2.1. IL-1 pellet implantation
Pellets impregnated with IL-1 β (100 ng of recombinant mouse IL-1 β (Sigma Chemical Co., St. Louis, MO)) and control pellets (without IL-1β impregnation) were obtained from Innovative Research of America (Sarasota, FL). As described earlier (Sheng et al., 2000
), these pellets were 1.5 mm in diameter and designed for controlled, slow release of IL-1 over a 21-day period. Forty male Sprague–Dawley rats, weighing 264 ± 6 g, were randomly assigned to three groups. Sixteen rats received implants of IL-1β-containing pellets (IL-1), 14 rats received pellets without IL-1 impregnation (sham), and 10 rats served as unoperated (normal) rats. The pellets were implanted 2.8 mm caudal to bregma, 4.5 mm right of the midline, and 2.5 mm deep to the pial surface. Twenty-one days following pellet implantation, rat brains were processed either for immunohistochemistry or for mRNA analysis.
2.2. Patients and tissues
Tissues from 12 demented patients, with postmortem neuropathological confirmation of Alzheimer’s disease according to CERAD criteria (Mirra et al., 1991
), were used in this study (four males, eight females; age 63–92 years; postmortem interval 2–13 h). Tissues from nine patients with no clinical or pathological evidence of neurological or psychiatric disease were used as controls (seven males, two females; age 50–93 years, postmortem interval 1–15 h). Parahippocampal tissue samples were obtained fresh at the time of autopsy from the left cerebrum. These were frozen in liquid nitrogen, and stored at −80°C until used for Western immunoblot analysis or for analysis of IL-1 mRNA. The right half of autopsied brains was fixed in 20% formalin for 7–10 days. Tissue blocks of hippocampus and adjacent mesial temporal cortex at the level of lateral geniculate nucleus were then obtained and embedded in paraffin for sectioning for immunohistochemical studies.
The polyclonal anti-human IL-1 antibody used here is specific for the IL-1α isoform (Cistron Biotechnology, Pine Brook, NJ). This antibody recognizes the 33-kDa (uncleaved) form of IL-1α, and reliably labels IL-1-expressing cells in tissues. The polyclonal anti-phospho-p38 MAP kinase antibody used here detects p38 MAP kinase only when activated by dual phosphorylation at threonine 180 and tyrosine 182 (New England Biolabs Inc, Beverly, MA). AT8 is a monoclonal antibody that recognizes tau protein only when phosphorylated at both serine 202 and threonine 205 (Goedert et al., 1995
). Monoclonal anti-Tau antibody (Zymed Laboratories Inc., South San Francisco) recognizes tau protein independent of phosphorylation state, at an epitope located within amino acids 404–441 of the rat sequence.
Single-label immunohistochemistry was performed on 10-μm sections cut from paraffin-embedded blocks of hippocampus and adjacent mesial temporal cortex. Paraffin sections were deparaffinized, rehydrated, permeabilized, and peroxidase blocked as described earlier (Sheng et al., 1997
). The primary antibody, either anti-IL-1 (diluted 1:20), anti-MAPK-p38 (1:300), or AT8 (1:300), was diluted in 2% normal goat serum in tris-buffered saline (TBS) and was incubated on the sections overnight at room temperature. The link antibody, either anti-mouse IgG or anti-rabbit IgG (Cappel), was diluted 1:50 in 2% normal goat serum in TBS and incubated on the sections for 30 min. The secondary antibody, either goat anti-rabbit or anti-mouse peroxidase-anti-peroxidase (diluted 1:300, DAKO, Carpinteria, CA), was incubated for 30 min. Immunoreactive structures were developed as a brown color using a diaminobenzidine kit (Zymed, South San Francisco).
Dual-label immunohistochemistry was performed (10 Alzheimer and seven control patients) using a commercially available kit (K1395, DAKO). Tissue sections were deparaffinized, rehydrated, and permeabilized as described above, and primary antibodies were diluted as described earlier (Sheng et al., 1997
). For IL-1/MAPK-p38 double labeling, anti-IL-1 antibody was applied directly to the tissue sections for overnight incubation. The sections were then washed in TBS (3 × 5 min), incubated in the kit peroxidase-labeled polymer (HRP from bottle 2) for 15 min, and covered with substrate-chromagen solution to produce a brown color reaction. The reaction was stopped with double stain block (bottle 4) for 3 min. The tissue sections were then incubated for 30 min with the second primary antibody, anti-MAPK-p38, followed by alkaline phosphatase-labeled polymer (bottle 5) for 30 min. Sufficient substrate-chromagen solution was applied to produce a red color reaction. MAPK-p38/AT8 double labeling was performed using a similar technique.
2.5. Quantification of immunoreactive cells
The numbers of cells immunoreactive for IL-1, for MAPK-p38, or for AT8 were counted using a CCD video camera attached to a Macintosh computer. Five 25 × microscopic fields (0.1 mm2 each) were analyzed for each patient.
2.6. Western immunoblot analysis
For analysis of tissue levels of total tau protein and AT8-immunoreactive (hyperphosphorylated) tau protein, aliquots of tissue homogenate (containing 20 μg of sample protein) from 12 Alzheimer and nine control patients were subjected to 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis. The gels were transferred to nitrocellulose membranes (0.45 μm; Schleicher and Schuell, Keene, NH) using 100 V × 1 h. Subsequently, the membranes were stained with Ponceau S (Sigma), then incubated overnight in 10 ml blotto (10% non-fat dry milk, 0.01% sodium azide) containing either anti-Tau (1:500) or AT8 (1:1000) antibodies, and washed for 1 h in blotto and phosphate-buffered saline (PBS) before incubation with 0.5 μCi of I125-labelled IgG. The membranes were then washed in PBS and exposed to Kodak-XAR film for either 42 h (for AT8) or 32 h (for anti-Tau). The autoradiographic images were analyzed using a MIRROR scanner and an Apple computer.
2.7. Reverse transcription-polymerase chain reaction analysis
Reverse transcription-polymerase chain reaction (RT-PCR) was carried out using an Advantage™ RT-for-PCR Kit (Clontech Laboratories, Inc., Palo Alto, CA). One microgram of total RNA was reverse transcribed in a 20 μl volume containing 20 pM oligo (dT)18 primer, 0.5 mM each dNTP, 200 U Moloney-Murine Leukemia Virus reverse transcriptase, 75 mM KC1, 3 mM MgCl2, and 50mM Tris–HCl, pH 8.3. After annealing the oligo (dT)18 primer at 70°C for 15 min, the samples were placed on ice and the remaining reaction mix was added. Following incubation at 42°C for 60 min, the reverse transcriptase was inactivated at 94°C for 5 min.
PCR was performed using an Advantage™ cDNA PCR Kit (Clontech). For the analysis of human tissue, a 30 μl reaction volume contained 5 μl template cDNA, 60 nM GAPDH (Clontech) and 0.2 μM IL-lα in 15 mM KOAc, 3.5 mM Mg(OAc)2, 75 μg/ml bovine serum albumin (BSA), and 40 mM Tricine–KOH, pH 9.2, supplemented with 0.2 mM dNTP, and 1 μl of Advantage™ cDNA Polymerase Mix (Klentaq-1 DNA polymerase and TaqStart Antibody). For the analysis of rat tissue, a similar amplification mix was used except the 30 μl reaction volume containing 2 μ (il template cDNA, 60 nM rat GAPDH and 0.2 μM MAPK-p38 primers (Ransom Hill Bioscience, Inc., Ramona, CA). Following addition of the Taq mix, cDNA was amplified using a Perkin-Elmer 2400 GeneAmp PCR System (Perkin-Elmer, Norwalk, CT). Linearity was established by generating a standard curve over a range of cycles for each primer and for pellet, sham, and control animals as well as for Alzheimer and control patients. Differences reported were evident over a range of cycles, demonstrating linearity. The mixture was subjected to 30 cycles of amplification for IL-1 and 29 cycles of amplification for MAPK-p38, with denaturation at 94°C for 1 min; annealing at 55°C for 1 min for IL-1 and at 60°C for 1 min for MAPK-p38; extension at 72°C for 1 min; and a final extension step at 72°C for 7 min.
Ten microlitres of amplified cDNA products (from 11 Alzheimer and seven control patients) were loaded onto each lane of a 1.5% agarose gel. The samples and a 1Kb ladder (Life Technologies, Gaithersburg, MD) were fractionated by size. The gel image was produced on a Stratagene Eagle Eye Digital Imaging system (Stratagene, La Jolla, CA) and saved for further analysis.
The statistical significance of differences in MAPK-p38 mRNA expression in experimental animals was assessed using ANOVA followed by Fisher’s test. The statistical significance of differences in numbers of IL-1-immunoreactive microglia, MAPK38-, and AT8-immunoreactive neurons between Alzheimer’s and control tissues was assessed using Student’s t-test. The significance of correlations between tissue IL-1 mRNA levels and numbers of IL-1-immunoreactive microglia, MAPK38-, and AT8-immunoreactive neurons were assessed using regression analysis.