Background
Traumatic brain injury (TBI) is the leading cause of morbidity and mortality among young people in the Western world. Patients with TBI sustain long-term neurological, cognitive and behavioral deficits leading to a greater requirement for institutional and long-term care. Despite intensive investigative efforts, there is a paucity of interventions designed to reduce morbidity and mortality associated with TBI [
1].
Immediately following TBI, there is a substantial excess release of neurotransmitters such as glutamate and signaling nucleotides such as adenosine. Excessive glutamate leads to hyperactivation of N-methyl-D-aspartate receptor (NMDAR) and subsequent excitotoxic neuronal injury. Recent data indicate that hyperactivation of glutamate receptors is short lived (< 1 hour), and there is a substantial reduction in NMDAR expression and signaling within 48 hours of injury [
2,
3]. Signaling pathways and molecules that are normally associated with neuronal survival (such as BDNF, TrkR, Src, ERK, cAMP and CREB) are reduced for several weeks following TBI [
2,
4,
5]. In addition to glutamate release and neuronal loss, TBI can also produce astro- and microgliosis and enhance the production of proinflammatory cytokines [
6‐
9]. This increased cytokine production can result in alterations in synaptic connections that can lead to additional neuronal loss. The latter effect can contribute to post-traumatic epilepsy (PTE) and long-term behavioral dysfunction with few therapies readily available [
10‐
13].
Membrane/lipid rafts (MLRs) are discrete regions of the plasma membrane enriched in cholesterol, glycosphingolipids and sphingomyelin, and the cholesterol binding and scaffolding protein caveolin (Cav). Three isoforms exist, with Cav-1 and Cav-2 usually co-expressed in a wide variety of tissues, while Cav-3 is canonically expressed in striated muscle [
14]. All three isoforms have been described in the central nervous system (CNS) [
15‐
17]. Cav-1 participates in the inflammatory response to the endotoxin lipopolysaccharide through toll-like receptor 4 (TLR4) and through negative regulation of endothelial nitric oxide synthase (eNOS) [
18]. Cav-3, normally associated with striated muscles, is not well studied in the CNS. We have recently shown that astrogliosis and microgliosis is increased in the brains of young Cav-1 knock-out (KO) mice [
19], and that Cav-1 and Cav-3 modulate microglia morphology [
20]. It is therefore conceivable that Cav-1 and Cav-3 might play an important role in the neuroinflammatory response in the brain following controlled cortical impact (CCI). To address this hypothesis, we first performed a variety of assays on wild-type (WT) mice with and without CCI (that is, histological, biochemical, electrophysiological, and by electron paramagnetic resonance (EPR)) to demonstrate establishment of the TBI model. We next conducted CCI and measured the neuroinflammatory response in the brains of WT, Cav-1 KO and Cav-3 KO mice subjected to CCI.
Materials and methods
Animal care
All animals were treated in compliance with the Guide for the Care and Use of Laboratory Animals (National Academy of Science, Washington, DC, USA). All animal-use protocols were approved by the Veterans Administration San Diego Healthcare System Institutional Animal Care and Use Committee (IACUC, San Diego, California, USA) prior to performed procedures. C57BL/6 WT and Cav-1 KO mice were purchased from Jackson Laboratories (Bar Harbor, ME, USA) and Cav-3 KO mice were a kind gift from Drs Ishikawa (Professor, Cardiovascular Research Institute, Yokohama City University School of Medicine, Yokohama, Japan) and Hagiwara (Professor, National Institute of Neuroscience, Kodaira, Tokyo, Japan) [
21].
Reagents
The following primary antibodies were used for Western blot (WB) and immunofluorescence microscopy (IF) analysis: Abcam (1 Kendall Square, Suite B2304, Cambridge, MA 02139-1517, USA) - A2AAR #ab79714, β3-tubulin #ab11314, Cav-3 #ab2912, MAP2 #ab32454; BD Transduction Labs (2350 Qume Drive, San Jose, CA 95131, USA) - NR2B #610417, TrkB #610102; Cell Signaling (3 Trask Lane, Danvers, MA, 01923, USA) - AMPAR #2460 s, Cav-1 #3267, NR1 #4204, NR2A #4205, PSD-95 #2507; Epitomics (863 Mitten Road, Suite 103, Burlingame, CA, 94010-1303, USA) - LDLR #1956-1, LRP-1 #2703-1; Imgenex (11175 Flintkote Ave, Suite E, San Diego, CA, 92121, USA) - GAPDH #IMG-5019A-1; Millipore (290 Concord Road, Billerica, MA, 01821, USA) - GFAP AB5541; Santa Cruz (10410 Finnell Street, Dallas, TX, 75220, USA) - A1AR sc-28995, A3AR sc-12938, TLR4 sc-30002, goat anti-mouse IgG-HRP sc-2031, goat anti-rabbit IgG-HRP, sc-2030 goat anti-rat IgG-HRP sc-2006; Stressgen (4243 Glanford Avenue, Victoria, BC, Canada) - HSP90 #SPA835; WAKO (1-2 Doshomachi 3-Chome, Chuo-Ku, Osaka, 540-8605, Japan) - Iba1 WB #016-20001, IF #019-19741; Molecular Probes (3175 Staley Road, Grand Island, NY, 14072, USA) - goat anti-rabbit-488 IgG (H + L) #A11008, goat anti-mouse-594 IgG (H + L) #A11005.
Controlled cortical impact model of traumatic brain injury
CCI was performed as described previously [
22]. Briefly, isoflurane (2% vol/vol) anesthetized mice were fixed into a stereotactic frame, maintaining basal temperature (37°C) throughout the procedure. A burr hole was made approximately 5 mm anterior to posterior (0 to −5 A-P) from the bregmatic suture and 4 mm laterally from the sagittal suture over the right hemisphere. Craniotomies were made with a portable drill over the right parietotemporal cortex and the bone flap was removed. Using a stereotaxic impactor (Impact One™;
myNeuoLab.com), a 3-mm tip was accelerated down to a 1 mm depth at a speed of 3 m/second with an 85 ms dwell time.
Histology (n = 4/group) and immunofluorescence (n = 3/group)
For histology, animals were transcardially perfused with 4% paraformaldehyde in 0.1 M PO
4 buffer then stored in the same buffer for 24 hours and processed for paraffin embedding. Serial sections through the hippocampus (two 5-μm sections per slide, 100 μm apart) were stained with Masson’s trichrome. Digital virtual slides obtained with Aperio Scanscope CS-1 scanner were used for extensive computer assisted morphometry in a Spectrum image analysis system (Aperio Technology Inc., 1700 Leider Lane, Buffalo Grove, IL, 60089, USA). Scanscope software and associated algorithms were applied for measurements of lesion volume and the count of dead or viable neurons in the impact zone, penumbra and relevant area of the contralateral hemisphere control (internal control) as described by Krajewska and colleagues [
23]. Whole brains were perfused with 4% paraformaldehyde, cryoprotected with 30% sucrose and frozen for cryostat sectioning in optimal cutting temperature embedding media. Free floating sections (50 μm) were washed in phosphate-buffered saline, blocked and incubated overnight with primary antibodies followed by species-specific secondary antibodies. Species-specific fluroconjugated Alexa® (3175 Staley Road, Grand Island, NY, 14072, USA) secondary antibodies were used at a 1:500 dilution with DAPI in 10% goat blocking solution. Sections were incubated for 1 to 2 hours at room temperature, gently rotating. We have previously characterized and optimized our immunofluorescence protocols for GFAP (glial fibrillary acidic protein), Iba1 (ionized calcium-binding adapter molecule 1) and MAP2 (microtubule associated protein 2) as previously described [
19,
20,
24,
25]. Incubation with 10% goat and no primary antibodies, with and without secondary antibodies, served as controls samples for these experiments. Coverslips or brain sections were mounted with an anti-fade solution and imaged; when appropriate, matched exposures were obtained. All other images were exposure and saturation optimized. All quantitation was done using NIH Image J.
Cognitive and motor tests (n = 20/group)
Male mice (2 to 3 months old) were subjected to CCI and monitored for an additional 3 months followed by a behavioral battery. Open field activity allows assessment of basic activity and general behavior/anxiety of the mouse. Locomotion was recorded and analyzed by a computerized video tracking system (Noldus XT 7.1, 1503 Edwards Ferry Road, Suite 310, Leesburg, VA, 201276, USA). Animals were habituated to the testing room; spontaneous locomotion was assessed in a white plexiglass open field box (41 × 41 × 34 cm enclosures) for 10 minutes. Recorded parameters were distance moved (cm), velocity (cm/second), and time spent in the center of the arena represented by 50% of the total arena (seconds). The wire grip test tests the ability of mice to hang on a metal rail [
26]. The metal wire is situated 40 cm from the ground and a soft surface is placed below the wire to prevent physical trauma to the mice. Latency to fall was timed and the test was repeated three times with an inter-trial interval of 30 seconds. The highest latency to fall was multiplied with the body weight to present the holding impulse (seconds × g). In the beam-walking test, mice traverse an elevated narrow beam to reach a platform. The protocol described here measures foot slips while crossing the beam. The apparatus was custom made according to a published protocol of Carter and colleagues [
27] with the height of the apparatus set at 50 cm. Continuous alternating T-maze test was used to assess the cognitive ability of the CCI mice; this enclosed apparatus is in the form of a T placed horizontally. Animals are started from the base of the T and allowed to choose one of the goal arms abutting the other end of the stem. Two trials are given in quick succession; on the second trial the rodent tends to choose the arm not visited before, reflecting memory of the first choice, termed as ‘spontaneous alternation’. We assessed this tendency in a test with 14 possible alternations according to plans and a protocol from a previously published method [
28,
29].
Electrophysiology (n = 4/group)
Transverse hippocampal slices were prepared as previously described [
30]. Mice were anesthetized with isoflurane before decapitation. The brain was quickly removed and immersed for 2 minutes in ice-cold artificial cerebrospinal fluid (ACSF) containing 119 mM NaCl, 2.5 mM KCl, 2.5 mM CaCl
2, 1.3 mM MgSO
4, 1 mM NaH
2PO
4, 26 mM NaHCO
3, 10 mM glucose, osmolarity 310 mOsm, continuously bubbled with carbogen (95% O
2-5% CO
2), pH 7.4. The hippocampus was extracted and cut in ice cold ACSF with a vibratome (Leica 1000, 1700 Leider Lane, Buffalo Grove, IL, 60089, USA) into 350 μm slices, which were allowed to recover in oxygenated ACSF at 35°C for 30 minutes, and then at room temperature for at least 1 hour prior to experimental recordings.
A slice was transferred into the submerged recording chamber and superfused with ACSF at a constant rate of 1.0 ml/minute at 32°C. To prevent de-oxygenation of ACSF in the recording chamber, the surface was continuously blown over by carbogen warmed to 32°C. Recording electrodes were made of borosilicate glass capillaries (1B150F, World Precision Instruments, Sarasota, FL, USA) and filled with ACSF (resistance 0.3 to 0.5 MΩ). Monopolar stimulating electrodes were made of Pt/Ir wires of diameter 25.4 μm (PTT0110, World Precision Instruments) and had 100 μm long exposed tips. The stimulating and recording electrodes were inserted under visual control perpendicular to the slice surface into the CA1 stratum radiatum 80 to 100 μm from the pyramidal layer, at a distance of 300 to 350 μm apart from each other. The magnitude of monosynaptic responses was evaluated as initial slope of field excitatory postsynaptic potential at latencies 0.1 to 0.9 ms, and the magnitude of polysynaptic responses as the averaged amplitude at latencies 12 to 45 ms after the stimulus. Testing stimuli (duration 100 μs, currents 60 to 80 μA) evoked field responses with amplitudes of 70 to 80% of maximum. Long-term potentiation was induced by tetanizations consisting of a single train of stimuli: 1 second at 100 Hz.
Superoxide measurements in synaptosomes by electron paramagnetic resonance (n = 3/group)
Brain NADPH oxidase (Nox) activity was assayed by detecting superoxide radical in synaptosomal isolations using EPR spin trapping spectroscopy according to a previously published protocol [
31]. Synaptosomal protein (0.2 to 0.5 mg) was mixed with 70 mM 5-(diethylphosphoryl)-5-methyl-1-pyrroline-N-oxide (Axxora, San Diego, CA, USA) and combinations of the substrates/inhibitors was loaded into a 50 μl glass capillary and introduced into the EPR cavity of a MiniScope MS300 Benchtop spectrometer (Louis-Bleriot-Str. 5, D-12487, Berlin, Germany) at a constant temperature of 37°C. Time evolution of the EPR spectra was recorded over 11 minutes from triggering Nox activity by adding appropriate combinations of substrates. For correlative analysis, the signals were quantified over the acquisition time of approximately 6 minutes (that is, the area under oxidative burst curves and normalized by the protein concentration). EPR conditions were as follows: microwave power, 5 mW; modulation amplitude, 2 G; modulation frequency, 100 kHz; MW frequency, 9.49 kHz; sweep width, 150 G centered at 3349.0 G; scan rate, 7.5 G/s and each spectrum was the average of 2 scans.
Cell culture
Primary cells were isolated using a Papain dissociation kit (#3150; Worthington Chemicals, Lakewood, NJ, USA) as previously described [
20,
24,
25]. Cultures were obtained from post-natal day 3 mouse pups. Mixed glia were separated from neurons according to manufacturer’s instructions and grown to confluence in T-75 flasks in Dulbecco’s modified Eagle’s medium with 10% fetal bovine serum.
Sucrose density fractionation and Western blot (n = 4/group)
Mouse cortex (50 to 100 mg) was homogenized using a carbonate lysis buffer (500 mM sodium carbonate, pH 11.0) containing protease and phosphatase inhibitors. Lysates were sonicated (three cycles for 15 seconds on ice). Protein was quantified by Bradford assay and normalized to 1 mg/ml. Sucrose was dissolved in MES buffered saline (25 mM MES (2-(
N-morpholino)ethanesulfonic acid) and 150 mM NaCl, pH 6.5) buffer to prepare 80%, 35% and 5% solutions [
25]. Sucrose gradients were prepared by adding 1 ml 80% sucrose followed by 1 ml sonicated sample with brief vortexing followed by 6 ml 35% sucrose followed by 4 ml 5% sucrose. Gradients were spun in an ultracentrifuge using an SW-41 rotor at 39 krpm at 4°C for 3 hours. Fractions (1 ml) were collected from the top of each tube starting at 4 ml to 12 ml. CCI samples were run as individual fractions and f4-6 (buoyant fractions; BF) and f10-12 (heavy fractions; HF) combined for WB. Samples were run on 10% or 4 to 12% bis-tris gels. After transfer to polyvinylidene fluoride membranes, samples were incubated with blocking buffer (3% bovine serum albumin in 20 mM Tris buffered saline containing 1% Tween) for 30 minutes and then incubated overnight with primary antibodies (in blocking buffer) at 4°C. Next day, membranes were washed (3 × 15 minute washes) and re-incubated with species-specific secondary antibodies conjugated to horseradish peroxidase from Santa Cruz at 1:5000 dilution in blocking buffer for 1 hour at room temperature. After extensive washing (4 to 5 × 15 minute washes) membranes were incubated with enhanced chemiluminescence reagent (Amersham Biosciences, PO Box 117, Rockford, IL, 61105, USA) and imaged with the UVP BioSpectrum Imaging System (UVP, 2066 W. 11th Street, Upland, CA, 91786 and saved as .tif files. Densitometric analysis was measured as previously described [
25].
MAGPIX cytokine multiplex assay (n = 6/group)
CCI or sham was performed on the WT, Cav-1 KO and Cav-3 KO mice (2 to 3 months old) and cytokine multiplex assay was performed on the cortex 4 hours post-CCI. Cortices were harvested and frozen 4 hours post-CCI separately from each hemisphere in liquid nitrogen. Frozen tissue was homogenized following the manufacturer’s instructions and 25 μl undiluted homogenate was added to 25 μl assay buffer. Magnetic beads (bead size = 6.45 μm) coated with specific antibodies (RCYTOMAG-80 K-PMX) were added to this solution and the reaction was incubated at 4°C for 24 hours. The beads were washed and incubated with 24 μl biotinylated detection antibody at room temperature for 2 hours. Completing the reaction, 25 μl streptavidin–phycoerythrin conjugate compound was added and allowed to incubate for 30 minutes at room temperature. Beads were washed and incubated with 150 μl sheath fluid for 5 minutes. Concentration of the samples was determined by Bio-Plex Manager version 5.0, after fluorescent capture, and MAGPIX xPONENT software (Millipore, 290 Concord Road, Billerica, MA, 01821, USA [
32]. The assays were run in triplicate to confirm the results. Samples were normalized to total protein concentration. Samples were analyzed for the following: IL-1α, IL-1β, IL-2, IL-4, IL-5, IL-6, IL-10, IL-12p70, IL-13, IL-17, IL-18, IFNγ, induced protein 10, chemokine C-C motif ligand (CCL)2 (previously known as monocyte chemoattractant protein 1; MCP-1), CCL3 (previously known as macrophage inflammatory protein 1 alpha; MIP-1α), CCL5 (also known as Regulated upon Activation Normal T-cell Expressed; RANTES), TNFα, vascular endothelial growth factor, eotaxin, growth related oncogene KC (keratinocyte chemoattractant) (CXCL1), leptin, granulocyte colony-stimulating factor, and granulocyte-macrophage colony-stimulating factor (GMCSF).
Statistical analysis
All data were analyzed by unpaired t tests or analysis of variance (ANOVA) with post-hoc Bonferroni’s multiple comparison or Student Neuman Keuls test as appropriate. Significance was set at P < 0.05. All data are presented as mean ± SEM. All statistical analysis was performed using Prism 6 (GraphPad Software, Inc., 7825 Fay Avenue, Suite 230, La Jolla, CA, 92037.
Discussion
The current findings are the first to demonstrate that loss of Cav isoforms produces isoform-specific effects on inflammation in a CCI model of TBI. The objective of the present study was to quantitatively assess neuroinflammation in the brain of Cav-1 KO and Cav-3 KO mice early after CCI. Many previously published studies have evaluated downstream signaling proteins involved in the induction of cytokines/chemokines after injury [
35,
36], but none have directly investigated the role of Cav and MLR-localized receptors and associated downstream signaling mediators on TBI-induced inflammatory responses. The loss of Cav-1, specifically, has been found to result in increased ischemic damage following transient middle cerebral artery occusion [
37]. One possible mechanism for increased injury is a lack of eNOS inhibition by Cav-1 leading to increased metalloproteinase activity and blood–brain barrier degradation [
38]. Because both microglia and astrocytes express Cav-1 and Cav-3, it is critical to understand how these proteins regulate receptor signaling, and secondary messengers such as NO, to induce or repress inflammation following CNS injury. Moreover, Cav-1 KO mice have previously been shown to exhibit enhanced anxiety and impaired spatial memory, demonstrating an important role for Cav-1 in normal neurological phenotype [
39]. Although it has yet to be determined which cell type contributes to these behavioral abnormalities, our previous work that demonstrates a reduction in MLR and MLR-localized synaptic proteins accompanied with reduced hippocampal synapses does indicate in part that loss of Cav-1 causes cellular morphological changes essential for normal brain physiology regardless of the cell type [
19].
Using a well-characterized CCI model of TBI, we detected glial reactivity in the ipsilateral hemisphere 4 hours post-injury and hippocampal neuronal death 24 hours post-injury. Behavioral studies revealed cognitive deficits in working memory, as determined by T-maze, 3 months post-injury with no motor deficits. Not surprisingly, the damage was not limited to the hippocampus, as extensive parietal cortical damage was also evident by 4 hours, which included enhanced neuroinflammation as indicated by the significantly elevated cytokine production in the ipsilateral cortex.
TBI can produce epileptogenesis, a neuropathological change that is frequently associated with depression, anxiety disorders and side effects from anti-epileptic treatments [
40]. PTE is a significant complication for the returning Veteran population with estimates that approximately 34% of returning Veterans who experienced moderate to severe head trauma are at risk for developing PTE. The findings from the current study show an increase in polysynaptic responses in the CCI hippocampal hemisphere, an indicator of increased pro-epileptic activity. Such a finding is a potential indicator of increased pro-epileptic activity because aberrant circuit formation is believed to be involved in epileptogenesis [
41,
42]. Therefore, these results (that is, enhanced polysynaptic responses) could be an important factor contributing to the post-TBI death of hippocampal neurons and development of epilepsy.
Another putative mechanism involved in the development of PTE is enhanced generation of reactive oxygen species [
43], as seen in the current study (Figure
2D,E). Previous studies have shown Nox activation leads to increased neurotoxic activation of microglia [
44]. Gene array studies have shown that changes in synaptic plasticity, glial proliferation and inflammatory reactivity occur before initial seizures manifest [
45,
46]. Anti-epileptic drugs, as a prophylactic intervention administered soon after TBI, have shown some efficacy in preventing early seizures (< 1 week), but are ineffective in preventing later, more devastating episodes of seizures [
47]. Therefore, more efficacious interventions that attenuate these initial key changes may alter the course of PTE development and potentially reverse the long-term cognitive changes that result from TBI.
We have previously shown a role for Cavs as regulators of neuronal survival [
19,
24,
25] and microglia activation [
20]. In an attempt to understand the potential role of Cavs in mediating the early inflammatory responses after TBI, 23 cytokines were measured 4 hours post-injury. Interestingly, 10 analytes were significantly elevated in both hemispheres of brains from either Cav-1 KO or Cav-3 KO mice. Common pro-inflammatory cytokines/chemokines, including IL-1β, IL-2, IL-6, IL-9, IL-10, IL-17, KC, MCP-1, MIP-1α, and GMCSF were upregulated in both Cav KO mice, yet only IL-6, KC, MCP-1, and MIP-1α were significantly elevated with CCI compared to the contralateral Cav. MCP-1 (CCL2) was significantly increased in the contralateral and ipsilateral hemisphere of both Cav KO mice; these results are in agreement with previously published work that demonstrated increased expression in a pilocarpine model of status epilepticus [
48]. Persistently elevated expression of MCP-1 in both Cav KO mice indicates a disruption in the normal signaling and trafficking of the MCP-1/CCR2 (MCP-1 receptor) complex, an interesting finding considering that previous work showed that MCP-1 KO mice have attenuated lesion size and less astrogliosis following TBI [
49]. Other studies have shown that Cav-1 plays a prominent role in astrocytic responses to MCP-1 by mediation of cellular signaling transduction through caveolae-localized CCR2 [
50,
51]. Therefore, interventions that increase Cav expression and restore normal CCR2 expression and function may be a potential therapeutic target. As a final Cav-mediated chemokine example from the multiplex analysis, MIP-1α (CCL3), a ligand for CCR5 (MIP-1α receptor), is significantly elevated after CCI in the ipsilateral hemisphere. Although many groups have found increased expression of MIP-1α following induced status epilepticus models, the role for MIP-1α, either protective or inflammatory, is still under debate [
52].
Various G-protein coupled receptors that are regulated by Cav, such as adenosine receptors, are involved in the complex process of microglia or astrocyte activation [
53‐
56]. The data from the current study demonstrated reduced expression of adenosine A
1AR and the anti-inflammatory A
3AR in both Cav-1 KO and Cav-3 KO brains. Evidence exists that the loss of A
1AR (A
1AR KO mice) results in an increased risk for epileptogenesis [
57,
58]. Because the current data show a reduction in A
1AR expression in Cav KO mice, loss of Cav isoforms due to injury (as shown in Figure
1D) may render the brain more susceptible to physiological changes (Figure
2C) and subsequent seizure development.
A
2AAR sits at the intersection of multiple control points for the development of neuropathology and neuropsychiatric conditions (reviewed in [
59,
60]). Activation of A
2AAR can negatively affect the functionality of A
1AR [
61], resulting in an enhanced inflammatory state. Additional evidence suggests that A
2AAR activation plays a major regulatory role in microglia-dependent neurotrophin release and subsequent microglia proliferation during neuroinflammation [
62]. The present findings demonstrate that both Cav KO mice have increased MLR localization of the pro-inflammatory A
2AAR compared to WT (Figure
3B). After injury, local adenosine concentrations greatly increase activating plasmalemmal localized A
2AAR receptors in microglia [
7,
63]. The present finding that Cav KO mice exhibit increased MLR-localized A
2AAR basally may in part explain the elevated cytokine/chemokine production in the brains of these mice both with and without CCI.
Cholesterol is a key component of MLR and for maintaining synaptic integrity. Because synaptic loss is one of the dynamic changes associated with the latency period for development of PTE [
64‐
67], changes in cholesterol homeostasis and MLR integrity may in part contribute to the etiology of PTE. Lipoprotein receptors are key players in cholesterol homeostasis [
68], and two important lipoprotein receptors in the brain, LRP-1 and LDL-R, are subcellularly localized to MLR [
33,
34]. Because Cav KO mice have reduced expression of LRP-1 and to a lesser extent LDL-R compared to WT (Figure
3C), events that cause decreased Cav expression in the brain (age or injury) may reduce cholesterol transport from glia to neurons and therefore increase the risk for synaptic loss, intercellular events we are presently investigating [
19].
Competing interests
The authors declare that they have no competing interests.
Authors’ contributions
IRN performed cell culture, biochemistry experiments (WB and IF), and participated in the draft of the manuscript. JMS assisted in behavioral analysis and participated in the draft of the manuscript. LAS performed cytokine array and assisted in analysis. SK and JAB performed CCI experiments. AK conducted electrophysiology experiments. WC assisted in CCI experiments and histology. AV performed behavioral studies. JAB and SK assisted in establishment of CCI model. SSA conducted EPR experiments and analysis. DMR participated in draft of the manuscript. HHP participated in the draft of the manuscript. PMP participated in establishment of CCI model and the draft of the manuscript. BPH participated in establishment of CCI model, study design, data analysis, and draft of the manuscript. All authors read and approved the final manuscript.