Lack of neuroinflammation in the HIV-1 transgenic rat: an [¹⁸F]-DPA714 PET imaging study

Experiments were carried out in male Tg (F344/Hsd) in three different age groups (3, 9, and 16 month-old) and male age-matched wild-type control rats (F344) (WT) purchased from Harlan Inc. (Indianapolis, IN). The total sample of animals used for PET imaging, blood metabolism experiments, and cytokine/chemokine measurements included 28 Tg and 27 WT rats. All rats were housed in a temperate-controlled environment with a 12-h light/dark cycle. The animals were allowed free access to food and water. All procedures were conducted during the light cycle. The rats were acclimated to careful handling prior to testing to minimize stress. Animal care and all experimental procedures were approved by the Institutional Animal Care and Use Committee (ACUC) of the National Institutes of Health (NIH).

Three WT rats weighing 210 ± 114 g were used for intrastriatal injection of quinolinic acid (QA). The animals were anesthetized with 1 mg/kg ketamine and xylazine (10:1) cocktail (Sigma) and then placed in a stereotaxic apparatus (Stoelting Wood Dale, IL, USA). Unilateral stereotaxic injections of 150 nmol in 1 μl of QA (Sigma; dissolved in 0.1 M phosphate-buffered saline (PBS), pH 7.4) in the right striatum were made over 5 min using a 10 μl Hamilton syringe fitted with a micropump (Stoelting Wood Dale, IL, USA). The syringe was fitted with a 26-gauge needle at the following coordinates according to Paxinos and Watson (1998): anteroposterior (AP) +1 mm, mediolateral (ML) −3.0 mm, dorsoventral (DV) −4.5 mm, from the bregma. The injection syringe was left in place for an additional 5 min to allow the QA to diffuse from the needle tip and avoid backflow. After removing the needle, the skin was sutured and the animals were allowed to recover before being returned to their cages. The animals were given buprenorphine (0.03 mg/kg) intramuscularly and examined daily until PET imaging. PET imaging studies were conducted either 7 days post-op (n = 1) or 3 days post-op (n = 2). All animal procedures were performed in accordance with the NIH ACUC guidelines.

Automated syntheses of [¹⁸F]DPA-714 were carried out using a slightly modified TRACERLab FX-FN module (GE Medical Systems, Germany). In brief, aqueous [18F]fluoride anions were sucked through the Chromafix® under vacuum. The trapped ¹⁸F-fluoride was eluted from the cartridge and transferred to the reaction vessel with an eluent solution containing K₂CO₃, acetonitrile, and Kryptofix-222. The reaction mixture was evaporated to dryness after addition. Tosylate substrate was then dissolved in dimethyl sulfoxide, and the mixture was transferred to the dry 18F-labeled KF-K222 complex and allowed to react at 165 °C for 5 min. On completion, the reaction mixture was diluted with semi-preparative HPLC solvent and passed through a Sep-Pak® light Alumina N cartridge (Waters Corporation, Milford, MA). The reaction vessel was rinsed with HPLC solvent and passed through the Sep-Pak® Alumina N cartridge by helium pressure. The combined crude solution collected in the collection flask was transferred to the HPLC injection loop and injected into a Phenomenex Luna 5 μ C18 semi-preparative reversed-phase HPLC column (250 × 10 mm), with a mobile phase of H₂O and acetonitrile (55/45, v/v) at a flow rate of 4.0 ml/min. The retention time (tR) of [¹⁸F]DPA-714 was determined to be 19 min. The radioactive fraction corresponding to [¹⁸F]DPA-714 was collected into a dilution flask. The final formulation of the tracer was performed automatically using a Sep-Pak® Plus C18-based system. The diluted fraction collected from the dilution flask was passed through the C18 cartridge to fix the tracer. The cartridge was washed with deionized water (10 ml), and the purified tracer was finally recovered by elution of the C18 cartridge with the ethanol (1.5 ml) from the reservoir. Yield and specific activity were determined at this stage (25 ± 3 %, specific activity 41–107 GBq/μmol, n > 10). The excess ethanol was removed through rotary evaporation and concentrated to 250–350 μl. The tracer was delivered and diluted with PBS for final formulation and animal administration.

The Tg (n = 3, 10 ± 1 months) and age-matched WT rats (n = 3, 10 ± 0.5 months) were anesthetized with 2–2.5 % isoflurane air/oxygen mixture and injected with 1–2 mCi (37–74 MBq) of [18F]DPA-714 (1.65 ± 0.43 mCi; 61.05 ± 15.91 MBq). Aliquots of blood (400 μl) were taken from each animal at 5, 15, 30, and 60 min post-injection and centrifuged at 17,000×g at 4 °C for 4 min. One hundred microliter of plasma was retrieved, and extraction was performed using 200-μl acetonitrile. The mixture was vortexed for 30 s and centrifuged at 17,000×g, 4 °C for 4 min. The supernatant was removed and 25 μl from each sample was applied on silica gel thin layer chromatography (TLC) plates. A mixture of chloroform (90%), methanol (9%), and ammonium hydroxide (1%) was used as the eluent. The dried TLC plates were placed overnight on a phosphorimaging plate with a pixel size of 25 μm (Fuji BAS-SR2025, Fujifilm, Japan). After exposure, the plates were scanned using a Fuji FLA-5100 and the data was analyzed using Image Gauge (Fujifilm, Japan). The radioactivity of the remaining plasma and pellets was determined by γ-counting (Perkin Elmer 2480 Wizard3) and used to calculate the percent parent uptake of the plasma, corrected for metabolites (differential uptake ratio, DUR = % injected dose normalized to animal weight and plasma volume). Finally, the percentages of unchanged [18F]DPA-714 in plasma as a function of time were fitted to an exponential decay equation (Fig. 1).

The 3-month-old group consisted of four Tg (237 ± 46 g) and four age-matched WT rats (263 ± 27 g). The 9-month-old group consisted of five Tg (344 ± 36 g) and five WT rats (410 ± 41 g). The 16-month-old group consisted of six Tg (392 ± 25 g) and three WT rats (464 ± 11 g).

Two to three rodents were scanned per day, and the order of scanning was counterbalanced: one Tg rat and one WT rat, in alternate order. The animals were anesthetized with 2–2.5 % isoflurane air/oxygen mixture. The intra-subject variability of the depth of anesthesia was monitored by measuring respiratory frequency periodically during the scan. The PET experiments were performed on a Bio PET/CT tomograph (Bioscan Inc., Washington, DC) with an axial field of view of 4.8 and 6.7 cm in diameter. Time coincidence window was set to 10 ns with an energy window of 250–700 keV. The lateral tail vein was cannulated for injection of radiotracer, and the cannula was then connected to a heparin lock and secured in place with medical tape. The animal was positioned prone with the head placed symmetrically in the center field of view (FOV) on the thermostatically heated bed supplied by the manufacturer (Bioscan Inc., Washington, DC).

[¹⁸F]DPA-714 injection of 1.66 ± 0.22 mCi (61.4 ± 8.1 MBq; 2.0 ± 0.9 nmol/rat) was then administered as a bolus injection (30 s) into the indwelling intravenous catheter followed by a 300-μl saline flush (maximum volume of injection = 600 μl). PET emission data was acquired for 60 min in list mode and then reframed into a dynamic sequence of 20 frames (6 × 20, 3 × 60, 11 × 300). The resultant emission Sinogram for each frame were then corrected for attenuation, scatter, ¹⁸F decay, randoms, and dead time. Dynamic images were reconstructed using OSEM-2D algorithm into 175 × 175 × 61 slices with a voxel size of 0.39 × 0.39 × 0.78 mm. The reconstructed images were co-registered to a template MR in stereotaxic space using a rigid body transformation model as previously described [27]. Time-activity curves were generated for volumes of interest (VOIs) that were drawn manually on the template MR image guided by an anatomical atlas of the rodent brain [28]. The VOI positions were evaluated by displaying the corresponding VOI on the co-registered PET images. Image analyses were performed using PMOD 3.4 kinetic modeling tool (PMOD Technologies Ltd., Zurich, Switzerland). Regional [¹⁸F]DPA-714 uptake was quantified as standardized uptake value equivalent to percentage of injected dose per cubic centimeter after correction for body weight (SUVc = % ID kg/cm³). The SUVs were calculated from the imaging frames obtained between 40 and 60 min after injection. Those frames were assumed to reflect “pseudo equilibrium” status since they had the lowest rate of change in the concentration activity curve (<5 %/h) [29]. The body weight was adjusted according to Kleiber laws [30] as previously described [31] to account for weight differences between our adult Tg and adult WT rat. For the QA injection group, we calculated the ratio of uptake in the ipsilateral striatum (site of injection) and cortex (needle track) to the contralateral striatum and cortex (reference region).

For the cytokine/chemokine data, multiple comparisons were performed using non-parametric ANOVA (Kruskal-Wallis) test followed by Dunn’s post hoc analysis. Simple comparisons were made using unpaired two-tailed Student’s t test for parametric data (with Welch correction) or Mann-Whitney U test for unpaired non-parametric data. A p value of <0.05 was considered significant.

There were significant differences in body weight between the Tg and age-matched WT rats in the 9-(p < 0.05) and 16-month-old (p < 0.01) groups; no differences in body weight were observed in the 3-month-old group. Due to the weight differences in the adult rats, we corrected the animal weight using the Kleiber method in which the metabolic activity is proportional to m ^0.74 with m being the animal’s body weight (in grams) [30]. We chose this method as a compromise between the total body weight method of correction that could underestimate the SUV in the brain and the lean body mass method of correction that could overestimate the SUV in the brain. This method has been used in another similar paper [31].

The chemical purity of our radiotracer was consistently >95%. There were no significant differences in specific activity (SA) values nor in the injected dose (ID) of [¹⁸F]DPA-714 in the 3-month-old (SA, 1.5 ± 0.6 and 1.3 ± 0.4 Ci/μmol; ID, 1.5 ± 0.3 mCi (55.5 ± 11 MBq) and 1.7 ± 0.2 mCi (62.9 ± 7.4 MBq), respectively), 9-month-old (SA, 2.1 ± 1.1 and 2.1 ± 0.9 Ci/μmol; ID, 1.9 ± 0.9 mCi (70.3 ± 33.3 MBq) and 1.9 ± 1.0 mCi (70.3 ± 37 MBq), respectively), or the 16-month-old Tg and age-matched WT rats (SA, 1.4 ± 0.5 and 1.4 ± 0.6 Ci/μmol; ID. 1.7 ± 0.1 mCi (62.9 ± 3.7 MBq) and 1.6 ± 0.1 mCi (59.2 ± 3.7 MBq), respectively). In the QA-lesioned rats, the SA was 1.6 ± 0.1 Ci/μmol and ID was 1.3 ± 0.4 mCi (48.1 ± 14.8 MBq).

No appreciable differences in blood metabolism patterns were observed between the Tg and WT rats. The parent molecule levels were similar in the Tg and WT rats at different time points: 92.40 ± 3.24 % compared to 95.81 ± 1.72 % at 5 min, 61.15 ± 22.49 % compared to 63.37 ± 15.52 % at 15 min, 35.74 ± 11.92 % compared to 37.14 ± 8.00 % at 30 min and 20.31 ± 4.97 % compared to 25.79 ± 6.70 % at 60 min (Fig. 1). Our metabolism results were very similar to previously reported values in the literature [32].

The time-activity curves (TACs) of the 60-min acquisitions were similar in shape between the Tg and WT rats in all regions. Representative TACs derived from the caudate nucleus region are shown in Fig. 2 and Additional file 1. The radioligand was rapidly taken up in the brain but displayed fast washout with only small activity concentrations measured at later time points. Pseudo equilibrium was reached at approximately 40 min after injection (Fig. 2).

In all the three age groups, the Tg rats generally showed slightly increased SUV values compared to the age-matched WT rats; however, this was not statistically significant in any group or any brain region (Table 1, Fig. 3b, c). In the QA-injected rats, on the other hand, there was a definite increase in [¹⁸F]DPA-714 binding ipsilateral to the injection site when compared to the contralateral side (Fig. 3a). The increased uptake was seen in the caudate at the site of the injection as well as along the tract of the needle in the cortex. The fold increase ranged from 1.2 to 2 (ipsilateral versus contralateral ratio).

The Iba1 cell density values (cells/mm²) were not significantly different between the Tg and WT rats in any of the evaluated brain regions, which included the cortex, striatum, and hippocampus (two-sample Student’s t test, p > 0.05).

For all the measured cytokine/chemokines in the brain lysates, there were no significant differences in cytokine/chemokine concentrations between the Tg and age-matched WT animals at any age (Fig. 4).

When we compared the general direction of change with age between the TG and WT rats, there were no appreciable differences with cytokine/chemokine concentrations generally following the same pattern of either increase, decrease, or no change in both groups.