Human eye and brain donors
Donor eyes were obtained from two sources: (1) Alzheimer’s Disease Research Center (ADRC) Neuropathology Core at the Department of Pathology in the University of Southern California (USC, Los Angeles, CA; IRB protocol HS-042071) and (2) National Disease Research Interchange (NDRI, Philadelphia, PA; IRB exempt protocol EX-1055). Both USC-ADRC and NDRI maintain human tissue collection protocols approved by a managerial committee and subject to National Institutes of Health oversight. For a subset of patients and controls we also obtained brain specimens from USC-ADRC. The histological work at Cedars-Sinai Medical Center was performed under IRB protocols Pro00053412 and Pro00019393. Sixty-two postmortem retinas were collected from 29 clinically and neuropathologically confirmed AD patients (age mean ± SD: 81.38 ± 13.79; range 40–98 years; 20 females and 9 males with different disease severities), 11 age- and gender-matched MCI patients (age mean ± SD: 86.45 ± 6.87; range 80–93 years; 5 females and 6 males with different disease severities), and 22 CN individuals (age mean ± SD: 78.18 ± 8.86; range 58–95 years; 13 females and 9 males showing neither clinical cognitive impairment/dementia nor brain pathology). The entire human cohort information is listed in Table 1. The groups had no significant differences in age, sex, or post-mortem interval (PMI) hours. All samples were deidentified and could not be traced back to tissue donors.
Table 1 Demographic data for all human eye donors Clinical and neuropathological assessments
The clinical and neuropathological reports provided by the USC ADRC Clinical Core included subjects’ neurological examinations, neuropsychological and cognitive tests, family history, and medication list; psychometric testing was performed by a trained psychometrist under the supervision of a licensed clinical neuropsychologist, following standard-of-care cognitive screening evaluations of patients in their respective neurology clinics, as previously described [21, 47]. NDRI reports provided the medical history of each subject. Most cognitive evaluations were performed annually, and, in most cases, less than 1 year prior to death. Cognitive testing scores from evaluations obtained closest to subjects’ death were used for this analysis. Two global indicators of cognitive status were used for clinical assessment: the Clinical Dementia Rating (CDR; 0 = Normal; 0.5 = Very Mild Dementia; 1 = Mild Dementia; 2 = Moderate Dementia; 3 = Severe Dementia) [60] and the Mini Mental State Examination (MMSE; normal cognition = 24–30, mild dementia = 20–23, moderate dementia = 10–19, severe dementia ≤ 9) [31]. In this study, the clinical diagnostic groups (AD, MCI, and CN) were determined by the source clinicians, based on a comprehensive battery of tests, including neurological examinations, neuropsychological evaluations, and the above-mentioned cognitive tests. For final diagnosis based on the neuropathological reports, the modified Consortium to Establish a Registry for Alzheimer’s Disease [65] was used as outlined in the National Institute on Aging (NIA)/Regan protocols with revision by the NIA and Alzheimer’s Association [39]. Aβ burden (diffuse, immature, or mature plaques), amyloid angiopathy, neuritic plaques, NFTs, neuropil threads, granulovacuolar degeneration, Lewy bodies, Hirano bodies, Pick bodies, balloon cells, neuronal loss, microvascular changes and gliosis pathology were assessed in multiple brain areas: hippocampus (CA1 and CA4), entorhinal cortex, frontal cortex, temporal lobe, parietal lobe, occipital lobe (primary visual cortex, area 17; visual association cortex, area 18), basal ganglia, brainstem (pons, midbrain), cerebellum and substantia nigra.
Amyloid plaques and tangles in the brain were evaluated using anti β-amyloid mAb clone 4G8, Thioflavin-S (ThioS), and Gallyas silver stain in formalin-fixed, paraffin-embedded tissues. Two neuropathologists provided scores based on independent observations of β-amyloid, NFT burden, and/or neuropil threads (0 = none; 1 = sparse 0–5; 3 = moderate 6–20; 5 = abundant/frequent 21–30 or above; N/A = not applicable), and an average of two readings was assigned to each individual. Final diagnosis included AD neuropathologic change (ADNC). Aβ plaque score was modified from Tal et al. (A0 = no Aβ or amyloid plaques; A1 = Thai phase 1 or 2; A2 = Thai phase 3; A3 = Thai phase 4 or 5) [74]. NFT stage was modified from Braak for silver-based histochemistry or p-tau IHC (B0 = No NFTs; B1 = Braak stage I or II; B2 = Braak stage III or IV; B3 = Braak stage V or VI) [15]. Neuritic plaque score was modified from CERAD (C0 = no neuritic plaques; C1 = CERAD score sparse; C2 = CERAD score moderate; C3 = CERAD score frequent) [58]. Neuronal loss, gliosis, granulovacuolar degeneration, Hirano bodies, Lewy bodies, Pick bodies, and balloon cells were evaluated (0 = absent; 1 = present) in multiple brain areas using hematoxylin and eosin (H&E) staining. Amyloid angiopathy was graded as follows: Grade I = amyloid restricted to a rim around normal/atrophic SMCs of vessels; Grade II = media replaced by amyloid and thicker than normal, but no evidence of blood leakage; Grade III = extensive amyloid deposition with focal vessel wall fragmentation and at least one focus of perivascular leakage; Grade IV = extensive amyloid deposition and fibrinoid necrosis, micro aneurysms, mural thrombi, lumen inflammation, and perivascular neuritis. For the correlation analyses against retinal parameters, we used the following CAA scoring system: no amyloid angiopathy was assigned ‘0’; grade I was assigned as ‘1’, grade I–II as ‘1.5’, grade II as ‘2’, and grade II–III as ‘2.5’.
Collection and processing of eyes and cortical tissues
Donor eyes were collected within 7 h, on average, from time of death and were either preserved in Optisol-GS media (Bausch & Lomb, 50,006-OPT) and stored at 4 °C for less than 24 h, fresh frozen (snap; stored at − 80 °C), or punctured once and fixed in 10% neutral buffered formalin (NBF) or 2.5% Paraformaldehyde (PFA) and stored at 4 °C. Brain tissues (hippocampus; occipital lobe – primary visual cortex, area-17, and frontal cortex, area-9) from the same donors were snap frozen and stored at − 80 °C. Parts from the fresh-frozen brain tissues were fixed in 4% PFA for 16 h following dehydration in 30% sucrose/PBS. Brain tissues were cryosectioned (30 μm thick) and placed in phosphate-buffered saline 1x (PBS) with 0.01% sodium azide (Sigma-Aldrich) at 4 °C. Irrespective of the human donor eye source, USC-ADRC or NDRI, the same tissue collection and processing methods were applied.
Preparation of retinal flatmounts and strips
Fresh-frozen eyes and eyes preserved in Optisol-GS were dissected with anterior chambers removed to create eyecups. Vitreous humor was thoroughly removed manually. Retinas were dissected out, detached from the choroid, and flatmounts were prepared [47]. By identifying the macula, optic disc, and blood vessels, the geometrical regions of the four retinal quadrants were defined with regard to the left and the right eye. Flatmount strips (2–3 mm in width) were dissected along the retinal quadrant margins to create four strips: superior-temporal—ST, inferior-temporal—TI; inferior-nasal—IN, and superior-nasal—NS, and were fixed in 2.5% PFA for cross-sectioning. In a subset of human eye donors, a second set of strips was prepared (5 mm in width) and stored at − 80 °C for protein analysis. Each strip was approximately 2–2.5 cm long from the optic disc to the ora serrata and included the central, mid, and far retinal areas. All the above stages were performed in cold PBS with 1 × Protease Inhibitor cocktail set I (Calbiochem 539,131). Eyes that were initially fixed in 10% NBF or 2.5% PFA were dissected to create eyecups, and the retinas were dissected free. Vitreous humor was thoroughly removed and flatmounts were prepared. As described above, a set of flatmount strips (ST, TI, IN, and NS) was dissected (2–3 mm in width), washed in PBS, and processed for retinal cross-sectioning.
Retinal cross-sections
Flatmount strips were initially embedded in paraffin using standard techniques, then rotated 90° horizontally and embedded in paraffin. The retinal strips were sectioned (7–10 µm thick) and placed on microscope slides that were treated with 3-Aminopropyltriethoxysilane (APES, Sigma A3648). Before immunohistochemistry, the sections were deparaffinized with 100% xylene twice (for 10 min each), rehydrated with decreasing concentrations of ethanol (100–70%), and then washed with distilled water followed by PBS.
Retinal vascular isolation and immunofluorescent staining
We modified the retinal vascular isolation method to use on human retinal tissues and immuno-fluorescently label pericytes and amyloidosis (illustrated in Fig. 1a). This trypsin-induced retinal digestion and vascular network isolation technique was originally developed in 1993 [51] and subsequently modified by replacing trypsin with commercially available elastase [77]. Our modified protocol is as follows: retinal strips from human donors or mouse whole retinas preserved in PFA were first washed in lukewarm running distilled water overnight, then digested in 40 U/ml elastase solution (Merck Millipore, Burlington, MA) for 2 h at 37 °C. After digestion, tissues were incubated in activation solution (Tris buffer at pH 8.5) overnight for extensive digestion. The next day, retinas were transferred to superfrost microscope slides with 1 × PBS, then carefully cleaned with rat whisker to remove unwanted tissues under a dissecting microscope. After cleaning non-vascular tissues, 1 × PBS was applied three times to wash the isolated vascular tissues. When we were able to observe clean vascular tree on slides under dissecting microscope, tissues were mounted on slides carefully without dehydration, then incubated in blocking buffer (Dako #X0909) for 1 h at room temperature (RT). Primary antibodies were applied to the tissue after blocking, then incubated at 4 °C overnight as listed (antibody information provided in Table 2): 4G8/lectin/PDGFRβ, 6E10/lectin/PDGFRβ, 11A50-B10/lectin/PDGFRβ, 12F4/lectin/PDGFRβ. Tissues were then washed three times by PBS and incubated with secondary antibodies against each species (information provided in Table 2) for 2 h at RT. After rinsing with PBS three times, vascular trees were mounted by Prolog Gold antifade reagent with DAPI (Invitrogen #P36935). For quantification purposes, images were taken on a Carl Zeiss Axio Imager Z1 fluorescence microscope (Carl Zeiss MicroImaging, Inc.) equipped with ApoTome, AxioCam MRm, and AxioCam HRc cameras (for more details see “Stereological quantification” below). For representative images, Z-stack images were repeatedly captured at same tissue thickness using a Carl Zeiss 780 Confocal microscope (Carl Zeiss MicroImaging, Inc.). Routine controls were processed using identical protocols while omitting the primary antibody to assess nonspecific labeling. Representative images of all negative controls are shown in Supplementary Fig. 1, online resource.
Table 2 List of antibodies used in the study Mice
The double-transgenic B6.Cg-Tg (APPSWE/PS1∆E9)85Dbo/Mmjax hemizygous (ADTg) mice strain (MMRRC stock #34832-JAX|APP/PS1) and their non-Tg littermates (as WT control non-AD) were used for retinal vascular isolation experiments. All mice are on the genetic background of B6. Mice were purchased from MMRRC and later bred and maintained at Cedars-Sinai Medical Center. The mouse experiments were conducted in accordance with Cedars-Sinai Medical Center Institutional Animal Care and Use Committee (IACUC) guidelines under an approved protocol. We used a total of nine 8.5-month-old mice (all males) divided into three groups: perfused WT (n = 3), perfused ADTg (n = 3), and non-perfused ADTg (n = 3) mice. Animals were deeply anesthetized under Ketamine/Xylazine (40–50 mg/kg) before being euthanized either by transcardial perfusion (0.9% ice-cold sodium chloride supplemented with 0.5 mM EDTA) or cervical dislocation (non-perfused group). Eyes were dissected and the retinas were immediately isolated. Using a 25-gauge needle, a hole is poked in the cornea and an incision is made along the ora serrata to remove the lens and cornea-iris. Next, a small incision is made in the sclera-choroid layers toward the optic nerve and using fine forceps, sclera and choroid is gently separated from the retina, which is cleanly snipped at its base from the optic nerve. Care is taken to isolate whole retina undamaged to preserve vasculature network. Following isolation, retinas were fixed in 4% PFA for 7 days. Retinas were then processed for retinal vascular isolation and immunofluorescent staining as described above.
Biochemical determination of Aβ1–40 levels by sandwich ELISA
Frozen human retinal flatmount strips from the temporal hemisphere (ST, TI) were weighed and placed in a tube with cold homogenization buffer [Tris/EDTA buffer pH 9 (DAKO, S2368), 1% Triton X-100 (Sigma, T8787), 0.1% NaN3 (Sigma, 438456) and 1 × Protease Inhibitor cocktail set I (Calbiochem 539131)], then homogenized by sonication (Qsonica Sonicator M-Tip, Amplitude 4, 6 W, for 90 s; sonication pulse was stopped every 15 s to allow the cell suspension to cool down for 10 s). The ultrasonic probe positioned inside the tube was placed in ice water. Next, retinal strip homogenates were incubated for 1 h at 98 °C in a water bath. After determination of the protein concentration (Thermo Fisher Scientific), retinal Aβ1–40 was determined using an anti-human Aβ1–40 end-specific sandwich ELISA kit (Thermo Fisher, KHB3481).
Immunofluorescent staining of retinal cross-sections
After deparaffinization, retinal cross-sections were treated with antigen retrieval solution at 98 °C for 1 h (PH 6.1; Dako #S1699) and washed in PBS. Retinal sections were then incubated in blocking buffer (Dako #X0909), followed by primary antibody incubation (information provided in Table 2) overnight in 4 °C with the following combinations: PDGFRβ (1:200)/lectin (1:200)/11A50-B10 (1:200), PDGFRβ (1:200)/lectin (1:200)/12F4 (1:200), CD31 (1:50)/JRF/cAβ 40/28 #8152 (1:2000), LRP-1 (1:200)/PDGFRβ (1:200)/lectin (1:200), cleaved caspase-3 (1:200)/PDGFRβ (1:200)/lectin (1:200). Alexa Fluor 488-conjugated tomato lectin was used to visualize blood vessel cells. Retinal sections were then washed three times by PBS and incubated with secondary antibodies against each species (1:200, information provided in Table 2) for 2 h at RT. After rinsing with PBS for three times, sections were mounted with Prolong Gold antifade reagent with DAPI (Thermo Fisher #P36935). Images were repeatedly captured at the same focal planes with the same exposure time using a Carl Zeiss Axio Imager Z1 fluorescence microscope (Carl Zeiss MicroImaging, Inc.) equipped with ApoTome, AxioCam MRm, and AxioCam HRc cameras. Images were captured at 20 ×, 40 ×, and 63 × objectives for different purposes (for more details see “Stereological quantification” below). Routine controls were processed using identical protocols while omitting the primary antibody to assess nonspecific labeling. Representative images of negative controls are shown in Supplementary Fig. 1, online resource.
Peroxidase-based immunostaining of Aβ
Fixed brain sections and retinal cross-sections after deparaffinization were treated with target retrieval solution (pH 6.1; S1699, DAKO) at 98 °C for 1 h and washed with PBS. In addition, treatment with 70% formic acid (ACROS) for 10 min at RT was performed on brain sections and retinal cross-sections before staining for Aβ. Peroxidase-based immunostaining was performed. For antibodies’ list and dilutions, see Table 2. Prior to peroxidase-based immunostaining, the tissues were treated with 3% H2O2 for 10 min, and two staining protocols were used: (1) Vectastain Elite ABC HRP kit (Vector, PK-6102, Peroxidase Mouse IgG) according to manufacturer’s instructions or (2) All Dako reagents protocol. Following the treatment with formic acid, the tissues were washed with wash buffer (Dako S3006) for 1 h, then treated with H2O2 and rinsed with wash buffer. Primary antibody (Ab) was diluted with background reducing components (Dako S3022) and incubated with the tissues for 1 h at 37 °C for JRF/cAβ 40/28 # 8152, or overnight at 4 °C for 11A50–B10 (Aβ40) mAbs. Tissues were rinsed twice with wash buffer on a shaker and incubated for 30 min at 37 °C with secondary Ab (goat anti mouse ab HRP conjugated, DAKO Envision K4000), then were rinsed again with wash buffer. For both protocols, diaminobenzidine (DAB) substrate was used (DAKO K3468). Counterstaining with hematoxylin was performed followed by mounting with Faramount aqueous mounting medium (Dako, S3025). Routine controls were processed using identical protocols while omitting the primary antibodies to assess nonspecific labeling. Representative images of negative controls are shown in Supplementary Fig. 1, online resource.
Transmission electron microscopy (TEM) analysis
Analyses of a retinal whole mount from an AD donor retina that was pre-stained with anti-Aβ42 mAb (12F4) and a high-sensitivity immunoperoxidase-based system with 3,3′ Diaminobenzidine (DAB) substrate chromogen were performed using transmission electron microscopy. Stained tissues were processed for electron microscopic imaging; the samples were dehydrated in serially graded ethanol and then infiltrated in Eponate 12 (Ted Pella, Inc. Redding, CA, USA) prior to embedding between two acetate sheets. Ultrathin sections of retina were cut into cross sections at a thickness of 70 nm, examined on a JEOL JEM 2100 (JEOL USA, Peabody, MA, USA), and photographed with the Orius SC1000B digital camera (Gatan, Pleasanton, CA, USA). Images were processed and colorized using Adobe Photoshop CS4 (Adobe Inc., San Jose, CA, USA).
TUNEL assay for detection of apoptotic retinal pericytes
Formalin-fixed paraffin-embedded retinal cross-sections after deparaffinization were washed with PBS and then incubated with Proteinase-K (Recombinant PCR grade, 15 µg/ml in 10 mM Tris/HCL pH 7.6; Roche Diagnostics GmbH; 03115836001) at 37 °C for 20 min. Next, slides were washed with PBS and incubated with TUNEL reaction mixture (50 µl on each slide; Roche Diagnostics GmbH; 11684795910) at 37 °C for 60 min, in a humidified chamber in dark (the samples were covered with parafilm to ensure a homogeneous spread of TUNEL reaction and to avoid evaporation loss). Afterward, slides were washed with PBS and fluorescent-based immunostaining was performed using blocking solution (DAKO X0909) for 45 min at RT. The tissues were incubated with primary antibody, goat anti PDGFRβ, overnight at 4 °C, then the secondary antibody, donkey anti goat Alexa 647, was applied for 1 h at RT. Then, the samples were washed with PBS and covered with ProLong™ Gold antifade mounting media with DAPI (Molecular Probes; #P36935). Negative and positive controls were included (see Supplementary Fig. 1, online resource) in this experimental setup: for TUNEL negative control the retinal tissues were incubated with only 50 µl of TUNEL label solution (without the TUNEL Enzyme solution-terminal transferase) instead of TUNEL reaction mixture. For TUNEL positive control the retinal tissues were incubated with DNase I (1000 U/ml in 50 mM Tris–HCL, pH 7.5; Worthington Biochemical Corp. Code D) to induce DNA strand breaks, prior to labeling procedure. The retinal tissue sections were then evaluated under fluorescent microscope.
Stereological quantification
For Fig. 1 of isolated retinal blood vessels, quantification was performed from 5 AD donors and 5 age- and sex-matched CN controls. The fluorescence of specific signals was captured using the same setting and exposure time for each image and human donor, with a Z-stack of 10 µm thickness using Axio Imager Z1 microscope (with motorized Z-drive) with AxioCam MRm monochrome camera ver. 3.0 (at a resolution of 1388 × 1040 pixels, 6.45 µm × 6.45 µm pixel size, dynamic range of > 1:2200 that delivers low-noise images due to Peltier-cooled sensor). Images were captured at 40 × objective, at respective resolution of 0.25 µm. Fifteen images were taken randomly from each region of central, mid-, and far-peripheral retina (five from each region) per subject. Acquired images were converted to grayscale and standardized to baseline using a histogram-based threshold in the NIH ImageJ software (version 1.52o). For each biomarker, total area of immunoreactivity was determined using the same threshold percentage from the baseline in ImageJ (with same percentage threshold setting for all diagnostic groups). The images were then subjected to particle analysis for lectin and Aβ to determine IR area. Pericyte number was based on 15 images, averaging the number in each microscopic visual field (covering 1.8 × 104 µm2 area), per human donor. We used the grid mode in ImageJ to manually count the number of pericytes. The ratio of Aβ to lectin was calculated by dividing Aβ IR area by lectin IR area in each of the 15 images (described above) and averaging the values per human donor. The sum of Aβ IR area from an identical number of randomly selected pericytes (n = 10) from each human donor was used to calculate Aβ in pericytes. An identical region of interest was used for the standardized histogram-based threshold technique and subjected to particle analysis.
For Figs. 2, 3, 4, 5, and 6 with analysis of retinal cross-sections and quantifications of PDGFRβ, vascular Aβ42, vascular Aβ40, Aβ40, LRP-1, cleaved caspase-3 and TUNEL, images were also acquired at the same setting and exposure time for each experiment, using the Axio Imager Z1 microscope, as described above. Images were captured at either 20 × or 40 × objectives, at respective resolutions of 0.5 and 0.25 µm. Three images were taken from central and far-peripheral retina and four images were taken from mid-peripheral retina (as shown in Fig. 2a, b). For each biomarker, the total area of immunoreactivity was determined using the same threshold percentage from the baseline in ImageJ (with same percentage threshold setting for all images), then subjected to particle analysis for each biomarker to determine their area or area percentage. For vascular PDGFRβ, vascular Aβ42 and Aβ40, and vascular LRP-1, area of blood vessels was chosen to acquire positive immunoreactive (IR) area percentage. For total retinal Aβ40 and total LRP-1 area, we chose the whole retina and documented total IR area of each biomarkers. Quantification of cleaved caspase-3+ and TUNEL+ pericytes was performed by randomly choosing 10–15 pericytes from each human donor, followed by manually counting using the grid in ImageJ. Then a percentage of cleaved caspase-3+ or TUNEL+ pericytes was calculated.
For vascular markers of Aβ42, Aβ40, and PDGFRβ, analysis was performed separately for longitudinal blood vessels and vertical blood vessels. Retinal cross-sections in this study were cut sagittally from flatmount strips, hence blood vessels were categorized by the shape of lectin stain: either as vertical blood vessels (≥ 10 µm in diameter) or longitudinal blood vessels (~ 10 µm in diameter). Note: for vertical blood vessels, the vascular wall area (determined by lectin) was selected for analysis, while excluding the blood vessel lumen. For longitudinal blood vessels, the total blood vessel including lumen and wall were selected for quantitative analysis. Dotted eclipse or rectangle frames were added to the representative images to highlight the area of quantification for both vertical blood vessels and longitudinal blood vessels.
Statistical analysis
GraphPad Prism 8.1.2 (GraphPad Software) was used for analyses. A comparison of three or more groups was performed using one-way ANOVA followed by Sidak’s multiple comparison post-hoc test of paired groups. Groups with two independent variables/factors were analyzed by two-way ANOVA followed by Sidak’s multiple comparison test to further understand interaction between the two independent variables. Two-group comparisons were analyzed using a two-tailed unpaired Student t test. The statistical association between two or more variables was determined by Pearson’s correlation coefficient (r) test (Gaussian-distributed variables; GraphPad Prism). Pearson’s r indicates direction and strength of the linear relationship between two variables. Required sample sizes for two group (differential mean) comparisons were calculated using the nQUERY t test model, assuming a two-sided α level of 0.05, 80% power, and unequal variances, with the means and common standard deviations for the different parameters. Results are expressed as mean ± standard error of the mean (SEM). P value less than 0.05 is considered significant.