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Pharmacological inhibition of the inflammatory receptor CCR2 … –

Apr 7th, 2023


All mice used in the current study were housed in the same rooms in Emorys animal facility during the course of the experiment, with one room designated for animal breeding and maintenance and an adjacent room solely for SE experiments. The lights were on a 12-h on/off cycle, and mice were fed and watered ab libitum. In Ccr2rfp/rfp KO mice, the Ccr2 open reading frame was disrupted with a cDNA encoding red fluorescent protein (RFP)20. The Ccr2rfp/rfp mice originally obtained were maintained on the C57BL/6 (Jackson Laboratories) inbred genetic background, and we subsequently backcrossed the mice to C57BL/6 (Charles River) for over 11 generations. To generate heterozygous Ccr2+/rfp mice, in which Ccr2-expressing monocytes are marked by RFP expression, male Ccr2rfp/rfp mice were bred to female C57BL/6 mice from Charles River.

After backcrossing for over 11 generations we submitted genetic material obtained from a Ccr2rfp/rfp mouse to Charles River Laboratories for strain background characterization. A total of 128 single nucleotide polymorphisms (SNPs) spread across the genome was analyzed to generate an allelic profile for comparison between the Jackson Laboratories and Charles River C57BL/6 substrains. Our Ccr2rfp/rfp mice were a 99.6% match to C57BL/6 inbred mice from Charles River Laboratories, making the mouse strain essentially congenic.

INCB3344 is a small molecule antagonist of the chemokine receptor, CCR2, and gifted from Pfizer through their compound sharing program. INCB3344 has favorable selectivity and specificity to CCR2 (target IC50 of 105nM; selectivity 300-fold against chemokine receptor CCR5 and CCR1, two close homologues of CCR2, and>100-fold selective against a broader panel of G protein coupled receptors)21.

Pharmacokinetics was performed by Sai Life Sciences Limited (India). Thirty male C57BL/6 mice were divided into two groups (n=15/group) and administered INCB3344 (30 or 100mg/kg, p.o.) formulation in 5% NMP, 5% Solutol HS-15, 30% PEG-400, and 60% citric acid (10mM). Blood samples were collected from retro orbital plexus under light isoflurane anesthesia at 0.5, 1, 2, 4, 8, 12, and 24h, and brains were collected at 2, 4, 8, 12, and 24h. Blood was obtained at the 0.5 and 2h time points from the same three mice, and these mice were sacrificed for brain collection at the 2h time point. Blood was obtained at the 1 and 4 time points from the same three mice, and these mice were sacrificed for brain collection at the 4h time point. Blood and brains were collected from three different sets of mice at the 8, 12, and 24h time points. Plasma samples were separated by centrifugation of whole blood and stored below -70C until bioanalysis.

Brain samples were homogenized using ice-cold phosphate buffer saline (pH-7.4) in a ratio of 2 (buffer):1(brain); and homogenates were stored below 7010C until analysis. All samples were processed for analysis by protein precipitation using acetonitrile (ACN) and analyzed with a fit for purpose LC/MS/MS method (LLOQ: 5.89ng/mL for plasma and 3.54ng/g for brain). Pharmacokinetic parameters were calculated using the non-compartmental analysis tool of Phoenix WinNonlin (Version 7.0).

Kainic acid (KA) was obtained from Tocris and dissolved at 3mg/ml in a 0.9% sterilesaline solution. Heterozygous Ccr2+/rfp mice were weighed and injected with a single dose of KA (30mg/kg, i.p.) at 10ml/kg. In mice, KA-induced seizures consisted of distinct motor behaviors, including forelimb and whole-body clonus, loss of posture, rearing, and falling. Animals that presented these behaviors with increased seizure intensity, duration, and frequency after the injection of KA were declared to be in SE, which is characterized in the KA model by periodic rearing and falling accompanied by whole-body clonic seizures. Behavior was scored using a modified Racine scale as previously described22. All mice that entered SE continued seizing for at least 60min; seizures usually persisted for several hours. The mice were group housed in a warm (28C) and humid environment for 1214h supplemented with wet food and hydrated with lactated Ringers solution i.p when needed, after which they were separated into individual cages.

After 24h, surviving mice were weighed, randomized into two groups, and received vehicle (5% NMP, 5% Solutol HS-15, 30% PEG-400, and 60% citric acid (10mM) or the CCR2 antagonist INCB3344 (100mg/kg, p.o.) at 24 and 48h after SE onset in a blinded design. The mice were sacrificed three days after SE onset, whole blood was obtained by cardiac puncture prior to perfusion, and the brain was processed for histology, Western analysis, and gene expression as described below.

The ability of a mouse to construct its nest between the second and third day after SE from a supplied nestlet square was assessed on a rating scale of 1523. 1: the nestlet is more than 90% intact; 2: nestlet is partially torn with 5090% untouched; 3: more than 50% nestlet is shredded without a nest site; 4: an obvious but flat nest was built; 5: perfect nest with walls higher than mouse body was built.

Three days after SE onset, mice were anesthetized deeply with isoflurane, perfused through the heart with PBS solution, and their brains rapidly removed from the cranium. The brain was immediately bisected through the midline, and the left hemisphere was fixed for 24h in 4% (wt/vol) paraformaldehyde at 4C. After 24h in 4% PFA, the left hemisphere was cryoprotected in 30% (w/v) sucrose in PBS solution. The cryoprotected left hemisphere was then frozen in 2-methylbutane chilled in dry ice and sectioned coronally at 25m using a freezing/sliding microtome. The hippocampus and cortex were dissected from the right half of the brain, immediately frozen on dry ice and stored at 80C for RNA isolation and Western blot analysis, respectively.

Numbers of Iba1+macrophages and RFP+monocytes were assessed on sets of every 12th systematically sampled 25m thick coronal immunostained section through the hippocampus. Thus, three sections were analyzed from each mouse. The sections were stained with rabbit polyclonal antibody to Iba1 (1:1000, Wako, 839,504) or RFP (1:1000, Abcam) and Vectastain Elite ABC Kits (Vector Laboratories). Stereological analysis was performed with the aid of the Stereologer software (Stereo Investigator 6, MBF Bioscience) and a motorized xyz stage coupled to a video microscopy system. The optical fractionator technique was used with 3D dissectors (area, 350350m2; height, 8m; guard height, 2m; counting frame, 5050m2)24. RFP+monocytes and Iba1+macrophages with complete soma within the dissector volume were counted.

For immunofluorescence staining, free floating sections were blocked for 45min in 5% goat serum and incubated overnight at 4C with antibodies against rabbit anti-Iba1 (1:1000, Wako, 839504) and rat anti-GFAP (1:1000, Invitrogen 143165. Primary antibody incubation was followed by extensive washing with PBS solution followed by incubation with fluorescent AlexaFluor secondary antibodies (1:400, Invitrogen) for 45min, and washing in PBS solution. Stained brain sections were mounted on slides and mounting medium with DAPI and antifade (Vector H-1200) was applied.

Fluorescent images of the neocortex overlying the hippocampus, amygdala, thalamus, and CA1 and CA3 hippocampus were acquired from Iba-1 and GFAP-stained brain sections with a Carl Zeiss Axio Observer A1 epifluorescence microscope equipped with an AxioCam Mrc5 camera. The camera settings for GFAP-stained sections were -0.09 (brightness), 8.56 (contrast), and 629ms (exposure time) with the Moments threshold setting used in NIH ImageJ Fiji software. The same camera settings and threshold levels were employed for all images stained with a given marker. Percentage area occupied by GFAP staining indicates the area above the threshold level. The percentage area of three sections were averaged to provide a single value for each animal. A meanSEM was calculated for each treatment group.

Cerebral cortices from saline perfused mice were homogenized in 10 volumes of RIPA buffer with protease and phosphatase inhibitors (Thermo Scientific). Brain homogenates were subsequently sonicated to shear DNA then centrifuged at 14,000g for 30min at 4C to remove nuclei and cell debris. Brain protein was run on a 420% Mini-PROTEAN TGX Gel and electroblotted onto PVDF membranes (Millipore). Membranes were blocked for one hour at room temperature with Odyssey blocking solution (Li-Cor), then incubated overnight at 4C with primary antibodies for albumin (1:1000; Cell Signaling) and GAPDH (1:10,000; Calbiochem), followed by incubation with polyclonal IRDye secondary antibodies 680LT and 800CW (1:15,000; Li-Cor). The blots were imaged by a Li-Cor imaging system using channels 700 and 800. Band intensity was measured and corrected for nearby background intensity by the Li-Cor software. The value of the albumin/GAPDH ratio for each sample was then determined. The average ratio for two groups was compared by t test. The fold-change for each group was referenced to the vehicle-treated group and plotted.

Sections were mounted on Superfrost Plus Microscope Slides (Fisher Scientific) and allowed to air-dry overnight. Sections were immersed in 0.06% potassium permanganate for 15min with gentle agitation, rinsed for one minute in distilled water, and then transferred to the Fluoro-Jade B staining solution (0.0001% wt/vol FJB in distilled water with 0.1% acetic acid) for 30min with gentle agitation in the dark. Sections were rinsed with three one-minute changes of distilled water and air-dried. The slides were immersed in xylene and then coverslipped with D.P.X. mounting media (Sigma-Aldrich). Three sections between bregma 1.34 and 2.40 were examined with a fluorescent microscope.

Following Fluoro-Jade B staining, images were obtained from three hippocampal areas (hilus, CA1, CA3) in each section. A researcher blinded to treatment and experimental conditions counted the number of Fluoro-Jade B-positive neurons in the hippocampus. Only positive neurons with a near-complete cell body shape and size were tabulated. Cell counts were expressed as the total number of Fluoro-Jade B-positive cells per section for each region.

Total RNA from mouse hippocampus was isolated by using TRIzol (Invitrogen) with the PureLink RNA Mini Kit (Invitrogen). RNA concentration and purity were measured by A260 value and the A260/A280 ratio, respectively. We typically recovered 515g RNA from each hippocampus with A260/280 ratio=2.12.2. First-strand cDNA synthesis was performed with 1.0g of total RNA, using qScript cDNA SuperMix (#95048, Quantabio) in a reaction volume of 20L at 25C for five minutes, then 42C for 30min. The reaction was terminated by heating at 85C for five minutes. qRT-PCR was performed by using eight L of 10x- or 50x-diluted cDNA, 0.10.5M of primers, and 2iQ SYBR Green Supermix (Bio-Rad Laboratories), with a final volume of 20L, in the iQ5 Multicolor Real-Time PCR Detection System (Bio-Rad). Cycling conditions were as follows: 95C for two minutes followed by 40 cycles of 95C for 15s and then 60C for one minute. Melting curve analysis was used to verify single-species PCR product. Fluorescent data were acquired at the 60C step. The geometric mean of the cycle thresholds for -actin, GAPDH, and HPRT1 was used as internal RNA-level control for relative quantification (Table1). Samples without cDNA served as the negative controls.

Whole blood was analyzed automatically by using the animal blood counter VetScan HM5 (ABAXIS).

The primary objective of the present study was to examine the consequences of systemic CCR2 antagonism after SE utilizing a small chemical molecule. Therefore, the effects of SE in mice subject to the CCR2 antagonist were compared to the effects of SE in vehicle-treated, aged-matched littermates. Prospective power analysis was based on preliminary data showing that CCR2 antagonism blocks monocyte brain entry to a similar degree encountered in Ccr2 KO mice after SE17, and the Ccr2 KO mice showed a 49% reduction in neuronal damage compared to CCR2-sufficient littermates post-SE resulting in an alpha<0.05 and power=0.69. Given these previous findings, the number of mice to achieve a desired power of 0.9 with similar attenuation of neuronal damage would be 19 mice/group. Twenty-four hours after SE, surviving mice were randomized and given CCR2 antagonist or vehicle in a blinded fashion. All assays were performed in a blinded fashion with the experimenter unaware of the treatment group. Just prior to performing statistical tests, potential outliers were identified by Grubbs test for removal. Unpaired t-tests were used for comparing two groups in Figs.2C, 3B, 5B, and 8D; an outlier was removed from 3B. Paired t-tests were used for Figs.7A,B, and 8B. An outlier was removed from 7A and another from 7B. The MannWhitney test was performed for evaluating nesting performance in Fig.2F. The KruskalWallis test followed by Dunns correction for multiple comparisons was used in Fig.6C after removing one statistical outlier from five of the six groups. All outliers, if retained, would not have changed the interpretation. The KruskalWallis test followed by Dunns correction for multiple comparisons was used in Figs.6D. One-way ANOVA followed by Sidak was used in Figs.3C, 7A,B, and 8E. Two-way ANOVA was used for Fig.2E.

For analysis of gene induction, the mean Ct values were compared between selected groups. After statistical analysis, individual Ct values from each sample group were converted to fold-change by 2Ct, and the fold-change from each group was plotted on a logarithmic scale. Results are expressed as mean valuesSEM. Statistical analysis was performed using GraphPad version 9 (GraphPad Software). For all analyses, the differences were considered to be statistically different if p<0.05.

Experimental procedures were performed in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals. The protocol (201900137) was reviewed and approved by the Institutional Animal Care and Use Committee of Emory University. The experiments reported here are in accordance with the ARRIVE guidelines.

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