In order to create a diabetic AD mouse model, we crossed the obese, diabetic Lepr
db/db (db/db) mice [11–13] with the APP
P264L/P264L (APP/PS1) knock-in model of AD [14, 15]. Because the homozygous db/db mice are infertile, heterozygous (Lepr
db/+) mice on a C57Bl/6 J background (Jackson Labs; Bar Harbor, ME) were bred with APP/PS1 mice on a CD-1/129 background (obtained from the breeding colony at the University of Kentucky). The resulting F1 mice heterozygous for all three alleles were then intercrossed to generate wild-type, heterozygous, and homozygous db mice that were either wild-type or homozygous for the AD knock-in genes. For most of the data presented here, we focused on four main genotypes: wild-type (WT; Lepr
+/+ × APP
+/+), db (Lepr
db/db × APP
+/+), AD (Lepr
+/+ × APP
P264L/P264L), and db/AD (Lepr
P264L/P264L). Some analyses included Lepr
+/+ and Lepr
db/+ × APP
P264L/P264L mice (noted where appropriate). Mice were housed under a 12 hour light–dark cycle and fed standard rodent chow ad libitum. Mice were euthanized by CO2 asphyxiation, followed by decapitation. All animal work was conducted with prior University of Kentucky (UK) IACUC approval, and was performed in accordance with USDA and PHS guidelines.
Tail snips were collected prior to weaning. For some of the db and APP genotyping, tail snips were sent to Transnetyx (Cordova, TN) for purification and analysis. For those analyzed in our lab, as well as PS1 genotyping, genomic DNA was isolated and purified from tail snips using the Promega Wizard Genomic DNA kit (Promega; Madison, WI). db genotyping was performed using a single nucleotide polymorphism Taqman® genotyping kit (Applied Biosystems Life Technologies; Grand Island, NY). APP and PS1 genotyping were performed by PCR as described previously  using GoTaq® Flexi DNA Polymerase (Promega).
The mice used for this study were broadly divided by age and will be referred to as young (1–4 months old: 3.0 ± 0.8 months), middle-aged (5–9 months old: 7.2 ± 1.6 months), and older (10–14 months old: 12.2 ± 1.0 months) based on the predicted lifespan of the db/AD mice (~15-16 months).
Glucose and insulin tolerance tests
Mice were fasted 3–6 hours prior to the start of the glucose tolerance test (GTT) or insulin tolerance test (ITT). All glucose measurements were obtained via tail bleed using a Bayer Breeze 2 glucometer and test strips (Bayer; Tarrytown, NY). For the GTT, a baseline measurement was obtained after which the GTT was initiated by intraperitoneal injection of dextrose (2 mg/g: Hospira; Lake Forrest, IL). Subsequent measurements were recorded at 15, 30, 60, and 120 minutes post-injection. For the ITT, a baseline glucose measurement was taken, after which insulin (0.75 U//kg: Eli Lilly; Indianapolis, IN) was injected intraperitoneally. Subsequent measurements were recorded at 15, 30, 60, and 120 minutes post-injection. Any glucometer reading of “HI” was set to 700 mg/dL for data analysis.
Blood pressure measurements
Blood pressure (BP) was measured using a Kent CODA 8 BP machine (Kent Scientific; Torrington, CT). Animals were allowed to acclimate to the tail blood pressure cuff for five minutes on a warming platform before recording BP measures. The BP measures consisted of 20 cycles of diastolic/systolic measures, with a 20 second rest period between cycles. After finishing the data collection, the mice were immediately released back into their home cages. The rodent restraints, cuffs, and warming platform were cleaned between animals; female animals were always run after male animals to avoid any possible irritation of the males. BP measures were performed at the same time each day to account for the possible influence of circadian rhythms.
Blood was collected upon decapitation in the presence of EDTA, centrifuged (1500 × g, 10 min.), and the plasma collected. Plasma leptin was measured by a commercially-available, species-specific ELISA (EMD Millipore; Billerica, MA), according to package instructions.
Frozen brain tissue was serially extracted in either PBS or HEPES (20 mM HEPES, 2 mM EDTA, 2 mM EGTA, 0.32 M sucrose) followed by 2% SDS, and 70% formic acid as previously described [17, 18]. Buffers were supplemented with protease inhibitor cocktail (Amresco; Solon, OH) and phosphatase inhibitor cocktail (EMD Millipore). The tissue was homogenized using an AHS200 PowerMax (VWR; Radnor, PA) homogenizer, the insoluble material was removed by centrifugation (PBS/HEPES/SDS: 20,800 × g, 30 minutes; formic acid; 20,800 × g, 60 min) and the supernatants frozen until use. Human-specific Aβ was measured by two-site sandwich ELISA as previously described . Oligomeric Aβ (mouse and human) was measured by single-site sandwich ELISA as previously described [19, 20]. Briefly, 384-well plates (Immulon 4HBX: Thermo Scientific; Waltham, MA) were coated with either 0.5 μg Ab42.5 (Aβtotal and Aβ1–40), Ab2.1.3 (Aβ1–42), or 4G8 (oligomers: Covance, Princeton, NJ)/well and blocked with Synblock (Serotec; Raleigh, NC) for two hours. PBS and SDS extracts were diluted in AC buffer (0.2 M sodium phosphate (pH7), 0.4 M NaCl, 2 mM EDTA, 0.4% Block Ace (Serotec), 0.4% BSA, 0.05% CHAPS, 0.05% NaN3) for analysis. Formic acid extracts were first neutralized with TP buffer (1 M Tris base, 0.5 M sodium phosphate: 20-fold dilution), then further diluted with AC buffer for analysis. Similarly, plasma was diluted in AC buffer for analysis. A standard curve was prepared from recombinant human Aβ1–42, Aβ1–40, or oligomeric Aβ diluted in AC buffer. Standards and samples were measured at least in duplicate. After incubation with the samples and standards, Aβ was detected with either biotinylated-4G8 (Aβtotal, Aβ1–42, and oligomers: Covance) or biotinylated-13.1.1 (Aβ1–40), followed by incubation with 0.1 μg/mL NeutrAvidin-HRP (Pierce Technologies; Rockford, IL). The plate was developed with 3′,3′,5′,5′-tetramethylbenzidine (Kirkeguard and Perry Laboratories; Gaithersburg, MD) and the reaction stopped with 6% o-phosphoric acid. The absorbance at 450 nm was measured with a BioTek (Winooski, VT) multiwell plate reader.
Protein levels of PS1, BACE1, BACE2, phosphorylated and total tau, endothelin-converting enzyme 1 (ECE1), and PSD95 were determined by Western or spot blot, using protein-specific antibodies (PS1 (EMD Millipore), BACE1 (Epitomics; Burlingame, CA), BACE2 (Abcam; Cambridge, MA), pTau (AT8: Sigma-Aldrich; St. Louis, MO), total tau (HT7: Pierce: [21, 22]), ECE1 (Acris Antibodies; San Diego, CA), PSD95 (D27E11; Cell Signaling; Danvers, MA)). Immunoreactive bands for PS1, BACE1, BACE2, tau, and ECE1 were visualized with Super Signal West Dura chemiluminescence HRP substrate (Pierce) after incubation with HRP-conjugated secondary antibodies and exposed to film. Densitometric analyses were performed using Image J software. Expression was standardized to β-actin (Sigma-Aldrich) or GAPDH (Abcam) expression in the same lane or spot, respectively. PSD95 and its GAPDH loading control (Abcam) were visualized with fluorescently-labeled secondary antibodies (LI-COR; Lincoln, NE) using an Odyssey Infrared Imager (LI-COR) for quantitation and analysis.
Tissue was homogenized in Trizol™ (Invitrogen; Grand Island, NY) in order to isolate RNA, followed by phenol/chloroform extraction. When needed, RNA was further purified by RNeasy columns (Qiagen; Valencia, CA). Expression of ECE1 and ECE2 were determined by two-step qRT-PCR, using iScript (BioRad; Hercules, CA) reverse transcription, followed by qPCR with PerfeCTa FastMix™ (Quanta BioSciences; Gaithersburg, MD). The geometric mean of the CT values for RPL30, cyclophilin, and RNA polymerase IIJ was used as an internal control to calculate and compare relative expression (2-ΔΔC
T). Gene specific primer sets were obtained from IDT (Coralville, IA).
Neprilysin and insulin degrading enzyme activity
Neprilysin (NEP) activity was measured as described . Briefly, hemibrains were homogenized in ice-cold Tris buffer (50 mM Tris–HCl and 150 mM NaCl, pH 7.2; 100 mg/mL) supplemented with 1 mM PMSF (Sigma-Aldrich), and 10 μM E-64 (RPI; Mt. Prospect, IL). The homogenate was centrifuged (1000 × g, 20 min., 4°C), followed by a high-speed centrifugation of the supernatant (100,000 × g, 1 hour, 4°C). The supernatant was removed, and the pellet resuspended in Tris buffer for the enzyme assay. NEP activity was measured using glutaryl-Ala-Ala-Phe-4-methoxy-2-naphthylamide (Sigma-Aldrich) as a substrate. Reactions were initiated with the addition of the membrane fraction, then fluorescent product formation was monitored (340 nm excitation, 425 nm emission, 37°C). Phosphoramidon (50 μM) and thiorphan (10 μM) were used to inhibit NEP activity and determine background fluorescence for each sample.
Insulin degrading enzyme (IDE) activity was measured using a commercially-available kit (EMD Millipore) according to manufacturer’s instructions. Briefly, hemibrains were homogenized in Tris buffer (100 mg/mL) supplemented with PMSF and E-64, centrifuged (20,800 × g, 30 min., 4°C), and the supernatant used for the activity assay. Samples were compared against rat IDE. Fluorescence was measured at an excitation wavelength of 320 nm and an emission wavelength of 405 nm.
T2*-MRI was performed using a horizontal bore Bruker Clinscan (7.0 T, 30 cm, 300 MHz: Billerica, MA) imager equipped with a triple-axis gradient (630 mT/m and 6300 T/m/s) and a helium-cooled 14 K quadrature head cryo-coil, cooled to 20°K. T2*-weighted images were acquired with a 2D GRE sequence with at 34 μm × 34 μm × 400 μm resolution, 15 mm FOV, 25 degree flip angle, 10 averages, TR 165 ms, and TE 15.3 ms. Mice were imaged under constant isofluorane anesthesia and their body temperature and respiration were continuously monitored. At least ten equally-spaced images were taken of each mouse brain. Asymmetrically-occurring dark spots on the images were considered indicative of vascular events (confirmed histologically, see below), whereas symmetrically-occurring dark areas were considered to be blood vessels and were excluded.
Vascular corrosion casting
Vascular corrosion casting was performed as described . Briefly, mice were anesthetized using pentobarbital (100 mg/kg), followed by transcardial perfusion with heparinized saline (0.9%). Following a brief perfusion with para-formaldehyde (4%), the brains were perfused with the polyurethane resin Pu4ii (4 mL/min: VasQtec; Switzerland). After allowing the resin to cure for at least two days, the brains were incubated in KOH (7.5%, 50°C, 48 h), followed by formic acid (5%, 50°C, 24 h). The tissue was subsequently frozen, then lyophilized to macerate the soft tissue. Finally, the casted brains were sputter-coated in palladium and viewed by scanning electron microscopy (Hitachi S-4300: Schaumburg, IL), using the middle cerebral artery as a landmark. Endothelial cell density was determined by endothelial cell nuclear imprints measured directly using Image J software. Aneurysm pathology was assessed on a 4 point scale based on clear data break points (0 = none; 1 = 1 possible; 2 = 1–3 definite; 3 = 4+ definite. Vascular density was determined by rank order of representative images using three blinded, independent reviewers. Images were scored from 1 (most dense) – 26 (least dense), and the ranks from the three reviewers averaged.
Tissue was harvested and fixed in PBS-buffered 10% formalin for at least 24 hrs. For Aβ immunohistochemistry, hemibrains were embedded in a matrix and sectioned (30 μm) by NeuroScience Associates (Knoxville, TN). For Prussian blue staining, hemibrains were embedded in paraffin and sectioned to 8 μm using a microtome. For free-floating sections, the hemibrains were incubated in sucrose (10%, 20%, 30% sequentially for 24 hours each) for cryoprotection, then sectioned on a sliding, freezing microtome to 25 μm.
Perl’s Prussian blue staining of hemosiderin was performed as described . Immunohistochemistry detecting Aβ was performed using antibody 4G8 (Covance) as described . Some Aβ immunohistochemistry was performed by NeuroScience Associates. Densitometry was performed on these sections using Image J software. Vascular Aβ was visualized by three different methods: 1) Congo red (0.2% in NaCl-saturated 80% ethanol), 2) Thioflavin S (1%: Sigma-Aldrich), and 3) resorufin (Sigma-Aldrich: ). Cerebral blood vessels were imaged in free-floating sections using a mouse anti-α-actin antibody (A5228: Sigma-Aldrich), followed by quantitation with Image J software. Triple labeling of free-floating sections was performed with the fluorescent Aβ-specific Amylo-Glo stain (Biosensis; Thebarton, Australia), rabbit anti-collagen IV (ab6586: Abcam), and rabbit anti-glial fibrillary acidic protein (G9269: Sigma-Aldrich).
Testing was performed by the UK Rodent Behavioral Core (http://www.rodentbehaviorcore.uky.edu/default.aspx/0_UK_Rodent_Behavior_Core). Mice were tested using the Morris Water Maze paradigm. The maze consisted of a circular pool (134.5 cm diameter) filled with 25°C water. A circular platform (11 cm diameter) was placed in the northeast quadrant 1 cm below the surface of the water so that it was not visible. Nontoxic tempura paint was used to create opaque water, thus obscuring the platform. The pool was placed behind dark curtains holding external maze cues. The cues were rotated each day. There were five consecutive training/acquisition days. On each-training day the animals swam four trials (rotating initial placement each time), lasting one minute each, with a five minute interval between trials. After a 30 minute rest upon the conclusion of training on the fifth day, we performed a probe trial where the platform was removed from the pool. The animal’s location in the pool was recorded for one minute and used to calculate the time spent in the target quadrant and the number of times crossing the platform area. After the completion of training, mice were tested for visual acuity during which the external cues were provided along with a visibly-raised platform. The mice were tested for visual acuity in four trials during one day. Water Maze data (e.g. swim speed, distance, latency to platform. etc.) were collected and analyzed using EthoVision XT software (Noldus Information Technology; Leesburg, VA).
A small number of db/AD mice (N = 7; 9–12 month old; 3 M/4 F) were injected intraperitoneally with Ab42.5 (300 μg in sterile saline) every two weeks for two months. Mice were imaged by T2* MRI prior to starting the treatment (baseline) and prior to death (endpoint). The majority of the brains (N = 6; 2 M/4 F) were extracted in RIPA buffer (50 mM Tris–HCl, 150 mM NaCl, 1% Triton X-100, 0.5% deoxycholate, 0.1% SDS; pH = 8.0) with protease inhibitor cocktail (Amresco) for Aβtotal ELISA measurement as described above. Brains from untreated, age-matched db/AD mice (N = 6; 2 M / 4 F) were also extracted in RIPA and used as controls. Endpoint MRI scans were compared against untreated, age-matched (11–14 months old) db/AD mice.
Weight data were analyzed by student’s t-test at each age using Microsoft Excel, and the probability adjusted using the Holm-Bonferroni method . All other data were analyzed with SPSS (Hewlett Packard; Palo Alto, CA) using the general linear model (GLM) module for ANOVA with the independent variables gender, db genotype, and AD genotype (for an explanation of this model, see http://pic.dhe.ibm.com/infocenter/spssstat/v21r0m0/index.jsp?topic=%2Fcom.ibm.spss.statistics.help%2Fidh_glm_multivariate.htm). Post-hoc multiple comparisons were conducted using Tukey’s test, Dunnett’s test, or similar. Chi-square analyses were performed on the visual acuity measurements for the Morris Water Maze. We performed correlation analyses using either Pearson’s r or Spearman’s ρ (parametric and nonparametric values, respectively), and adjusted probability using the Holm-Bonferroni method. For nonparametric comparisons, we used a Kruskal-Wallis ANOVA, or Mann–Whitney U test, where appropriate. For most presented statistics, we note an overall effect of genotype (db or AD) across the data set. In some cases, we also present a direct comparison between two different genotypes.