Unless otherwise stated, all chemicals used in this study were purchased from Sigma (St. Louis, MO, USA). Monoclonal mouse antibodies against β-amyloid precursor protein (APP), glial fibrillary acidic protein (GFAP), and α-synuclein were purchased from Millipore (Billerica, MA, USA; APP and GFAP) and BD Bioscience (San Jose, CA, USA; α-synuclein). Polyclonal rabbit antibodies against tyrosine hydroxylase (TH) and ionized calcium binding adapter molecule 1 (Iba1) were obtained from Millipore and Wako Chemicals USA (Irvine, CA, USA), respectively. Polyclonal rat antibodies against dopamine transporter (DAT) and polyclonal goat antibodies against COX-2 were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA). [3H]Dopamine ([3H]DA, specific activity 34.6 Ci/mmol) was purchased from PerkinElmer (Boston, MA, USA). Enzyme-linked immunosorbent assay (ELISA) kits for immunoassay of rat interleukin-1β (IL-1β) and tumor necrosis factor-α (TNFα) were purchased from R&D Systems (Minneapolis, MN, USA).
Timed pregnant Sprague–Dawley rats arrived in the laboratory on Day 19 of gestation. Animals were maintained in a room with a 12-h light/dark cycle and at constant temperature (22 ± 2°C). The day of birth was defined as postnatal Day 0 (P0). After birth, the litter size was adjusted to 12 pups per litter to minimize the effects of litter size on body weight and brain size. All procedures for animal care were conducted in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals and were approved by the Institutional Animal Care and Use Committee at the University of Mississippi Medical Center. Every effort was made to minimize the number of animals used and their suffering.
Injection of LPS (2 mg/kg i.p., from Escherichia coli, serotype 055: B5) was performed in five-day-old Sprague–Dawley rat pups of both sexes. The control rats were injected with the same volume of sterile saline (0.1 mL). All animals survived the injection. Both LPS- and saline-injected animals were further divided into two groups: one received an i.p. injection of celecoxib (20 mg/kg), and the other group received an i.p. injection of vehicle. Celecoxib (20 mg/kg) was dissolved in 20% dimethyl sulfoxide (DMSO) in normal saline
[17, 18] and administered immediately after the LPS injection. Thirty rats (15 male and 15 female pups) from each group were used in the present study. Behavioral tests were conducted in 12 rats from each group from P5 to P6. Rats were sacrificed on P6. Twenty-four rats from each group were sacrificed by decapitation to collect fresh brain tissue for Western blot analysis (six rats for each group), determination of the mitochondrial complex I activity (six rats for each group), ELISA assay (six rats for each group), and [3H]DA uptake study (six rats for each group). Six additional rats from each group were sacrificed by transcardiac perfusion with normal saline followed by 4% paraformaldehyde for brain section preparation. Free-floating coronal brain sections of 40-μm thickness were prepared in a freezing microtome (Leica, SM 2000R, Wetzlar, Germany) for immunohistochemistry staining.
Behavioral tests were performed as described by Fan et al., with modifications. The developmental test battery that was used was based on previously documented tests for neurobehavioral toxicity
[19, 20]. Behavioral tests, including the righting reflex and negative geotaxis test, were performed for all rat pups from P5 to P6.
This test is believed to be a reflection of muscle strength and subcortical maturation
[20, 21]. Pups were placed on their backs, and the time required to turn over on all four feet and touch the platform was measured. The cut-off time was 60 s.
This test is believed to test reflex development, motor skills, vestibular labyrinth and cerebellar integration
[19, 20]. Rats were placed on a 15° incline with their heads pointing down the slope and had to turn to face upward and begin to crawl up the slope. Each pup was given three trials a day, and the time spent to make a turn of 180° upward was recorded. The cut-off time was 60 s.
Brain injury was estimated based on the results of immunohistochemistry in consecutive brain sections prepared from rats sacrificed one day (P6) after LPS injection. For immunohistochemistry staining, primary antibodies were used in the following dilutions: TH and Iba1, 1:500; GFAP, 1:200; and APP and COX-2, 1:100. TH was used to detect dopaminergic neurons in the substantia nigra (SN). The amount of APP, a membrane-spanning glycoprotein, in normal axons and neurons is not enough to be detected, but the accumulation of APP can be detected as an early sign of axonal and neuronal lesions
[22, 23]. Microglia were detected using Iba1 immunostaining, which recognizes both resting and activated microglia. GFAP was used to detect astrocytes. COX-2 provided selective staining of inducible cyclooxygenase. Sections were incubated with primary antibodies at 4°C overnight and further incubated with fluorescence-conjugated secondary antibodies (Alexa Fluor 555, 1:500 or Alexa Fluor 488, 1:200; Jackson Immunoresearch, West Grove, PA, USA) for 1 h in the dark at room temperature. DAPI (4′,6-diamidino-2-phenylindole) (100 ng/mL) was used simultaneously to identify nuclei in the final visualization. Sections incubated in the absence of primary antibodies were used as negative controls. When double-labeling was required, primary antibodies from different hosts were used in combination with appropriate secondary antibodies, which were raised against the immunoglobulin from the corresponding host. The resulting sections were examined under a fluorescent microscope (BX60, Olympus America Inc., Center Valley, PA, USA) at appropriate wavelengths.
Protein expression of DAT and α-synuclein was determined in P6 rat brains by Western blotting according to the methods of Fan et al.[7, 8] and Hadlock et al., with modifications. One day after LPS injection (P6), brains were quickly removed, and tissues were frozen in liquid nitrogen and stored at −80°C. Tissues were homogenized in an extraction buffer (Biosource, Camarillo, CA, USA), and a mixture of protease inhibitors (Calbiochem, La Jolla, CA, USA) and 1 mM phenylmethylsulfonyl fluoride (PMSF) was added, accompanied by application of a Sonic Dismembrator (Fisher Scientific, Suwanee, GA, USA) three times for 10 s each. Protein levels of homogenates were determined by the Bradford method. The homogenates were diluted 1:2 (v/v) with Laemmli sample buffer. Equal quantities of protein (10 μg/10 μL) were loaded into each well of a 4% to 20% SDS-polyacrylamide gradient gel (MINI-PROTEAN TGX, 4 to 20%, Bio-Rad Laboratories, Hercules, CA, USA). The separated proteins were transferred electrophoretically to polyvinylidene difluoride (PVDF) membranes (Bio-Rad Laboratories) at 100 V for 1 h. The blots were incubated with a blocking solution containing 5% nonfat milk and 0.1% Tween-20 in Tris-buffered saline (TBS) for 1 h before incubation with the primary antibody (1:1,000) in the blocking solution overnight at 4°C. The blots were then incubated with peroxidase-conjugated antibodies in blocking solution (1:4,000) for 1 h at room temperature. Immunoreactivity was detected by the Enhanced Chemiluminescence Plus or Advanced ECL system (GE Healthcare, Piscataway, NJ, USA). Images were acquired with the Chemidoc MP Imaging System followed by quantification using Image Lab software (both from Bio-Rad Laboratories). To ensure that equal amounts of protein were applied to the immunoblot, the membranes were stripped with a stripping buffer (Thermo Scientific, Rockford, IL, USA) and re-probed for β-actin (1:4,000, Sigma) to normalize the results.
Synaptosomal [3H]DA (dopamine) uptake
Uptake of [3H]DA was determined according to the methods of Hadlock et al. and Nickell et al., with modifications. One day after LPS injection (P6), brain tissues were homogenized in ice-cold 0.32 M sucrose (50 mM Tris buffer, pH 7.4) and centrifuged (800 × g for 12 minutes; 4°C). The supernatant (S1) was then centrifuged (20,000 × g for 15 minutes; 4°C), and the resulting pellets (P2, synaptosomes) were resuspended in ice-cold water at concentrations of 2 mg/mL to lyse the synaptosomal membranes. Synaptosomal fractions were chilled at 4°C until DA uptake experiments commenced. Assays were performed in duplicate with a final volume of 250 μL. Aliquots of 25 μL synaptosomal fractions (50 μg of P2 protein) were added to tubes containing assay buffer (126 mM NaCl, 4.8 mM KCl, 1.3 mM CaCl2, 16 mM NaH2PO4, 1.4 mM MgSO4, 11 mM glucose and 1 mM ascorbic acid, pH 7.4) and 1 μM pargyline and then incubated at 37°C for five minutes. Nonspecific uptake was determined in the presence of 10 μM nomifensine. Samples were placed on ice, and 25 μL of 0.1 μM [3H]DA (10 nM final concentration) was added to each tube, after which accumulation was permitted to proceed for five minutes at 37°C. DA concentration and time of uptake were chosen based on the reports by Hadlock et al. and Nickell et al.. The reaction was terminated by the addition of 250 μL ice-cold assay buffer and subsequent filtration, followed immediately by the washing two times of ice-cold assay buffer. Radioactivity retained by the filters was counted using a liquid scintillation counter (PerkinElmer). Nonspecific uptake, defined as DA uptake in the presence of 10 μM nomifensine, was subtracted from total uptake to define DAT-mediated specific uptake.
Determination of mitochondrial complex I activity
Complex I activity was determined by a spectrophotometric assay based on the quantification of the rate of oxidation of the complex I substrate NADH to ubiquinone as described by Champy et al. and Hoglinger et al., with minor modifications. Brain tissues from each pup were collected at 6 or 24 h after LPS injection. The frozen brain tissue was homogenized mechanically, sonicated on ice in 10 mM Tris–HCl buffer (pH 7.2) containing 225 mM mannitol, 75 mM saccharose and 0.1 mM EDTA, and then centrifuged (600 × g) for 20 minutes at 4°C, to obtain post-nuclear supernatants. The optical density of the supernatants (40 μg sample protein) in 1 mL an assay mixture was spectrophotometrically recorded at a wavelength of 340 nm for 200 s at 37°C. The assay mixture was a potassium phosphate buffer (25 mM, pH 7.5) containing 2 mM potassium cyanide, 5 mM magnesium chloride, 2.5 mg/mL bovine serum albumin, 2 μM antimycin A, 100 μM decylubiquinone and 300 μM NADH. The proportion of NADH oxidation sensitive to an excess of rotenone (10 μM) was attributed to the activity of complex I. This procedure minimizes the dissociation of rotenone from complex I because of the use of small buffer volumes, maintenance at low temperatures, and rapid analysis. The specific activity (nmol NADH oxidation/min/mg protein) of complex I (NADH-ubiquinone oxidoreductase) was calculated using a molar extinction coefficient ε340nm = 6.22 mM-1 cm-1. Enzyme activities were expressed as nmol/min/mg of brain tissue. Complex I activity was calculated as follows: Complex I activity = (Rate (min-1)/ ε340nm (6.22 mM-1 cm-1))/ 0.040 mg.
Determination of IL-1β and TNFα protein by ELISA
Two major pro-inflammatory cytokines, IL-1β and TNFα, were determined by ELISA as previously described
[6, 29]. Briefly, brain tissues from each pup were collected 24 h after LPS injection, when the LPS-stimulated increase in inflammatory cytokines in the rat brain reached a peak value
. Brain tissues were homogenized by sonication in 1 mL ice-cold PBS (pH 7.2) and centrifuged at 12,000 × g for 20 minutes at 4°C. The supernatant was collected, and the protein concentration was determined by the Bradford method. ELISA was performed following the manufacturer’s instructions, and data were acquired using a 96-well plate reader (Bio-Tek Instruments, Inc., Winooski, VT, USA). The cytokine contents were expressed as pg cytokine/mg protein.
Quantification of data and statistics
Brain sections at the bregma level and the midbrain sections at a level one-third rostral from the lambda to the bregma were used for determination of the most pathological changes. Most immunostaining data were quantified by the counting of positively stained cells. When the cellular boundary was not clearly separated, numbers of DAPI-stained nuclei from the superimposed images were counted as the cell number. Three digital microscopic images were randomly captured in each of the three sections, and the number of positively stained cells in the three images was counted and averaged (cells/mm2). The mean value of cell counts from three brain sections was used to represent one single brain. For convenience of comparison among the treatment groups, results were standardized as the average number of cells/mm2. APP or COX-2 staining was quantified using National Institutes of Health (NIH) image software to determine the percentage area containing APP- or COX-2-positive staining in the entire area of the captured image
. In response to LPS challenge, the number of Iba1+ microglia and GFAP + astrocytes increases, and the soma of these cells become larger. In addition to cell density, Iba1 or GFAP immunoreactivity was also quantified by calculating the percentage area of the whole image containing Iba1 or GFAP immunostaining
The behavioral data were presented as the mean ± SEM and analyzed by one-way ANOVA followed by the Student-Newman-Keuls test. Data from immunostaining, immunoblotting analysis, [3H]DA uptake, mitochondrial complex I activity and ELISA assay were presented as the mean ± SEM and analyzed by one-way ANOVA followed by the Student-Newman-Keuls test. Results with a P-value of less than 0.05 were considered statistically significant.