Background
Traumatic brain injury (TBI) causes cell death and neurological dysfunction through both direct physical disruption of tissue or pathways (primary injury), as well as delayed and potentially reversible molecular and cellular pathophysiological mechanisms (secondary injury) resulting in progressive white matter and grey matter damage [
1]. Such delayed injury begins within seconds to minutes after the insult and may continue for days, weeks or potentially months to years [
2]. These processes are characterized by neuronal cell death, as well as infiltration and activation of blood-borne immune cells, such as macrophages and lymphocytes, and activation of resident microglia [
3].
After TBI, microglia become activated and undergo marked changes in cell morphology and behavior. Upon activation, microglia contract their processes and transform from a ramified to an ameboid cellular morphology, followed by proliferation and migration towards the site of injury [
4]. Activated microglia can secrete a large number of factors, including cytokines, chemokines and other pro-inflammatory substances (for example, nitric oxide, prostaglandins and superoxide) that are toxic to neurons [
5,
6]. Microglial-mediated inflammation has been implicated as an important mechanism contributing to progressive neurodegeneration in multiple chronic neurological disorders [
7]. Animal and clinical studies indicate that post-traumatic neuroinflammation persists for months to years after brain injury [
8‐
11], and may contribute to chronic neurodegeneration and related significant neurological deficits [
12‐
15].
We have previously identified a cluster of microglial associated genes and associated proteins that are chronically expressed after central nervous system (CNS) trauma [
16], including membrane subunits of the reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidase enzyme. NADPH oxidase amplifies pro-inflammatory gene expression in activated microglia and promotes microglial-mediated neurotoxicity [
17], and may be a key driving factor for the chronic progression of neurodegenerative disease [
18]. More recently, we demonstrated that microglia in culture and in the rodent CNS express receptors for metabotropic glutamate receptor 5 (mGluR5). Activation of mGluR5 using the specific agonist (
RS)-2-chloro-5-hydroxyphenylglycine (CHPG) inhibits microglial activation and the release of inflammatory factors, in part by inhibiting NADPH oxidase [
19‐
21].
In the present studies we show that mGluR5 is chronically expressed in reactive microglia following controlled cortical impact (CCI) induced TBI in mice, and that single dose CHPG administration one month after TBI inhibits subsequent chronic post-injury inflammation and reduces the number of highly activated microglia that express NADPH oxidase. Such markedly delayed treatment also significantly reduces late tissue loss after TBI and improves long-term sensorimotor and cognitive recovery. As this effect is blocked by concurrent systemic administration of a selective mGluR5 receptor antagonist, the therapeutic actions of CHPG reflect actions at mGluR5 receptors.
Methods
Controlled cortical impact injury
All surgical procedures were carried out in accordance with protocols approved by Georgetown University Medical Center Institutional Animal Care and Use Committee. Our custom-designed CCI injury device [
22] consists of a microprocessor-controlled pneumatic impactor with a 3.5 mm diameter tip. Male C57Bl/6 mice (20 to 25 g) were anesthetized with isoflurane evaporated in a gas mixture containing 70% N
2O and 30% O
2 and administered through a nose mask (induction at 4% and maintenance at 2%). Depth of anesthesia was assessed by monitoring respiration rate and pedal withdrawal reflexes. Mice were placed on a heated pad and their core body temperature was maintained at 37°C. The head was mounted in a stereotaxic frame, and the surgical site was clipped and cleaned with Nolvasan and ethanol scrubs. A 10-mm midline incision was made over the skull, the skin and fascia were reflected, and a 4-mm craniotomy was made on the central aspect of the left parietal bone. The impounder tip of the injury device was then extended to its full stroke distance (44 mm), positioned to the surface of the exposed dura, and reset to impact the cortical surface. Moderate-level injury was induced using an impactor velocity of 6 m/s and deformation depth of 2 mm as previously described [
23]. After injury, the incision was closed with interrupted 6-0 silk sutures, anesthesia was terminated, and the animal was placed into a heated cage to maintain normal core temperature for 45 minutes post-injury. All animals were monitored carefully for at least 4 hours after surgery and then daily. Sham animals underwent the same procedure as injured mice except for the impact.
Drug treatment
One month post-injury, mice received a single intracerebroventricular (icv) injection of CHPG or equal volume vehicle. A 10 mM solution (saline with 1% dimethyl sulfoxide) was injected into the left ventricle (coordinates from bregma = anteroposterior: -0.5, lateral: -1.0; ventral: -2.0) using a 30 gauge needle attached to a Hamilton syringe at a rate of 0.5 μL/min, with a final volume of 5 μL, or 50 nmols of CHPG. Fifteen minutes prior to CHPG administration, one group of mice received the selective mGluR5 antagonist 3-((2-Methyl-4-thiazolyl)ethynyl)pyridine (MTEP; 10 mg/kg in saline) by intraperitoneal injection. All other mice received an equal volume vehicle intraperitoneal injection. Dosages were based upon prior investigations in spinal cord injury and TBI models [
20,
24].
In vivo magnetic resonance imaging
One, two and three months after CCI, brain lesion volume was assessed using T2-weighted magnetic resonance imaging (MRI) as previously described [
23] (n = 6 per group). Briefly, anesthetized animals were placed in a heated plexiglass holder and a respiratory motion detector positioned over the thorax to facilitate respiratory gating. The plexiglass holder was then placed in the center of the 7 Tesla magnet bore (Bruker Medical Inc., Billerica, MA, USA) where a 72-mm proton-tuned birdcage coil was positioned. Field homogeneity across the brain was optimized and a sagittal scout image was acquired (rapid acquisition relaxation enhancement (RARE) pulse sequence image; field of vision, 4 × 4 cm; 128 × 128 resolution; repetition time (TR) to echo time (TE), 1,500/10 ms with a RARE factor of 8, making the effective TE 40 ms). Multi-slice, multi-echo T2-weighted images were acquired using the following parameters: field of vision, 3 × 3 cm; 256 × 256 resolution; TR to TE, 1,500/10 ms; RARE factor of 8; TE, 40 ms; 10 × slices; slice thickness, 0.75 mm. Lesion volume was quantified from the summation of areas of hyperintensity on each slice, multiplied by slice thickness, for both the ipsilateral and contralateral hemispheres. Contralateral volumes were subtracted from ipsilateral volumes to obtain TBI-induced lesion volumes.
Ex vivo diffusion tensor imaging
Ex vivo diffusion tensor imaging (DTI) was performed four months post-injury on formalin-perfused brain (n = 5 per group). DTI imaging was performed with a 9.4 Tesla NMR spectrometer (Bruker Biospin, Billerica, MA, USA) equipped with a Micro2.5 gradient system (100 G/cm maximum gradient strength). We used our recently developed diffusion weighted gradient and spin echo sequence [
25] with the following parameters: TE, 33 ms; TR, 900 ms; number of radio frequency pulses (Nrf) = 4; bandwidth, 100 kHz; and four signal averages. The imaging field of view and matrix size were 13.0 × 10.0 × 18.4 mm and 128 × 96 × 180 mm respectively, and the native resolution was approximately 100 × 100 × 100 μm
3. The spectral data were apodized by a symmetric trapezoidal function with 10% ramp widths and zero-filled before Fourier transformation. For DTI, eight diffusion directions (
b-value 1700 s/mm
2), and two non diffusion-weighted images were acquired with δ = 3 ms, ∆ = 15 ms. The total imaging time was 11 hours. The signal-to-noise ratios in the corpus callosum measured in the non diffusion-weighted images were greater than 40 for all experiments.
Image processing
For the
ex vivo results, an average diffusion-weighted image (aDW) was obtained by averaging the six diffusion-weighted images. Signals from skull tissues in the aDW images were manually removed. The diffusion tensor was calculated using a log-linear fitting method [
26,
27]. The fractional anisotropy (FA), primary eigenvector (
v
1), parallel diffusivity (λ
║, the primary eigenvalue) and perpendicular diffusivity (λ
┴, the average of the secondary and tertiary eigenvalues) were calculated on a voxel-by-voxel basis from diffusion tensor using DTIStudio
http://www.mristudio.org[
28]. The
in vivo and
ex vivo aDW images were first rigidly aligned to our
ex vivo MRI based atlases [
29], respectively, using the six-parameter rigid registration function in the automated image registration package [
30,
31].
Motor and cognitive function testing
Motor performance was assessed one, two and three months post-injury using the beam walk task, as previously described [
22] (n = 7 (vehicle), 7 (CHPG), 4 (MTEP + CHPG)). Briefly, mice were trained to cross a narrow wooden beam 6 mm wide and 120 cm in length, which was suspended 300 cm above a 60-mm thick foam rubber pad. The number of foot-faults for the right hind limb was recorded over 50 steps and a basal level of competence at this task was established before surgery with an acceptance level of < 10 faults per 50 steps. Spatial learning and memory was assessed using an acquisition paradigm of the Morris water maze (MWM) test three and a half months post-injury as described [
32] (n = 4 (naïve), 8 (vehicle), 8 (CHPG), 8 (MTEP + CHPG)). A white circular pool was divided into four quadrants using computer-based AnyMaze video tracking system (Stoelting Co., Wood Dale, IL, USA) and the platform was hidden in one quadrant (south-west) 35.56 cm from the side-wall. Spatial learning and memory performance was assessed by determining the latency (seconds) to locate the sub-merged hidden platform with a 90-second limit per trial. On the day after the MWM acquisition test reference spatial memory was assessed by a probe trial; the time spent (seconds) within a 60-second limit in the quadrant where the platform had been hidden during acquisition phase was recorded. A visual cue test was performed using a flagged platform placed on the platform in one of the quadrants (with a 90-second limit per trial) and latency (seconds) to locate the flagged platform was recorded. In addition, the Water maze search strategies employed by each animal during the acquisition trials of the MWM test were analyzed as previously described [
33]. Briefly, three strategies were identified and categorized: spatial strategy was defined as swimming directly to the platform in no more than one loop or swimming directly to the correct target quadrant and searching; systematic strategy was defined as searching the interior portion of or entire tank without spatial bias, and searching incorrect target quadrant; and looping strategy was defined as circular swimming around the tank, swimming in tight circle, and/or swimming around the wall of tank.
Unbiased stereological assessment of cortical microglia
At the indicated time points, mice were anesthetized (100 mg/kg sodium pentobarbital, intraperitoneal injection) and transcardially perfused with 100 mL of 0.9% saline followed by 300 mL of 4% paraformaldehyde (10% buffered formalin solution, Fisher Scientific, Pittsburg, PA, USA) (n = 5 per group). The brain was removed and post-fixed in 4% paraformaldehyde overnight and cryoprotected in 30% sucrose. Coronal sections were cut (three × 60 μm followed by three × 20 μm sections) and serially collected throughout the injured brain, starting at +1.78 mm from the bregma. Sections were mounted onto glass slides for immunohistochemical analysis. Microglia were immunostained with anti-ionized calcium binding adaptor molecule 1 (Iba-1) (1:1000; Wako Chemicals, Richmond, VA, USA) for 1 hour, washed in PBS and incubated with biotinylated anti-rabbit immunoglobulin G antibody (Vector Laboratories, Burlingame, CA, USA) for 2 hours at room temperature. Sections were placed in avidin-biotin-horseradish peroxidase solution, diluted according to the manufacturer's instructions for 1 hour (Vectastain elite ABC kit, Vector Laboratories) and then reacted with 3,3'- diaminobenzidine (Vector Laboratories) for color development. Sections were counterstained with cresyl violet (FD NeuroTechnologies, Baltimore, MD, USA), dehydrated and mounted for analysis.
Stereoinvestigator software (MBF Biosciences, Williston, VT, USA) was used to count the number of cortical microglia in each of the three microglial morphological phenotypes (ramified, hypertrophic and bushy) using the optical fractionator method of unbiased stereology. The sampled region was the perilesional ipsilateral cortex between -1.22 mm and -2.54 mm from the bregma. Every fourth 60-μm section was analyzed beginning from a random start point. Sections were analyzed using a Leica DM4000B microscope (Leica Microsystems, Exton, PA, USA). The optical dissector had a size of 50 × 50 μm in the x and y-axis with a height of 10 μm and guard zone of 4 μm from the top of the section. Dissectors were positioned every 150 μm in the x and y-axis. Microglial phenotypic classification was based on the length and thickness of the projections, the number of branches and the size of the cell body as previously described [
34]. Neurolucida software (MBF Biosciences) was used to trace and quantify the size of microglial cell bodies and dendrites at different stages of activation following injury. Microglia were outlined using the live image setting so that the width of the dendrites could be traced while focusing on the section. The cell body was outlined using the contour tool followed by the tracing of the individual dendrites, using the dendrite line tool. A quick measure tool quantified the average thickness of each microglial branch. Neurolucida explorer software was used to determine the cell body area and volume, and dendrite length and branching number. Ramified microglia possessed long thin processes (> 650 μm in length), had a small cell body volume (< 10 μm
3) and many branches (20 to 30). Hypertrophic microglia possessed medium length processes (300 to 550 μm in length), had larger cell body volumes (50 to 75 μm
3) and many branches (20 to 30). Bushy microglia possessed short thick processes (< 200 μm in length), had a larger cell body volume (80 to 100 μm
3) and very few branches (< 10). The volume of the region of interest was measured using a Cavalieri estimator method with a grid spacing of 100 μm. The estimated number of microglia in each phenotypic class was divided by the volume of the region of interest to obtain the cellular density expressed in cells per cubic millimeter.
Unbiased stereological counting of surviving hippocampal neurons
The stereoinvestigator software was used to count the total number of surviving neurons in the Cornu Ammonis (CA) 1, CA3 and dentate gyrus subfields of the hippocampus using the optical fractionator method of unbiased stereology. The sampled region for each hippocampal subfield was demarcated in the injured hemisphere and cresyl violet neuronal cell bodies were counted. The volume of the hippocampal subfield was measured using a Cavalieri estimator method. The estimated number of surviving neurons in each field was divided by the volume of the region of interest to obtain the cellular density expressed in cells per cubic millimeter.
Immunohistochemistry
Immunohistochemistry was performed on 20-μm sections and standard immunostaining techniques were employed. The following primary antibodies were used: rabbit anti-Iba-1 (1:500, cat. no. 019-19741, Wako Chemicals), mouse anti-mGluR5, clone 464823 (1:100; cat. no. MAB45141, R&D Systems, Minneapolis, MN, USA), rat anti-ED1 (1:200, cat. no. MCA1957G, AbD Serotec, Raleigh, NC, USA), and mouse anti-gp91phox (1:200, cat. no. 611415, BD Transduction Inc., Franklin Lakes, NJ, USA). Counterstaining was performed with 4', 6-diamidino-2-phenylindole (1 μg/mL; Sigma-Aldrich, St. Louis, MO, USA). Fluorescence microscopy was performed using a LEICA (TCS SP5 II) confocal microscope system (Leica Microsystems).
Statistical analysis
Lesion volume, functional data and unbiased stereological analysis were performed by an investigator blinded to treatment group. Quantitative data were expressed as mean ± standard errors of the mean (SEM). Functional data for beam walk and acquisition phase of MWM were analyzed by repeated measures (trial and time) one-way (groups) analysis of variance (ANOVA) to determine the interactions of post-injury trial and groups, followed by post-hoc adjustments using Student-Newman-Keuls test. Search strategy analysis was analyzed using a chi-square analysis. The T2-weighted MRI analysis was analyzed by two-way ANOVA, followed by post-hoc adjustments using Bonferroni t-test. Remaining data were analyzed using Mann-Whitney U test, Student's t test or one-way ANOVA, where appropriate. The functional data was analyzed using SigmaPlot 12 (Systat Software, San Jose, CA, USA). All other statistical tests were performed using the GraphPad Prism Program, Version 3.02 for Windows (GraphPad Software, San Diego, CA, USA). P < 0.05 was considered statistically significant.
Discussion
Taken together, our data demonstrate chronic microglial activation after experimental TBI, which may contribute to the observed progressive neurodegeneration and tissue loss that is associated with functional impairments. Furthermore, they demonstrate that the temporal window for neuroprotective intervention after TBI is significantly longer than generally believed. These observations are consistent with prior experimental work demonstrating progressive cortical damage and increased NFκB activation in macrophages and/or microglia up to one year after trauma [
12,
38,
39]. Prolonged microglial activation has been demonstrated many months after TBI in humans [
9] and increased microglial activation has also been observed up to four years after TBI in human post-mortem tissue [
11].
We previously reported that CHPG treatment, acting through mGluR5, reduces microglial activation and the associated release of free radicals and pro-inflammatory cytokines in microglial cell culture models (both primary cultures and a mouse microglial cell line) after stimulation with the classical activators lipopolysaccharide or interferon-γ [
19,
21]. CHPG treatment also abolished the neurotoxic potential of activated microglia and reduced NADPH oxidase activity in such
in vitro models. These effects of CHPG were blocked by knockout of the mGluR5 receptor or by addition of the selective mGluR5 antagonist MTEP, and reduced by co-incubation with siRNAs directed against either of the two membrane subunits of NADPH oxidase [
19]. The current study supports these findings, as MTEP administration prior to CHPG treatment blocked its protective actions.
Examining the injured cortex one month after TBI, we demonstrated that mGluR5 expression was up-regulated in activated microglia that co-expressed the phagocytic marker, ED1, and exhibited a hypertrophic or bushy cellular phenotype. The observed post-traumatic expression of mGluR5 in activated microglia is consistent with previous
in vivo studies demonstrating microglial mGluR5 expression at the lesion site in spinal cord injury and excitotoxic brain injury models [
20,
40]. In response to trauma, there is both microglial proliferation and activation, along with migration to the site of injury [
41,
42]. In contrast to the ramified appearance of resting microglia, activated microglia undergo substantial morphological changes, with reduction and thickening of processes leading to a hypertrophic or bushy appearance [
34]. Although microglia are believed to have both neurotoxic and neuroprotective properties [
3,
5,
43], considerable experimental data suggest that post-traumatic inflammation can contribute to delayed cell and tissue loss [
35,
44,
45]. Indeed, microglial activation and release of associated inflammatory factors has been proposed as an important contributing factor for many acute and chronic neurodegenerative disorders [
7,
46].
Here we demonstrate that delayed CHPG administration significantly reduced the number of microglia showing the reactive bushy or hypertrophic phenotypes associated with a pro-inflammatory and potentially neurotoxic state [
47]. Moreover, activated microglia expressed the NADPH oxidase sub-unit, gp91
phox four months post-injury, suggesting the chronic expression of NADPH oxidase in these cells; these data are consistent with the chronic up-regulation of expression of NADPH oxidase sub-units in a microglial-associated gene cluster six months after spinal cord injury [
16]. Activated microglia cause neuronal cell death in culture through mechanisms that involves NADPH oxidase activation, and this process is inhibited by CHPG treatment [
19,
21]. Activated microglia also have been implicated in chronic functional deficits after TBI in humans [
9]. Recently, inhibition of NADPH oxidase has been shown to reduce microglial activation in the post-ischemic brain [
48]. Notably, single dose CHPG administration one month after TBI reduced the expression of NADPH oxidase in reactive microglia at four months post-injury. Together, these data suggest a positive activation feedback loop for neuroinflammation that contributes to delayed neurodegeneration and related functional deficits. Interruption of this positive feedback loop may explain why even a single injection of CHPG administered one month post-injury inhibits chronic neuroinflammation and limits functional loss in our model.
mGluR5 is also expressed in other cell types of the CNS, such as neurons, astrocytes and oligodendrocytes [
49,
50]. In addition to their anti-inflammatory effects, group I mGluR agonists also reduce neuronal apoptosis [
51] as well as oligodendrocyte cell death [
52]. Although it is possible that anti-apoptotic effects of CHPG on neurons or oligodendrocytes may have contributed to the recovery observed in the current study, such apoptotic processes so late after injury are likely limited and, given the half life of the compound, modulation of such events would probably contribute minimally at best to the improved outcome observed. In order to clarify the mechanism underlying mGluR5-mediated neuroprotection after TBI, future studies will require cell specific mGluR5 knockout in neurons, astrocytes, oligodendrocytes and microglia.
It is widely accepted that the therapeutic window for limiting post-traumatic neurodegeneration after acute brain injury is limited [
53]. Indeed, most experimental treatment studies for TBI have focused on the first hours after injury [
1]. With the recognition that more delayed apoptotic mechanisms may contribute to injury [
54,
55], the potential therapeutic window has been expanded to perhaps 24 to 72 hours. Although it has been shown experimentally and, more recently, clinically that tissue loss after TBI may progress for months or longer [
12,
14,
15,
56,
57], there have been few attempts to pharmacologically modify such markedly delayed neurodegeneration. Here we demonstrate that single dose treatment one month after trauma significantly reduced both histological changes and behavioral dysfunction over a subsequent period of months. Considerable tissue sparing was observed in hippocampal and cortical regions after CHPG treatment, which was associated with significant improvements in both sensorimotor (beam walk) and cognitive (MWM) function.
Chronic neurological deficits are characteristic of moderate to severe TBI, although such deficits may stabilize or improve over time - likely reflecting endogenous plasticity. The DTI studies extend the observations from T2-weighted MRI by demonstrating significantly better preservation of white matter tracks in the brain at four months in the CHPG-treated TBI group as compared to controls. DTI detects directionality of water diffusion. After TBI, FA increases, whereas mean diffusivity, or the directed diffusion of water, typically along white matter tracks, is reduced [
58,
59]. These techniques have been used to reflect the integrity of white matter tracks (tractography) in the CNS [
58]. FA has been shown to be negatively correlated with deficits in memory performance [
59], consistent with our observations that CHPG treatment reduced this measure while improving cognitive performance.
Conclusions
In summary, we demonstrate that markedly delayed treatment with CHPG one month after TBI significantly reduces subsequent lesion progression and white matter damage, with marked improvement in sensorimotor and cognitive function. Such delayed CHPG treatment attenuates neurodegeneration in the CA3 and dentate gyrus subfields of the hippocampus, areas that show significant neuronal loss after TBI and are associated with cognitive impairment after injury [
35,
60,
61]. CHPG administration significantly attenuates the activation state of microglia in the injured cortex, although it did not alter the total number of microglia. The fact that co-administration of an mGluR5 antagonist blocked the protective effects of CHPG indicates that its therapeutic actions were specific and mediated by mGluR5 receptors, consistent with our previously published
in vitro data [
19,
21].
This study significantly extends the currently accepted therapeutic window for neuroprotection after head injury. It also provides mechanistic experimental support for microglial-mediated chronic neurodegeneration after TBI and introduces a novel therapeutic approach for brain trauma. Given the therapeutic efficacy of delayed CHPG treatment, and its ability to modify chronic neuroinflammatory processes after traumatic injury, similar strategies might prove beneficial for other chronic neurodegenerative disorders that show a significant inflammatory component.
Competing interests
KRB, DJL and AIF are listed as inventors for a use patent from Georgetown University for mGluR5 agonists in the treatment of neuroinflammation.
Authors' contributions
KRB carried out the TBI surgeries, mouse behavior and T2-weighted MRI. DJL performed MRI analysis. DJL and BAS carried out imaging and stereology studies. JZ carried out the ex vivo DTI. KRB, DJL and BAS designed and coordinated the study and wrote the manuscript. AIF conceived the study and wrote the manuscript. All authors read and approved the final manuscript.