Healthy male Sprague-Dawley rats (n=64, aged 8-10weeks, weighed between
200 and 250 g) were used in this study. All rats were purchased from the
Experimental Animal Center of Chongqing Medical University, where rats were
housed in cages on a 12-h light-dark schedule with food and water ad libitum. All
experiments were performed in accordance with the international guidelines
Principles of Laboratory Animals Care and were approved by the Animal Care
Committee of Chongqing Medical University.
Induction of type 2 diabetes mellitus
After one week of adaptive feeding, 40 rats were randomly assigned to 2 groups,
Normal and DM group. Rats in the Normal group were fed with the laboratory regular
chow, while those in the DM group with a high-fat and high-glucose diet. Four weeks
later, an intraperitoneal injection of low-dose STZ (30 mg/kg) were given to rats in
the DM group, while an equivalent amount of citrate buffer (pH 4.5) alone in the
Normal group. At 72 h after injection, blood glucose level greater than 16.7mmol/L
was taken as the successful establishment of type 2 DM model. Maintenance of the
diabetic state was confirmed by the measurements of blood glucose from tail vein
every week. Rats that failed to meet those requirements together with those dead ones
were excluded from our study. Six weeks after successful diabetes induction, rats
were treated according to the study design. At the end of the treatment, animals were
sacrificed with overdose pentobarbital. Rat eyeballs were enucleated and immediately
put into 40 g/L paraformaldehyde for hematoxylin and eosin (H-E) staining or
immunohistochemistry. Retinas were isolated carefully and snap frozen in liquid
nitrogen for western blot or enzyme-linked immunosorbent assay (ELISA).
Intravitreal treatment of Cs-A/HMGB-1
This study comprised of two experimental parts. First, rats in the Normal and DM group were deeply anesthetized by pentobarbital injection, and topical ocular anesthesia was achieved with 0.4% benoxinate hydrochloride of eye drop. After pupils were dilated satisfactorily, rats were injected with Cs-A (42 ng/2 μL Dimethyl sulfoxide (DMSO)) intravitreally in the left eye with a 30-gauge micro-injector under a dissecting microscope. For the control, 2μL DMSO was injected into the right eye of the same rat. According to the treatment, rat eyes could be categorized into four groups: Normal, Normal+Cs-A, DM and DM+Cs-A group. Rats with operational complications such as vitreous hemorrhage, retinal detachment or death were excluded from our study. Forty-eight hours after administration of Cs-A, rats were anesthetized and sacrificed for further experiments.
The second part of the experiment was that twenty-four normal healthy rats
(Sprague-Dawley, non-diabetic, 200 - 250 g) were subjected to intravitreal injections
into the left eyes using the method described above. Rats were divided into three
groups according to the injection substances: Normal control group (n=8) received
5μL sterile phosphate buffer saline (PBS); Normal+HMGB-1 group (n=8) received
sterilized solution of recombinant HMGB-1 (5 ng/5 μL PBS; R&D Systems,
Minneapolis, MN); and Normal+HMGB-1+Cs-A group (n=8) received a combination
of HMGB-1(5 ng/5 μL PBS) and Cs-A (42 ng/2 μL DMSO).
H-E staining of the retinal tissues
Rat eyeballs were put into 4% paraformaldehyde solution at 4℃ overnight with
anterior segments removal and then dehydrated transparentized and embedded in
paraffin blocks. Sections of five-micron thickness were cut and transferred to
triethoxysilane-coated slides. Then these slides were stained with hematoxylin–eosin,
followed by being dehydrated and cemented with neutral resins. Finally,
histopathological abnormalities of retinal tissues were observed under a light
microscope, and photographs were taken with an Olympus BX60 microscope
(Olympus Optical Co Ltd, Tokyo, Japan).
Immunohistochemistry and Western blot
For immunohistochemistry, the paraffin-embedded retinal sections (5μm) were
dewaxed and dehydrated. Endogenous peroxidase was quenched with 3% H2O2
blocker for 10 minutes at room temperature. Then, the sections were incubated with
anti-HMGB-1 antibody (1:500, Epitomics, Cambridge, U.K.) at 4 ℃ overnight. After
extensive washing with PBS, the sections were incubated with horseradish peroxidase
-streptavidin immunoglobulin (1:500, Abcam, Cambridge, UK) for 30 min, developed with diaminobenzidine for 3 min and counterstained with hematoxylin. Quantitative image analysis was performed with Image-Pro Plus 6.0 software (Media Cybernetics Inc, Bethesda, Maryland MD, USA). Ten fields per retina were randomly selected and the densitometry mean values of HMGB-1 immunostaining were determined.
For western blot, retinas were sonicated in 200 μl of lysis buffer (PBS containing
10 mM EDTA, 1% Triton X-100 and the protease inhibitor cocktail). Then, lysates
were centrifuged to gain supernatants and protein concentrations were determined
with the Bio-Rad method (Bio-Rad, Richmond, CA). Protein samples were separated
on polyacrylamide gel electrophoresis and transferred to a polyvinylidene difluoride
membrane. The membrane was blocked with 5% dry milk in Tris-buffered saline with
0.1% Tween 20 (TBST) for 1 h and incubated with anti-HMGB-1 antibody(1:500, Epitomics, Cambridge, U.K.), anti- IL-1β antibody (1:500,Epitomics, Cambridge, U.K.), anti-TNF-α antibody (1:500, Abcam, Cambridge, U.K.) or anti-actin antibody (1:1000, Sigma, St. Louis, MO, USA) at 4 ℃ overnight. Then, the membranes were rinsed three times with TBST buffer followed by incubation with the secondary horseradish peroxidase-conjugated antibody (1:1000, Abcam, Cambridge, U.K.) for 1h at 37℃. The immunocomplexes were visualized by the ECL chemiluminescence method. Subsequently, semi-quantitative analysis was performed using the quantity one software (Bio-Rad, Richmond, CA). The amount of target proteins was quantified relative to the level of β-actin.
ELISA Assay of IL-1β and TNF-α
At the end of the treatment, each retina was dissected and homogenized in 100 μL
of lysis buffer supplemented with protease inhibitors (Beyotime). Samples were
centrifuged at a speed of 12,000 rpm for 10 min at 4 °C and the supernatants were
collected. After protein concentrations were assessed with the Bio-Rad method
(Bio-Rad, Richmond, CA), samples were subjected to corresponding ELISA kits
(R&D Systems, Minneapolis, MN) for the determination of IL-1β and TNF-α levels
according to the manufacturer’s instructions. The absorbance at 450nm was read on
an automated plate reader (Spectra Max Gemini UVmax; Molecular Devices,
Sunnyvale, CA). All measurements were performed in triplicate and the tissue sample
concentration was calculated from a stand curve and corrected for protein
RNA extraction and RT-PCR
The retinas were separated from eyeballs. The whole RNA of murine retinas was isolated with TRIzol reagent (Invitrogen, Paisley, UK) according to the manufacturer’s instructions. Each RNA extract (2 mg) was reverse-transcribed into cDNA using RevertAidTM First Strand cDNA Synthesis Kit (Mbi, Glen Burnie, MD, USA). PCR was performed in 50 μl of a solution containing Taq DNA polymerase, dNTP, RT products, IL-1β primer, TNF-α primer and GAPDH primer. More details are shown in Table 1.
After pre-incubation for 5 min at 94°C, 35 cycles of amplification (94°C for 30 sec, 55°C for 30 sec, and 72°C for 30 sec) were performed. Amplified products were run
in a 1.5% ethidium bromide agarose gel, band intensities were captured with a ChemiDoc system and LabWorks software (UVP, Upland, CA, USA), and values were transferred to an Excel spreadsheet for calculation of means and standard errors.
Table 1 Primer sequence
The data were expressed as mean ± standard deviation (SD). Data were analyzed
using the one-way analysis of variance (ANOVA) followed by Bonferroni's post
hoc test. All statistical analyses were performed with GraphPad Prism software
(version 4.0, GraphPad Software, San Diego, Calif.). P value less than 0.05 was
considered statistically significant.