular dose field. Rats were anesthetized using ketamine (80 mg/kg) and xylazine (8 mg/kg) and positioned horizontally with their heads at the midpoint of this field (centered 15 cm above the base and 6 cm forward of the collimator face) to optimize uniformity of the dose distribution. Secondary lead shielding (1 cm thick) was used to limit radiation exposure to structures outside the brain including the jaw, pharynx, nose, and eyes. The head was oriented such that the radioactive source was lateral to the midline, with the 2 cm dose field dimension encompassing the anterior-posterior extent of the brain. To compensate for the affects of tissue attenuation, the prescribed radiation dose was administered bilaterally in two consecutive half-dose fractions. The measured dose rate at the time of irradiation was approximately 3.2 Gy/minute.
Ramipril treated rats (RAM subgroups) received a daily ramipril dose of approximately 1.5 mg/kg delivered by addition of the compound to their drinking water, whereas untreated rats (RAD subgroups) received bottle changes according the same schedule. The ramipril concentration in the drinking water was based on animal weight and the average ad libitum water consumption rate of approximately 20 ml/day measured among our experimental animals. Ramipril is an ester-containing prodrug that is rapidly absorbed after oral intake and its absorption is not affected by food. Upon absorption, ramipril is metabolized by the liver and converted into its active form, ramiprilat, a potent ACE inhibitor. The bioavailability of ramipril is highly predictable and the stability of the drug in drinking water is superior to other ACE inhibitors. The drug also has the demonstrated ability to cross the blood-brain barrier, unlike many other clinically available ACE inhibitors [32
Rats were sacrificed under deep pentobarbital anesthesia (80 mg/kg) by transcardial perfusion with saline (300 ml) followed by 10% neutral buffered formalin (300 ml). Brains were removed and post-fixed overnight at 4°C in 10% neutral buffered formalin, coronally sectioned into 2 mm blocks, and processed for paraffin embedding. Groups of four serial sections (7 μm thickness) were cut at 50 μm intervals along the rostral caudal axis of the hippocampus. Within each of these groups, one section was stained with hematoxylin-eosin (H&E) for routine histological assessment, whereas the three remaining sections were stained immunohistochemically using antibodies for Ki-67 (1:100, 60 min, Thermo Fisher Scientific, Fremont, CA,), a selective marker of cellular proliferation; doublecortin (DCX; 1:100, overnight at 4°C, Santa Cruz Biotechnology, Santa Cruz, CA), a selective marker of immature neurons; and CD68 (1:200, 30 min, AbD Serotec, Oxford, UK) a selective marker of activated microglia. Immunohistochemically stained sections were counter-stained using either hematoxylin (CD68) or DAPI (Ki-67 and DCX), as appropriate. For immunohistochemical processing, sections were deparaffinized and rehydrated, boiled for 10 minutes in 10 mM citrate buffer, incubated with primary antibodies, and labeled with DAB (CD68; 4+ detection/Betazoid DAB: Biocare Medical) or Cy3 (Ki-67 and DCX; 1:250 Alexa 555 secondary, Invitrogen) per manufacturer's instructions.
All analyses were performed by individuals naïve to the experimental conditions using previously established methods [6
]. Cell counts were performed bilaterally and exhaustively at 400× within the designated fields. Counts of Ki-67+
proliferating cells were performed within the SGZ (defined as the region extending 25 μm on either side of the border between the hilus and the GCL), and were restricted to cells with uniform cytoplasmic staining of a clearly demarcated spherical or elliptical structure containing a spherical DAPI-stained nucleus. Counts of DCX+
immature neurons were performed within the GCL and SGZ, and were restricted to cells with uniform cytoplasmic staining of a clearly demarcated spherical or pyramidal structure (often with dendritic processes extending through the GCL toward the molecular layer) containing a spherical DAPI-stained nucleus. Counts of CD68+
activated microglia were performed within the GCL and SGZ, and were restricted to cells with cytoplasmic staining of a small cell body and/or microglial processes associated with an elliptical nucleus. The length of the SGZ at the GCL-hilar boarder and the volume of the SGZ and GCL were calculated for each section from which counts were obtained. SGZ lengths were used to standardize Ki-67 and DCX counts as linear densities. The SGZ and GCL volumes were used to normalize the CD68 counts as volume densities [34
]. (Fig. )
Figure 1 Representative images of immunohistochemical staining for Ki-67+ progenitors (red), DCX+ immature neurons (green), and CD68+ activated microglia (brown) in the SGZ and GCL, obtained at 400× from CLT-SHAM, 10 Gy, and 10 Gy-RAM group rats sacrificed (more ...)
The average linear density of Ki-67+
cells residing within the SGZ and inferior margin of the GCL was used as measure of granule cell progenitor proliferation near the time of sacrifice for each rat. The average linear density of DCX+
cells residing within the SGZ and GCL was used as a measure of neurogenic potential near the time of sacrifice for each rat. The average volume density of CD68+
cells residing within the SGZ and GCL was used as a measure of microglial activation for each rat. Group means and standard deviations were calculated from these data and, when necessary, log transformations were performed prior to analysis to adjust for unequal variances. These averages were statistically analyzed using analysis of variance (ANOVA) and Student's t to test whether: 1. Granule cell progenitor proliferation and/or neurogenesis were differentially affected in the Control group by RAM (relative to vehicle only); 2. Granule cell progenitor proliferation and/or neurogenesis were decreased in the 10 Gy- and 15 Gy-WBI Radiation Treatment groups relative to Control; 3. Decreases in granule cell progenitor proliferation and/or neurogenesis in the 10 Gy- and 15 Gy-WBI Radiation Treatment groups were reduced by RAM (relative to vehicle only); and 4. Microlglial activation was increased in the 10 Gy- and 15 Gy-WBI Radiation Treatment groups relative to Control and whether these increases were reduced by RAM (relative to vehicle only) [37