Effects of phosphodiesterase 3A modulation on murine cerebral microhemorrhages

All animal procedures followed the “Principles of Laboratory Animal Care” (NIH Publication No. 85-23), were approved by the University of California, Irvine, Institutional Animal Care and Use Committee, and followed the ARRIVE Guidelines for animal experiment reporting. To study the effects of PDE3A inhibition on CMH development, two approaches—genetic deletion and pharmacological inhibition of the PDE3A pathway—and two mouse models—the inflammation-induced mouse model of CMH and an amyloid precursor protein transgenic (APP/Tg2576) mouse model that develops CAA and spontaneous CMH—were used.

PDE3A knockout (KO) mice were used in the inflammation-induced mouse model of CMH. Heterozygous PDE3A^+/− KO breeding pairs (generously provided by Dr. Vincent Manganiello [7, 18]) were bred in-house to maintain a colony of homozygous PDE3A^−/− KO and wild-type (WT) littermate controls. All mice used in the study were 16–18 months, and the average weight of the WT and PDE3A^−/− KO mice was 44 ± 1.9 and 41 ± 1.5 g, respectively.

Lipopolysaccharide (LPS) derived from the gram-negative bacterium Salmonella typhimurium (Sigma-Aldrich, St. Louis, MO) was used to induce CMH development, as described previously [16]. Briefly, PDE3A^−/− KO mice (n = 14) and WT littermate controls (n = 16) were given LPS (3 mg/kg) intraperitoneally (i.p.) at 0, 6, and 24 h. Control PDE3A^−/− KO mice (n = 6) and control WT littermates (n = 4) received an equal volume of sterile saline i.p. (Teknova, Hollister, CA). Mice fed and drank ad lib and received 1 mL sterile saline subcutaneously (s.q.) on an as-needed basis to prevent dehydration. Mice were monitored for 48 h, after which they were sacrificed (Fig. 1a). Mice that died prematurely were excluded from histochemical and biochemical analysis, but were used for survival analysis.

The effect of pharmacological inhibition of PDE3A by cilostazol, a selective PDE3A inhibitor [19], on CMH development was studied in both the inflammation-induced and CAA-associated APP/Tg2576 (Tg) mouse models. To determine the achievable plasma cilostazol concentration, 4-month-old Tg mice and WT littermates were fed with mouse chow formulated with 0.03, 0.3, or 3% of cilostazol (Harlan, Madison, WI) (six WT and six Tg mice in each group), for 2 weeks. Plasma was collected before introduction of the cilostazol diet (T0) and after 2 weeks on the diet (T1). Cilostazol plasma levels were measured on HPLC by Sumika Analysis & Evaluation Service (Shanghai) Ltd.

To study the effect of cilostazol on CAA-associated CMH, 22-month-old Tg mice and WT littermates were housed under a 12-h light–dark cycle with free access to chow and water. Mice were divided into four groups: Tg mice fed regular chow (Tg; n = 6); Tg mice fed 0.3% cilostazol diet formulated in regular chow (Tg-CIL; n = 8); WT mice fed regular chow (WT; n = 8); and WT mice fed 0.3% cilostazol formulated in regular chow (WT-CIL; n = 7), for 10 weeks (Fig. 1b). Mice that died prematurely during the course of the 10 weeks were excluded from histochemical and biochemical analysis, but were used for survival analysis.

Nine weeks after initiation of the cilostazol or regular diet regimen, LPS derived from the gram-negative bacterium S. typhimurium was administered (1 mg/kg dose of i.p. at 0, 6, and 24 h) to a separate series of WT mice fed regular chow (WT-LPS; n = 10) and WT mice fed 0.3% cilostazol formulated in regular chow (WT-CIL-LPS; n = 8) to study the effect of pharmacological inhibition of the PDE3A pathway on inflammation-induced CMH development. WT mice fed regular chow and treated with an equal volume of PBS (i.p. at 0, 6, and 24 h) served as control mice (WT-PBS; n = 8). Mice were kept under standard vivarium conditions for 7 days after the first LPS or PBS injection and observed twice daily (Fig. 1c). Mice that died prematurely during the course of the 7 days were excluded from histochemical and biochemical analysis, but were used for survival analysis.

Forty-eight hours after the first LPS or saline injection (for genetic manipulation studies) or 10 weeks after initiation of cilostazol or regular diet treatment (for pharmacological inhibition studies), animals were anesthetized with a lethal dose of Nembutal (150 mg/kg, i.p.), cardiac perfusions were performed using ice-cold PBS for 5 min to flush the cerebral vasculature, and the brains were harvested immediately. The left cerebral hemispheres were flash-frozen using dry ice and stored at −80 °C for biochemical analyses. The right hemispheres were fixed in 5 mL of 4% paraformaldehyde (BM-154, Boston BioProducts, Ashland, MA) at 4 °C for 72 h for histological analyses.

Fixed mouse hemi-brains were examined for grossly visible surface CMH using a ×10 magnifying glass. Surface microhemorrhages were quantified and photographed using Canon PC2054 20 megapixel (Canon, Tokyo, Japan). Fixed brains were placed in an agarose mold and sectioned into 40-μm coronal sections with a vibratome (Technical Products International, Inc., St. Louis, MO). Every 4th, 5th, 6th, and 7th section was collected, and approximately 30 sections were analyzed per mouse brain.

The effect of PDE3A KO (genetic manipulation of the PDE3A pathway) on acute CMH development was assessed by examining the formation of hematoxylin and eosin (H&E)-positive CMH in a 2-day study, as described previously [20]. Prussian blue (PB) staining to detect CMH was not used for the PDE3A KO acute study based on previous studies from our lab [20]. For H&E staining, every 6th section (approximately 30 sections per mouse brain) was stained with H&E by the research service core at UCI Medical Center’s Department of Pathology and Laboratory Medicine. CMH were identified as a collection of red blood cells (RBC) that appear red-orange under H&E staining using a light microscope with a ×20 objective by an observer blinded to the experimental groups. All brain sections were examined, and all identified CMH were photographed using an Olympus BX40 microscope and CC-12 Soft-Imaging System with Olympus Microsuite™-B3SV software. Digitized images were analyzed using NIH ImageJ version 1.46r to determine CMH number, size (μm²), and positive area (sum of the area occupied by all the individual H&E-positive CMH expressed as a percentage of the total area analyzed) per mouse by an observer blinded to the experimental groups. To determine the total area analyzed, H&E-stained slides were scanned using the Canon MP250 scanner (Canon, Tokyo, Japan) under 600 dots per inch (DPI). The total area of each brain section was quantified using NIH ImageJ software and added to determine the total tissue area that was analyzed per mouse. The total CMH positive area was then normalized to the total tissue area for each mouse and expressed as a percentage of the total area analyzed, as mentioned above.

To study the effect of the PDE3A inhibitor cilostazol on CAA-associated CMH development, the PB method for hemosiderin (a marker of subacute CMH) was used [17, 20]. Briefly, three coronal sections per brain with coordinates −1.2, −2.2, and −3.2 mm posterior to the bregma selected according to The Mouse Brain in Stereotaxic Coordinates were stained with PB and counterstained with nuclear fast red [21]. All brain sections were examined, images of microscopic hemorrhages were taken using Olympus BX-UCB microscope with a ×20 objective, and CMH were analyzed using NIH ImageJ software by an investigator blinded to the experimental groups. Eclipse tool was used as a convex hull to encircle the CMH to determine the average size and total positive area of the PB-positive lesion in square micrometers based on the incorporated scale bar. For the total PB-positive area, individual PB-positive lesions for all the three brain sections were summed. The number, average size, and total area of PB-positive CMH per section were reported as described previously [17].

In the genetic manipulation studies, blood samples were obtained before and 26 and 48 h after LPS or saline injection via the retro-orbital sinus using heparinized capillary tubes (Fisher Scientific, Hampton, NH). For this, mice were briefly anesthetized using 3% isoflurane. Blood samples were collected on ice and centrifuged at 8000 × g for 15 min at 4 °C. For TNF-α (a marker of systemic inflammation) ELISA, supernatant was collected and assayed using a TNF-α ELISA kit (Life Technologies, Carlsbad, CA) as per the manufacturer’s protocol. For β-thromboglobulin (β-TG; a marker of platelet activation) ELISA, blood was collected in 1-mL Eppendorf tubes prefilled with a plasma collection solution (21% by volume of collected blood) to prevent in vitro platelet activation and assayed using a β-TG ELISA kit (US Biological, 023697, Salem, MA) as per the manufacturer’s instructions. The plasma collection solution for β-TG comprised acid–citrate–dextrose (ACD) National Institutes of Health formula A (Sigma-Aldrich, St. Louis, MO), aspirin (Sigma-Aldrich, St. Louis, MO), and prostaglandin E1 (Sigma-Aldrich, St. Louis, MO) at a 100:8:1 ratio, respectively [22].

Immunohistochemistry was performed for ICAM-1 (inflammation-inducible protein intercellular adhesion molecule-1; a marker of endothelial cell activation), IgG (a marker of BBB damage), Iba-1 (ionized calcium-binding adaptor molecule 1; a marker of microglia/macrophages), and GFAP (glial fibrillary acidic protein; a marker of astrocytes). Immunohistochemical analyses [16] were performed using one coronal 40-μm section per mouse approximately −2 to −2.4 mm posterior to the bregma, and identical coordinate sections per staining from the analyzed groups were incubated in 0.5% hydrogen peroxide in 0.1 M PBS (pH 7.4) containing 0.3% Triton X-100 (PBST) for 30 min at room temperature to block endogenous peroxidase activity. After washing with PBST, sections were incubated for 30 min with PBST containing 2% bovine serum albumin (BSA) to block non-specific protein binding. Sections were then incubated overnight at 4 °C with a rabbit anti-mouse IgG antibody (1:200 dilution; Jackson ImmunoResearch, West Grove, PA), rabbit monoclonal antibody against ICAM-1 (1:500 dilution; Abcam, Cambridge, MA), rabbit antibody against Iba-1 (1:200 dilution; Wako Chemicals USA, Richmond, VA), or rabbit antibody against GFAP (1:2000 dilution; Abcam, Cambridge, MA). After washing with PBST, sections were incubated at room temperature for 1 h with biotinylated anti-rabbit IgG (1:500 dilution; Jackson ImmunoResearch, West Grove, PA), followed by 1 h incubation at room temperature with ABC complex according to manufacturer instructions (Vector Laboratories, Burlingame, CA). Sections were developed with 3,3′-diaminobenzidine (DAB) (Vector Laboratories, Burlingame, CA). Sixteen images per brain section from the frontal cortex, hippocampus, and thalamus areas were acquired randomly at ×20 magnification, and the total positive immunoreactive area (expressed as % of the total area analyzed) was quantified using NIH ImageJ software by an observer blinded to the experimental groups.

Data were represented as mean ± SEM, and all statistical analyses were performed using GraphPad Prism 5 (GraphPad Software Inc., La Jolla, CA). To compare more than two groups, one-way ANOVA with Bonferroni’s post hoc test was used for normal data and the Kruskal-Wallis test with Dunn’s post hoc test for non-normal data. The Student t test was used to compare two groups with normally distributed data. Survival curves were analyzed using the Log-rank test. One-sample t test was used to compare group means with a hypothesized mean = 0 when the values in the control group were zero (e.g., surface microhemorrhages and TNF-α levels in the control mice). A p value of <0.05 was considered statistically significant.

While studying the effect of genetic deletion of PDE3A on CMH development, all the control mice (control PDE3A^−/− KO (n = 6) and control WT littermates (n = 4)) and LPS-treated WT mice (n = 16) survived the duration of the study. The LPS-treated PDE3A^−/− KO mice had a significantly lower (p < 0.01) survival (9 of 14) compared with the LPS-treated WT mice (Fig. 2a).

While studying the effect of pharmacological inhibition of PDE3A with cilostazol on CAA-associated CMH development, the survival of mice was as follows: 8/8 in the WT group, 6/7 in the WT-CIL group, 5/6 in the Tg group, and 8/8 in the Tg-CIL group (Fig. 2b). No significant difference in the survival was observed between any experiment groups. While studying the effect of pharmacological inhibition of PDE3A with cilostazol on inflammation-induced CMH development, the survival of mice was as follows: 8/8 in the WT-PBS group, 5/10 in the WT-LPS group, and 5/8 in the WT-CIL-LPS group (Fig. 2c). LPS treatment caused a significant reduction (p < 0.05) in survival in the WT and WT-CIL treated mice compared with the PBS-treated WT mice.

No grossly visible CMH were visible on the brain surface of mice treated with saline, in either WT or PDE3A^−/− KO mice. A 3 mg/kg dose of LPS at 0, 6, and 24 h induced the development of grossly visible surface microhemorrhages 48 h after the first LPS injection, in both WT (9.5 ± 1.9 per brain; p < 0.05) and PDE3A^−/− KO (8.3 ± 2.9 per brain; p < 0.05) mice compared with saline controls (Fig. 3a). Genetic deletion of PDE3A had no significant effect on LPS-induced surface microhemorrhage development, and no significant difference between LPS-treated WT and LPS-treated PDE3A^−/− KO mice was observed.

Negligible H&E-positive acute parenchymal CMH were observed in saline-treated WT (0.021 ± 0.008 per brain section) and PDE3A^−/− KO (0.06 ± 0.03 per brain section) mice. A 3 mg/kg dose of LPS at 0, 6, and 24 h significantly increased the formation of H&E-positive acute parenchymal CMH in both the WT (0.71 ± 0.16 per brain section) and PDE3A^−/− KO (1.2 ± 0.46 per brain section) mice compared with their respective saline-treated controls (Fig. 3b). There was a trend toward a higher H&E-positive CMH number in the LPS-treated PDE3A^−/− KO mice compared with the LPS-treated WT mice (p > 0.05).

LPS treatment produced H&E-positive CMH that were significantly larger in size compared with the saline controls, in both the WT and PDE3A^−/− KO mice. However, the mean CMH size did not differ significantly between LPS-treated WT and LPS-treated PDE3A^−/− KO mice (Fig. 3c). Similarly, the total CMH load (or the CMH positive area) was significantly higher in the LPS-treated WT and LPS-treated PDE3A^−/− KO mice compared with their respective saline-treated controls, but no difference between LPS-treated WT and LPS-treated PDE3A^−/− KO mice was observed (Fig. 3d). Representative images of H&E-positive CMH in the saline- and LPS-treated WT and PDE3A^−/− KO mice are shown in Fig. 3e. Overall, genetic deletion of PDE3A did not significantly alter H&E-positive inflammation-induced acute CMH development in the current study.

TNF-α levels were close to the detection limit in the saline-treated WT and PDE3A^−/− KO mice (data not shown). Two hours after the last LPS injection (or 26 h after the first LPS injection), serum TNF-α levels were significantly elevated in both the WT (265 ± 60 pg/mL) and PDE3A^−/− KO (131 ± 35 pg/mL) mice and the TNF-α levels were significantly higher in the WT mice compared with the PDE3A^−/− KO mice (Fig. 4a). TNF-α levels continued to be significantly elevated at 48 h after the first LPS injection compared with saline controls; however, no significant difference was observed between LPS-treated WT (61 ± 8 pg/mL) and LPS-treated PDE3A^−/− KO (62 ± 11 pg/mL) mice (Fig. 4a). Serum TNF-α levels were significantly associated (Spearman r = 0.48, p < 0.05) with the CMH load in the LPS-treated WT mice (Fig. 4b), but no significant correlation was observed in the LPS-treated PDE3A^−/− KO mice. No significant difference was observed in the extent of platelet activation as measured by plasma β-TG between the groups (data not shown).

The plasma cilostazol readings were negligible in mice before the diet introduction (0.15 ± 0.15 ng/mL) and increased in the 0.03% (1175 ± 303.2 ng/mL), 0.3% (3676 ± 495.6 ng/mL), and 3% (4088 ± 670.4 ng/mL) groups. These levels are comparable to the plasma levels of cilostazol in humans following a 200 mg dose [23]. The mean cilostazol plasma levels were similar in the 0.3 and 3% groups and significantly higher than in the control and 0.03% groups. Based on this, a 0.3% cilostazol supplemented diet was used for the pharmacological inhibition studies.

In the studies examining the effect of cilostazol on CAA-associated spontaneous CMH development, we found that APP/Tg2576 (Tg) mice had a significantly higher number of spontaneously developed CAA-associated PB-positive CMH (12 ± 1.1 CMH per brain section; p < 0.01) compared with the WT littermates (3 ± 0.6 CMH per brain section) (Fig. 5a). Ten weeks of treatment with cilostazol, however, did not significantly alter the number of PB-positive CMH in both the Tg (8 ± 2 CMH per brain section) and WT (5 ± 2 CMH per brain section) mice as shown in Fig. 5a. Similarly, the average size and total area of the PB-positive lesions were significantly higher in the Tg mice compared with the WT littermates, and cilostazol treatment did not affect the size and area in WT or Tg mice (Fig. 5b, c). The PB-positive lesion size and total area was 701 ± 189 and 2608 ± 970 μm² in the WT mice and 497 ± 161 and 2086 ± 800 μm² in the WT-CIL mice, respectively. Similarly, the PB-positive lesion size and total area was 5040 ± 1206 and 61,971 ± 15,561 μm² in the Tg mice and 9918 ± 2412 and 91,594 ± 30,473 μm² in the Tg-CIL mice, respectively. Representative images showing PB-positive lesions in the WT and Tg mice with and without cilostazol treatment are shown in Fig. 5d. Overall, Tg mice had significantly higher CAA-associated PB-positive CMH development compared to WT mice, and 10-week cilostazol treatment did not alter PB-positive CMH development in the WT and Tg mice.

In the studies examining the effect of cilostazol on inflammation-induced PB-positive (subacute) CMH development, we found that the PB-positive lesion number, size, and area were significantly higher in the LPS-treated WT mice fed regular diet compared with the PBS controls fed regular diet (Fig. 6a–c). Cilostazol treatment did not alter the PB-positive lesion number, size, or total area in the LPS-treated WT mice (Fig. 6a–c). Representative images showing PB-positive lesions in the WT, WT-LPS, and WT-LPS-CIL mice are shown in Fig. 6d. Overall, 10-week cilostazol treatment did not alter PB-positive inflammation-induced CMH development.

Figures 7 and 8 show the effect of PDE3A modulation on the various markers of neuroinflammation and BBB function in the inflammation (LPS)-induced and CAA-associated CMH mouse models.

In the inflammation-induced CMH mouse model used for the PDE3A KO studies, LPS treatment produced a significant increase in brain levels of ICAM-1 (a marker of endothelial activation) (Fig. 7a), markers of neuroinflammation (Iba-1 and GFAP) (Fig. 7b, c), and BBB function (IgG, fibrinogen, and claudin-5) (Fig. 7d–f) in the WT and PDE3A^−/− KO mice compared with saline-treated controls. With genetic deletion of PDE3A, there was a significantly reduced ICAM-1 positive immunoreactive area in both the presence and absence of LPS. GFAP positive immunoreactive area was significantly lower in the LPS-treated PDE3A^−/− KO mice compared with the LPS-treated WT littermates (Fig. 7a, c). Genetic deletion of PDE3A did not alter microglial activation (Iba-1) or BBB function (IgG, claudin-5, and fibrinogen), either in the presence or absence of LPS.

In the CAA-associated CMH mouse model used for the pharmacological inhibition studies with cilostazol, the ICAM-1 and Iba-1 positive immunoreactive areas were significantly higher in the Tg mouse brains compared with the WT littermates, while increases in the GFAP positive immunoreactive area in the Tg mice reached statistical significance only in those mice treated with cilostazol (Fig. 8a–c). Markers of BBB function (brain parenchymal IgG, fibrinogen, and claudin-5) did not differ significantly between the WT and Tg mice (Fig. 8d–f). Cilostazol treatment had no significant effect on any marker of endothelial activation, neuroinflammation (except GFAP), or BBB function in either WT or Tg mice compared with their respective regular feed controls (Fig. 8a–f). In the inflammation-induced CMH mouse model, cilostazol treatment did not reduce markers of endothelial, astrocyte, or microglial activation, or injury to BBB (data not shown).