Pharmacological antagonism of interleukin-8 receptor CXCR2 inhibits inflammatory reactivity and is neuroprotective in an animal model of Alzheimer’s disease

Human brain tissue

Preparation of human ND and AD sections

The procedures used to isolate postmortem tissue have been described 20]. Entorhinal cortical sections from six ND cases (ages from 60 to 85 years, postmortem
intervals, 6–24 h) and six AD cases (ages from 64 to 87 years, postmortem intervals,
5–10 h) were obtained from the Kinsmen Laboratory brain bank at the University of
British Columbia (UBC, Vancouver, British Columbia, Canada). Average age of individuals
and mean postmortem delay did not differ significantly between AD and ND cases. The
ND cases exhibited no clinical or pathological history of dementia or other neurological
disorders. Five of the ND cases were scored as Braak stage I with one case scored
as Braak stage II 21]. All cases of AD met the clinical criteria and postmortem confirmation for AD 22] and were characterized by high levels of plaque density and neurofibrillary tangles.
The AD cases were rated as Braak V (one case) or VI (five cases).

Immunohistochemical staining and analysis in human ND and AD sections

For immunofluorescent staining, free-floating sections (30 ?m) from ND and AD tissues
were washed in phosphate-buffered saline (PBS) with Triton X-100 (PBST; 0.01 M PBS,
pH 7.4, containing 0.3 % Triton X-100) and transferred into 5 % skim milk in PBST
for 1 h. Sections were then incubated for 48 h at 4 °C with antibodies for CXCR2,
HLA-DR, or GFAP and then rinsed in PBST and incubated with Alexa Fluor 488-conjugated
goat anti-rabbit IgG (1:200; Invitrogen) for 1 h at room temperature. After washing
in PBST, sections were mounted on glass slides and coverslipped with Prolong Gold
anti-fading agent (Invitrogen). For double-immunofluorescence staining 23], free-floating sections were incubated for 48 h at 4 °C with a mixture of two primary
antibodies: CXCR2/HLA-DR and CXCR2/GFAP. After incubation with the indicated primary
antibodies, sections were rinsed in PBST and incubated for 1 h at room temperature
with a mixture of Alexa Fluor 488 goat anti-rabbit IgG (1:200; Invitrogen) and Alexa
Fluor 594 goat anti-mouse IgG secondary antibody (1:200; Invitrogen).

In vivo studies using intrahippocampal injection of A? peptide

Surgical procedures. All animal procedures were approved by the UBC Animal Care Ethics
Committee, with adherence to guidelines of the Canadian Council on Animal Care. Male
Sprague Dawley rats (Charles River Laboratories, Montreal, QC, Canada) weighing 280–300 g
were used for in vivo studies. In brief, rats were injected intraperitoneal (ip) with
an anesthetic mixture of ketamine hydrochloride (100 mg/kg; Bimeda-MTC, Cambridge,
ON, Canada) and xylazine hydrochloride (10 mg/kg; Bayer Inc., Etobicoke, ON, Canada)
and were placed in a stereotaxic apparatus (David Kopf Instruments, Tujunga, CA, USA).
Animals received stereotaxic injection of A?
_1–42
or controls (PBS or reverse peptide A?
_42–1
) as previously described 6], 24]–26]. Following skin incision to expose the skull, peptides (California Peptides, Napa,
CA, USA) were slowly injected (0.2 ?l/min) into the dentate gyrus region of rat hippocampus.
Injection coordinates were as follows: anterior-posterior (AP), ?3.3 mm; medial-lateral
(ML), ?1.6 mm; dorsoventral (DV), ?3.2 mm; all measurements from bregma.

Preparation and administration of chemicals

Amyloid peptide. The procedures for preparation of amyloid-beta peptide for intrahippocampal injection
have been described 6], 25], 26]. Full-length A?
_1–42
or reverse peptide A?
_42–1
(California Peptide, Napa, CA, USA) was first dissolved in 35 % acetonitrile (Sigma)
and further diluted to 500 ?M with incremental additions of PBS with vortexing. The
peptide solution was subsequently incubated at 37 °C for 18 h to promote fibrillization
and aggregation and stored at 20 °C 11], 24]. Peptides (2 nmol) were injected for durations of 1, 3, and 7 days in this work.

SB332235. This compound was kindly donated by GlaxoSmithKline (709 Swedeland Road,
King of Prussia, PA, USA). The compound was dissolved in a saline solution and applied
by ip injection at a single dose of 1 mg/kg at the time of peptide injection. SB332235
has been characterized as a specific antagonist for CXCR2-mediated functional responses
15], 27].

Immunohistochemical staining of rat brain

Animals were transcardially perfused with heparinized cold saline followed by 4 %
paraformaldehyde under ketamine/xylazine anesthesia. Brains were then removed, postfixed,
cryoprotected, and cut into 40-?m sections 6]. Free-floating sections were processed for immunohistochemistry as described previously
6], 24], 26]. Briefly, sections were incubated in PBS containing 1 % bovine serum albumin, normal
goat serum (NGS), and 0.2 % Triton X-100 (Sigma-Aldrich, St Louis, MO, USA) for 1 h.
Sections were incubated overnight at 4 °C with the following primary antibodies: anti-glial
fibrillary acidic protein, a marker for astrocytes (GFAP; 1:1000; Sigma-Aldrich),
anti-neuronal nuclei (NeuN; 1:500; Chemicon, Temecula, CA, USA), and two specific
microglial antibodies (anti-ionized calcium-binding adapter molecule 1 (Iba-1; 1:500;
Wako Chemicals, Richmond VA, USA) and HLA-DR (1:1000; Dako, Mississauga, ON, Canada).
Other antibodies used included ones for CXCR2 (1:500; Santa Cruz Biotechnology, Santa
Cruz, CA, USA), A?
_1–42
(1:100; Dako), and 4-hydroxynonenal (4-HNE, 1:500 Jaica, Shizuoka, Japan). Sections
were rinsed in PBS with 0.5 % BSA and incubated with secondary antibodies conjugated
with Alexa Fluor 488 or 594 (1:200; Invitrogen, Burlington, ON, Canada) for 1 h in
the dark.

In this work, double immunostaining was also carried out for microglial and astrocytic
CXCR2 expression. In the former case, since Iba-1 antibody was raised in rabbit, mouse
OX-42 (1:500; Serotec, Oxford, UK) was used for staining of receptor in microglia.
CXCR2 association with astrocytes used respective antibodies for receptor/cell of
CXCR2/GFAP. Sections were rinsed in PBS with 0.5 % BSA and incubated with a mixture
of secondary antibodies (Alexa Fluor 488 and 594; 1:100; Invitrogen).

To determine production of reactive oxygen species (ROS), peptide-injected animals
received ip injection of 1 mg/kg hydroethidine (HEt; Molecular Probes) which is oxidized
to ethidium bromide in the presence of superoxide radicals 28]. At 3 h following HEt injection, animals were killed by transcardiac saline perfusion
and brains were removed and frozen. Coronal sections (40-?m thickness) of hippocampus
were examined under a Zeiss Axioplan 2 fluorescent microscope equipped with an ethidium
filter and digital video camera (DVC) system (Diagnostic Instruments, Sterling Heights,
MI, USA).

Immunohistochemical analysis of rat brain

Quantification of immunohistochemical staining followed published procedures 25], 26], 29]. Digitized images were obtained with a Zeiss Axioplan 2 fluorescent microscope equipped
with a DVC system. Quantitative image analysis for the immunostained rat hippocampal
sections was performed on three equally spaced sections through the level of the injection
site. In each stained section, hippocampal boundaries were outlined with the granule
cell layer (GCL) denoted as the superior blade of dentate gyrus. The molecular layer
(ML) was then defined as the region between GCL border and hippocampal fissure. Neuronal
viability and lipid peroxidation were measured in GCL, and glial responses and superoxide
production were measured in adjacent ML. Digitized images were analyzed using Northern
Eclipse software (Empix Imaging, Mississauga, ON, Canada).

RT-PCR in peptide-injected rat hippocampus

The specific protocols for Reverse Transcription PCR (RT-PCR) closely followed those
outlined in previous work from this laboratory 26], 29], 30]. Anesthetized animals were killed by decapitation at 1, 3, and 7 days after peptide
injection. The control animals were killed at 3 days after PBS or reverse peptide
A?
_42–1
injection. Brains were removed, and hippocampal tissues were freshly dissected onto
cold metal tissue matrices (Harvard Apparatus) and quickly frozen in liquid nitrogen.
Total RNA was extracted using Trizol reagent (Invitrogen) and processed using reverse
transcriptase; cDNA products were amplified by PCR using a GeneAmp thermal cycler
(Applied Biosystems, Foster City, CA, USA) with Taq polymerase. PCR primers (?-actin
was used as a reaction control) were as follows: CXCR2: forward, 5?-GTC AGG ATC CAA
GTT TAC CTC AAA AAT GG-3?; reverse, 5?-CTT AGG TCG ACG GTC TTA GAG AGT AGT GG-3?.
The primers for IL-8 were as follows: forward, 5?-ACT GAG AGT GAT TGA GAG TGG AC AC-3?;
reverse 5?-AAC CCT CTG CAC CCA GTT TTC-3?. Relative mRNA levels (stimulated values
normalized to controls) were obtained using NIH ImageJ software 1.24 (National Institute
of Health, Bethesda, MD, USA).

Western blot for CXCR2

Total protein from rat hippocampal tissue was used for Western blot analysis. Protein
samples (50 ?g) were subjected to SDS-PAGE prior to transfer onto a PVDF membrane
(Millipore, Bedford, MA, USA), blocked with either 5 % skim milk or bovine serum albumin,
and probed with anti-CXCR2 (1:200; Santa Cruz Biotechnology) and ?-actin (1:5000;
Abcam, Cambridge, MA, USA). HRP-conjugated secondary antibodies (GE Healthcare biosciences,
Piscataway, NJ, USA) were used to develop immunoblots which were processed using enhanced
chemiluminescence (ECL) detection (GE Healthcare Biosciences). Band intensities were
quantified using ImageJ software (NIH).

Statistical analysis

Results are presented as mean?±?SEM. The statistical analysis was performed using
a one-way ANOVA, followed by the Student–Newman–Keuls multiple comparison test or
Student’s t test (GraphPad Prism 3.0; Graph Pad) with significance level set at p??0.05.