CD200-CD200R dysfunction exacerbates microglial activation and dopaminergic neurodegeneration in a rat model of Parkinson's disease

Specific monoclonal antibodies against CD200R (CD200R-blocking antibody, BAb), CD11b, MHC II and isotype control mouse IgG1 (Control antibody, CAb) were obtained from Serotec (Indianapolis, IN, USA). The ELISA kit for rat-TNFα was obtained from R&D Systems (Minneapolis, MN, USA). The ELISA kit for rat-IL-6 was purchased from BD (San Diego, CA, USA). Elite ABC kit and 3,3'-diaminobenzidine tetra-hydrochloride (DAB) substrate were purchased from Vector (Vector Laboratories, Burlingame, CA, USA). The BCA Protein Assay Kit was from Thermo Fisher Scientific (Rockford, IL, USA). High-performance liquid chromatography (HPLC)-grade methanol was obtained from BDH Laboratory (Poole, UK). All other chemicals were obtained from Sigma-Aldrich (St. Louis, MO, USA).

All animal experiments were performed according to the NIH Guide for the Care and Use of Laboratory Animals and were approved by the Shanghai Jiao Tong University School of Medicine Animal Care and Use Committee (2009087). Male Sprague-Dawley rats (10-12 weeks old, weighing 220-260 g at the start of the experiment) were provided by the Shanghai Institutes of Biological Sciences animal house, and were caged in groups of 5 with food and water given ad libitum. The animals were kept in a temperature-controlled environment at 22 ± 2°C on a 12:12 light-dark cycle.

For stereotaxic surgery, rats were anesthetized with an intraperitoneal injection of pentobarbital (50 mg/kg). When the animals were deeply anesthetized, they were placed in a stereotactic apparatus. Subsequently, the rats were injected with BAb (1 μg/μl, 5 ul for each site) or CAb (1 μg/μl, 5 ul for each site) into the right striatum (anterior lesion site: AP: 1.0 mm anterior to the bregma, L: 2.6 mm from the midline, D: 4.5 mm from the dura; posterior lesion site: AP: 0.3 mm posterior to the bregma, L: 3.5 mm from the midline, D: 4.5 mm from the dura). The sham groups were injected with vehicle (10 mM PBS, 5 μl for each site, Veh). The next day, each group was injected with 6-OHDA (4 μg/μl in 0.9% saline with 0.02% ascorbic acid, 2 μl for each site) into the right ascending medial forebrain bundle (MFB) (one 4.2 mm posterior to bregma, 1.2 mm lateral to the midline, and 7.8 mm below the dura, and another 4.4 mm posterior to bregma, 1.7 mm lateral to the midline, and 7.8 mm below the dura). The microinjection coordinates used were obtained from a rat brain atlas by Paxinos and Watson. The injection was made at a rate of 1 μl/min using a 10 μl Hamilton syringe with a 26-gauge needle. At the end of each injection, the syringe needle was left in place for 5 min, and then was slowly withdrawn to prevent reflux of the solution.

At 21 days post 6-OHDA-injection, animals were deeply anesthetized with pentobarbital (100 mg/kg, i.p.) and perfused through the aorta with 150 ml of 0.9% saline, followed by 250 ml of a cold fixative consisting 4% paraformaldehyde in 100 mM phosphate buffer (PB). Brains were then dissected out (3-4 mm in thickness) and postfixed for 24 hours with paraformaldehyde in 100 mM PB before placed into 30% sucrose solution in phosphate-buffered saline for 24-72 hours at 4°C. Brains were then cryosectioned coronally on a Leica1650 cryostat (cut thickness: 25 μm) with a random start, and including sections before and after both anatomical regions to confirm the entire structure was quantified. Sections were collected serially throughout the SN and placed into PBS for further experiments.

Free-floating sections were pretreated with 0.3% H₂O₂ in 0.1 M PBS (pH 7.2-7.5) for 10 min at RT (60 rpm) to block endogenous peroxidase activity, then washed with 0.1 M PBS for 3 times. The tissue was then blocked with diluted blocking serum (Elite ABC kit, Vector Laboratories, Burlingame, CA, USA) for 20 minutes at room temperature. Sections were then incubated with the primary antibody to TH (mouse anti-TH, 1:4000, Sigma), CD11b (mouse anti-CD11b, 1:1000, serotec) or MHC II (mouse anti-MHC II, 1:1000, serotec) overnight at 4°C. The following day the sections were washed and then incubated with diluted biotinylated secondary antibody (Vector laboratories) for 30 min at room temperature. The secondary antibody was amplified using avidin-biotin complex (Vector laboratories) for 30 min at room temperature. Finally the sections were developed with 3,3'-diaminobenzidine tetra-hydrochloride (Vector Laboratories). Sections were then mounted onto glass slides and dried overnight. The next day the slides were passed through a gradient of ethyl alcohol and xylene to dehydrate the tissue. The slides were then coverslipped using permount mounting medium.

Unbiased stereological estimates of DA (TH-positive cell) neuron numbers were performed using StereoInvestigator analysis software (MicroBrightField, Williston, VT), combined with a Nikon Eclipse E600 microscope, and the optical fractionator method according to previously published reports [42, 43]. Boundaries in the SN were defined according to previously defined anatomical analysis in the rat [44] and cells were counted from every sixth 25-μm section (~24 sections) along the entire SN (to ensure coefficient of errors <0.1, the rostral-caudal length of the SN was 4 mm), by investigators blinded to treatment history, with a 60 × objective. In brief, optical dissectors (area of counting frame, 64,000 mm³; guard height, 2 μm; spaced 300 μm apart in the x-direction, and 200 μm apart in the y-direction) were applied to each section in the series throughout the entire SN (including pars reticulata and compacta; estimates are reflective of two sides; n = 5 for each group). We show the percent of neurons remaining on the ipsilateral side compared to those on the intact contralateral side. Values are expressed as the mean ± S.E.M. of all animals in each group.

Microglial quantification similarly used adjacent (8 sections) serial sections. An observer blind to sample identity counted numbers of CD11b-immunoreactive (CD11b-ir) positive cells in the SN on each side (Nikon microscope at a 40 × magnification). Here the X-Y step length used was between 300-400 mm in order to count 100-200 CD11b-ir cells in each side of the SN. A positive cell was defined as a nucleus covered and surrounded by CD11b immunostaining. The stage of cells was identified by their morphology. For quantitation of MHC II immunoreactive (MHC II-ir) cells, cells in stage 4 were identified by their morphology on MHC II staining under 40× magnification and counted in every sixth 25-μm-thick serial section of the SN of each rat using a two-blinded procedure. Graphs show the number of MHC II-ir cells in the SN.

Animals (n = 5 each of the following groups: 6-OHDA/Veh, 6-OHDA/CAb, and 6-OHDA/BAb) were sacrificed by CO₂ and their brains were quickly removed and placed on ice. The left and right striatum were freshly dissected out, weighed, frozen in liquid nitrogen, and stored at -80ºC for later use.

Each sample was homogenized by sonication in ice-cold 0.1 mol/L perchloric acid and then centrifuged at 12,000 rpm for 30 minutes at 4ºC. The supernatants (20 μl) were injected into a high-performance liquid chromatography (HPLC) system coupled to an electrochemical detection device (Coularray; ESA, Chelmsford, MA) for measuring dopamine (DA), 3,4-dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA). The protein contents were determined in pellet fractions by the method described by Lowry [45], and expressed as ng/g wet weight of tissue (ng/g WW).

We adapted a classification system for microglial activation according to Kreutzberg [46]:

Stage 1: Resting microglia. Rod-shaped soma with fine and ramified processes.

Stage 2: Activated ramified microglia. Elongated cell body with long and thicker processes.

Stage 3: Amoeboid microglia. Round body with short, thick and stout processes.

Stage 4: Phagocytic cells. Round cells with vacuolated cytoplasm; no processes can be observed at the light microscopy level.

Stages of microglia activation were confirmed by observation by at least two blinded observers. Black circles in Figure 2 show examples of microglia in different stages. All of these cell types are CD11b-ir, and MHC II stained only activated microglia but not resting microglia.

Apomorphine-induced rotational behaviour was assessed at 7 and 21 days after 6-OHDA-injection. Rotational behaviour was tested in rotometer bowls [47]. Five minutes after intraperitoneal administration of apomorphine (0.5 mg/kg diluted in 0.9% saline), the total number of full 360° rotations in the contralateral direction was counted for 30 min.

Rats were killed by CO₂ overdose followed by cervical dislocation and decapitation at 21 days after injection of 6-OHDA. The brain was removed and immediately transferred to ice and cut at the level of the infundibular stem forming a hindbrain block containing the SN. The SN were dissected, snap-frozen in liquid nitrogen and stored at -80°C. Tissue was homogenized on ice in 400 μl of Tris-HCl buffer (pH = 7.3) containing protease inhibitors (10 mg/ml aprotinin, 5 mg/ml peptastin, 5 mg/ml leupeptin, 1 mM PMSF). Homogenates were centrifuged at 10,000 g at 4ºC for 10 min and then ultracentrifuged at 40,000 r.p.m. for 2 h. Supernatants were aliquoted and stored at -80ºC until use. BCA protein assays were performed to determine total protein concentration in each sample. Commercially available rat TNF-α (R&D, Minneapolis, MN, USA and rat IL-6 kits (BD, San Diego, CA, USA) with high sensitivity were used to quantify these cytokines according to the manufacturers' instructions (7.8 pg/ml for rTNF-α and 20 pg/ml for rIL-6). Three animals per group were analyzed and each sample was analyzed in duplicate.

Statistical analysis of the data was performed using GraphPad Prism version 5.00 for Windows (GraphPad Software, San Diego California, USA, http://​www.​graphpad.​com). The results are reported as mean ± SEM. Two-way ANOVA followed by Bonferroni's test was applied to determine significant differences among data of rotational experiments with two time points. Univariate one-way ANOVA and Tukey-Kramer post-hoc test were used to analyze data from other experiments between treated groups. The criterion for statistical significance was P < 0.05.

Unilateral injection of 6-OHDA into medial forebrain bundle (MFB) induces the loss of dopaminergic cell in the ipsilateral SNpc and was used as a hemiparkinsonian animal model in this study. To investigate the role of CD200-CD200R dysfunction in 6-OHDA-induced neurotoxicity, we employed a CD200R monoclonal antibody to block CD200-CD200R engagement, which was first used by Wright, G. J. [32], and later by many other investigators [33, 41, 48‐51]. In the present study, BAb, CAb or Veh was injected into striatum one day before 6-OHDA injection. Then, apomorphine-induced rotational behaviour was analyzed to assess unilateral degeneration of presynaptic dopaminergic neuron terminals at 7 and 21 days after 6-OHDA injection. Although rats that had been microinjected with 6-OHDA/Veh could contralaterally rotate to the site of 6-OHDA lesion with apomorphine administration (1.5 ± 0.6 at 7 days, 3.7 ± 1.3 at 21 days), apomorphine-induced rotation was significantly increased in 6-OHDA/BAb rats at both time points (7.7 ± 2.6 and 18.3 ± 2.3 respectively, p < 0.0001) (Figure 1). Pretreatment with BAb not only exacerbated but also accelerated (as early as 7 days) motor deficits in hemiparkinsonian rats (Figure 1). Animals that responded to apomorphine treatment with at least 7.0 turns/min could be regarded as successfully induced hemiparkinsonian rats [43, 52]. The rats that received 6-OHDA/Veh treatment showed only 1.5 ±0.6 turns/min at 7 days and 3.7 ±1.3 turns/min at 21 days induced by apomorphine administration, and both of these values are less than 7.0 turns/min. So the dose of 6-OHDA (16 μg) used in this experiment could be considered as a sub-toxic dose. However, rats treated with 6-OHDA/CAb did not show a significant increase in contralateral rotational number, 2.7 ± 1.1 turns/min at 7 days and 4.7 ±1.7 turns/min at 21 days, compared to the rats treated with 6-OHDA/Veh (Figure 1).

To confirm that the phenotype of our PD rats is consistent with dopaminergic neuron loss in SN, we stained midbrain coronal sections with an antibody against tyrosine hydroxylase (TH) and performed non-biased stereological estimation of TH-immunoreactive (TH-ir) neurons in SN. We observed that a sub-toxic dose of 6-OHDA was able to induce moderate but not overt dopaminergic neurodegeneration in SN (55.0 ± 6.0% of contralateral) (Figure 2A). Intrastriatal injection of BAb resulted in a significant decrease in TH-ir neurons in whole SN in animals treated with 6-OHDA/BAb (5.2 ±2.0%, P < 0.0001). However, no dramatic decrease in TH-ir cells was observed between groups treated with 6-OHDA/Veh and 6-OHDA/CAb (Figure 2A). These results indicate an exacerbating effect of BAb on the degeneration of dopaminergic neurons. At higher magnifications, we observed that treatment of 6-OHDA/BAb not only decreased the number of TH-ir cells but also their arborisation or fibers. TH-ir fibers (Figure 2 arrowheads) were less densely spread amongst TH-ir cell bodies (Figure 2 arrows) in the SN in 6-OHDA/BAb-treated rats (Figure 2G), compared to either control group (6-OHDA/Veh, 6-OHDA/CAb) (Figure 2E-F). There were no marked morphological differences in numbers of TH-ir cells in SN between rats pretreated with CAb (6-OHDA/CAb) and Veh (6-OHDA/Veh) (Figure 2E-F). Furthermore, BAb administration had a significant effect on DA and its metabolites in ipsilateral striatum. Twenty-one days after 6-OHDA injection, the DA content of right striatum in 6-OHDA/Veh-treated rats was 1765 ± 236 ng/g wet weight of tissue (ng/g WW) (n = 5) (Table 1). Protein levels of DA metabolites in this group were 894 ± 95 ng/g WW (n = 5) and 599 ± 104 ng/g WW (n = 5) for DOPAC and HVA respectively (Table 1). Injection of CAb prior to 6-OHDA-lesion did not cause significant changes in DA or its metabolites in the right striatum of rats in comparison to vehicle control animals (Table 1), while the contents of DA and its metabolites in 6-OHDA/BAb group were significantly lower than that in the 6-OHDA/Veh group. These values were 38 ± 8 ng/g WW (n = 5), 24 ±3 ng/g WW(n = 5) and 17 ±2 ng/g WW (n = 5), respectively (Table 1).

The direct effect of BAb is destruction of the balance between CD200 and its receptor. CD200R receptor is expressed only on microglia [24, 53, 54].

Signal transferred from CD200 to its only known receptor, CD200R, has been shown to be critical for restraining microglial activation [30]. Thus, we studied microglial activation and possible neuroinflammation in SN at 21 days post-6-OHDA-injection by immunohistochemistry as described in Methods.

First, we studied morphological changes and quantification of microglia using the microglia-specific marker CD11b (a constitutive marker of microglia). CD11b recognizes complement receptor type 3 (CR3), the expression of which is greatly increased in hyperactive microglia compared with resting microglia. In our study, profound microglial responses were observed in ipsilateral SN in rats following treatment with 6-OHDA/BAb (Figure 3F,I). Round and amoeboid cells (Stage 4) became predominant in the core of the SN and were mingled with rod-shaped (stage 3) or highly ramified (stage 2) microglia near the boundary (Figure 3I). Twenty one days post-6-OHDA injection there was an increase in CD11b-ir cells in all groups. In addition, we found that the total number of CD11b-ir cells in SN was significantly increased in 6-OHDA/BAb-treated rats (716 ± 23%, P = 0.0002) versus 6-OHDA/CAb-treated rats (318 ± 20%) and 6-OHDA/Veh-treated rats (273 ± 27%) (Figure 3A). No significant difference was found between 6-OHDA/Veh and 6-OHDA/CAb groups (P = 0.2519) (Figure 3A). Furthermore, we analysed the quantification of microglia in different stages. Four cellular patterns (Figure 1, 2, 3, 4) were defined according to Kreutzberg's classification [46]. We observed that stage 4 cells constituted over 85% of the microglia population in the 6-OHDA/BAb-treated group, while less than 10% of them were found in the other two groups (Figure 3B). The majority of cells presenting in the core lesion of the SN in the other two groups were stage 3 cells or stage 2 cells. In the 6-OHDA/Veh group, stage 3 cells constituted about 34% of the population, while 39% of population turned out to be stage 2 cells. In the 6-OHDA/CAb group, 33% of population were stage 3 cells, while 38% were stage 2 cells. Statistically significant differences were found between the 6-OHDA/BAb-treated group and the two control groups (P < 0.01), but no difference was found between the two control groups. We also investigated the expression of MHC II, a marker for activated microglia, which is practically undetectable in resting microglia. MHC II-ir cells showed similar morphology to that of stage 4 microglia. MHC II-ir microglia were found scattered throughout the SN in the 6-OHDA/Veh and 6-OHDA/CAb groups (Figure 3M-N), while mainly stage 4 MHC II-ir microglia were visualized in the core lesion of SN from the 6-OHDA/BAb treated group (Figure 3L,O). The number of stage 4 MHC II-ir microglia was dramatically increased in 6-OHDA/BAb-treated rats compared with the two control groups (n = 5, P < 0.001) (Figure 3C). No difference was detected between the two control groups.

Taken together, these data suggest that BAb administration shifts stage 2 or stage 3 microglia to stage 4 in SN. These results also show a distinct population of activated microglia (MHC II-ir), which correlates with levels of neurodegeneration and motor deficit in the 6-OHDA/BAb group.

To further confirm a relationship between CD200-CD200R signalling and neuroinflammation in PD, we assayed several molecules that would be secreted by activated microglia in the proinflammatory stage [10, 55‐59]. We detected the expression profile of two most-important cytokines, TNF-α and IL-6, in SN of rats from each group at 21 days post-6-OHDA-injection. This is the time point at which dopaminergic neurodegeneration, rotational behaviour and microglial activation were investigated. Interestingly, we found significant increases in the induction of TNF-α and IL-6 expression in rats treated with 6-OHDA/BAb in comparison with the other treatments (6-OHDA/CAb, 6-OHDA/Veh) (Figure 4, P < 0.001). No difference was noted between the two control groups (Figure 4). Thus we speculate that these two cytokines, TNF-α and IL-6, might be involved in the exacerbating effects observed in the 6-OHDA/BAb-treated animals.