Histone deacetylase inhibitor ITF2357 is neuroprotective, improves functional recovery, and induces glial apoptosis following experimental traumatic brain injury

Abstract

Despite efforts aimed at developing novel therapeutics for traumatic brain injury (TBI), no specific pharmacological agent is currently clinically available. Here, we show that the pan-histone deacetylase (HDAC) inhibitor ITF2357, a compound shown to be safe and effective in humans, improves functional recovery and attenuates tissue damage when administered as late as 24 h postinjury. Using a well-characterized, clinically relevant mouse model of closed head injury (CHI), we demonstrate that a single dose of ITF2357 administered 24 h postinjury improves neurobehavioral recovery from d 6 up to 14 d postinjury (improved neurological score vs. vehicle; P≤0.05), and that this functional benefit is accompanied by decreased neuronal degeneration, reduced lesion volume (22% reduction vs. vehicle; P≤0.01), and is preceded by increased acetylated histone H3 levels and attenuation of injury-induced decreases in cytoprotective heat-shock protein 70 kDa and phosphorylated Akt. Moreover, reduced glial accumulation and activation were observed 3 d postinjury, and total p53 levels at the area of injury and caspase-3 immunoreactivity within microglia/macrophages at the trauma area were elevated, suggesting enhanced clearance of these cells via apoptosis following treatment. Hence, our findings underscore the relevance of HDAC inhibitors for ameliorating trauma-induced functional deficits and warrant consideration of applying ITF2357 for this indication.—Shein, N. A., Grigoriadis, N., Alexandrovich, A. G., Simeonidou, C., Lourbopoulos, A., Polyzoidou, E., Trembovler, V., Mascagni, P., Dinarello, C. A., Shohami, E. Histone deacetylase inhibitor ITF2357 is neuroprotective, improves functional recovery, and induces glial apoptosis following experimental traumatic brain injury.

Under normal conditions, stability of cellular acetylation homeostasis of histones and other proteins is preserved by maintenance of an appropriate balance between discrete sets of enzymes, which facilitate forward and backward modifications (1, 2). In contrast, disruption of acetylation homeostasis has been reported in various neuropathological states (3). Particularly, several studies demonstrated that neurodegeneration is associated with a global decrease in histone acetylase (HAT) activity resulting in relative overdeacetylation (4, 5).

Subsequently, histone deacetylase (HDAC) inhibitors were tested for ameliorating neurodegenerative diseases in animals, yielding promising findings in models mimicking disorders such as Huntington’s disease, amyotrophic lateral sclerosis, and experimental autoimmune encephalomyelitis (6,7,8,9). It should be noted in this context that several HDAC inhibitors are presently used clinically for other indications, and long-term use reveals a broad safety margin. Valproic acid (VPA) is an HDAC inhibitor used to treat epilepsy (10); butyrate is used to treat sickle-cell anemia and thalassemia (11, 12), and suberoylanilide hydroxamic acid (SAHA) is used to treat T-cell lymphoma (13).

Given the involvement of neurodegeneration in ischemic brain injury, some HDAC inhibitors were examined for a possible neuroprotective property in rodent stroke models. These investigations demonstrated that HDAC inhibitors reduce lesion size and improve functional recovery after brain ischemia (14,15,16). Despite the growing body of evidence pointing to benefits brought about by HDAC inhibitors in stroke models, data relating to the potential use of this line of treatment following traumatic impact is lacking. Specifically, the overall functional value of such therapy on traumatic brain injury (TBI) has not been established, greatly limiting the interpretation of recently reported molecular effects (17). As the ability to effectively intervene in post-trauma pathological processes is hindered by a narrow time frame for efficacious drug administration and belated arrival of injured individuals to hospital (18), the ability to administer HDAC inhibitors within a clinically feasible timeframe after TBI must also be addressed in the scope of evaluating their potential clinical value. Finally, it would be preferable to conduct such evaluations in an experimental model that does not involve preinjury manipulations, which may compromise its relevance to the human pathological setting.

To address these questions, the current study examined the functional and molecular consequences of treatment with the HDAC inhibitor ITF2357 in a well-established mouse model of closed head injury (CHI). ITF2357 is a potent hydroxamic acid-containing pan-HDAC inhibitor, as assessed by enzyme activity (19). We chose to focus on this compound in light of its favorable safety profile in humans, as in a phase II clinical trial in children with active systemic onset juvenile idiopathic arthritis, an oral dose of this agent at 1.5 mg/kg/d was well tolerated, exhibited no organ toxicity, and significantly reduced the systemic feature score and number of painful joints (20).

MATERIALS AND METHODS

Animals and trauma model

The study was performed according to the guidelines of the Institutional Animal Care Committee of the Hebrew University (Jerusalem, Israel). Male Sabra mice of the Hebrew University strain, weighing 35–45g, were used in this study. Animals were kept under standard conditions of temperature and light and were provided with food and water ad libitum. Experimental CHI was induced using a modified weight-drop device developed in our laboratory (21). Following isoflurane anesthesia, a midline longitudinal incision was made and the skull exposed. A Teflon-tipped cone (2-mm diameter) was placed 1–2 mm lateral to the midline in the midcoronal plane. The head was manually held in place, and a 95-g weight was dropped on the cone from a prefixed height, resulting in a focal injury to the left hemisphere. After recovery from anesthesia, the mice were returned to their home cages with postoperative care and free access to food and water. Sham controls received anesthesia and skin incision only.

Neurobehavioral evaluation

The Neurological Severity Score (NSS), previously developed and validated in our laboratory (22, 23), was used to evaluate post-traumatic functional impairment. NSS consists of 10 individual tasks, including open-field performance, beam walk, balance, and hemiparesis evaluations, which reflect motor function, alertness, and behavior (23). One point is given for failure to perform a task and 0 for success. The NSS at 1 h post-trauma reflects the initial severity of injury. Thus, the extent of recovery (ΔNSS) is calculated as the difference between the initial NSS score at 1 h postinjury and at any subsequent time point.

Drug treatment protocol

ITF2357 (Italfarmaco, Cinisello Belsamo, Italy) was dissolved in DMSO. Prior to use, the compound was diluted in sterile saline, heated to boiling for complete dissolution, and cooled to ∼30°C before injection. Control for these studies was 0.5% DMSO saline solution. ITF2357 solutions were freshly prepared for each experiment. To initially substantiate a functional effect of ITF2357 on neurobehavioral outcome after brain trauma, the drug was given either as pretreatment (a single injection at 30 min prior to the induction of injury) or administered following the impact. Postinjury injections were carried out at either at 1 or 24 h after trauma. In each group of mice, animals with a similar initial severity of injury (NSS at 1 h after trauma; n≥9 mice/group) were dosed i.p. with 100 μl containing either 10 mg/kg of ITF2357 or vehicle. This dose was selected in accordance with previous studies utilizing ITF2357 in mice (24), and no adverse effects or mortality were observed among treated mice in all experiments included in the current report.

NSS scores at 1 h after injury were similar in all groups (vehicle and treated) confirming comparable and moderate severity of injury. Mice were subsequently subjected to NSS evaluations over time. As evaluations were conducted on separate groups of mice, each ITF2357-treated group was tested concurrently and compared to an equivalent vehicle-treated group (containing littermates held and transported together) as to control for interexperimental differences due to external environmental parameters. Because the functional benefit of ITF2357 was most pronounced when administered 24 h postinjury, this protocol was used for subsequent experiments, including immunohistochemical studies, lesion volume measurements, and Western immunoblotting.

Brain pathology immunohistochemistry

Animals were sacrificed (n=4/group) with transcardial perfusion using 4% ice-cold paraformaldehyde in phosphate-buffered saline (PBS; pH 7.4). Brains were removed, postfixed in the same fixative overnight, cut in 5 predetermined brain levels, and embedded in paraffin. Serial 6-μm-thick coronal sections were obtained.

For assessment of gross trauma pathology and degenerative cells, sections were deparaffinized, hydrated in alcohol-graded solutions, rinsed with Tris-buffered saline (TBS), and subjected to hematoxylin-and-eosin (H&E) and Fluoro-Jade B (FJB) staining. Immunohistochemical stains for light microscopy were performed on paraffin sections using the following antibodies: antiglial fibrillary acidic protein (GFAP; 1:2500 polyclonal rabbit-anti-GFAP; Dako Cytomation, Glostrup, Denmark), anti-active caspase-3 (1:2000 polyclonal rabbit-antiactive caspase-3; R&D Systems, Minneapolis, MN, USA) and lectin (3 mg/ml Lycopersicon esulentum tomato; Sigma Chemical Corp., St. Louis, MO, USA). Antigen retrieval was performed by incubating deparaffinized sections for 60 min in citrate buffer (pH 6) in a food-steamer device (Braun, Kronberg/Taunus, Germany). Endogenous peroxidase was blocked with 0.3% H₂O₂ in methanol and was followed by incubation in blocking buffer (10% normal goat serum and 0.3% Triton-X in TBS) for 30 min. Antibodies were diluted in commercial antibody diluent (Dako Cytomation), and sections were incubated overnight at 4°C. Immunoreactions were visualized using the EnVision System-HRP kit (Dako Cytomation) for GFAP and caspase-3 and the LSAB-2 System-HRP (Dako Cytomation) for lectin staining. Labeling was visualized using 3,3′-diaminobenzidine-tetrahydrochloride (DAB; Dako Cytomation). All but FJB-stained sections were counterstained with hematoxilin. Sections were evaluated under light or fluorescent microscopes (Zeiss Axioplan 2; Carl Zeiss, Oberkochen, Germany) as appropriate.

Double immunofluorescence was performed on paraffin sections using the following antibodies: antiactive caspase-3 (1:1000; polyclonal rabbit-antiactive caspase-3; R&D Systems) and anti-MAC-3 (1:200; monoclonal rat anti-mouse-MAC-3; BD Biosciences, Erembodegem, Belgium). Sections were deparaffinized, hydrated in graded alcohol solutions, rinsed with TBS, and incubated in blocking buffer (Dako antibody diluent; Dako Cytomation) for 60 min. Antibodies were diluted in blocking buffer, and sections were incubated overnight at 4°C. Immunoreactions were visualized using fluorescent secondary antibodies (1:200 anti-rabbit Alexa Fluor 594 and 1:200 anti-rat Alexa Fluor 488; Invitrogen Molecular Probes, Carlsbad, CA, USA). Nuclei were visualized with 4,6-diamidino-2-phenylindole 4,6-diamidino-2-phenylindole (DAPI), and sections were studied using confocal microscopy (Nikon Instruments, Melville, NY, USA) for detection of marker coexpression.

Neuropathological evaluation was performed on three slides per staining containing sections spaced ∼50 μm apart by two blinded investigators under either light or fluorescent microscopes (Zeiss Axioplan 2) with the aid of a charge-coupled device camera (Nikon DS-5Mc-L1). Thus, we avoided counting the same cell or process in more than one section. Gross trauma pathology was studied using H&E-stained sections. Astrocytic reactions, microglia/macrophages, and caspase-3-labeled cells were studied on d 3 and 21 after injury under an ×20 optical field using a 610- × 610-μm prefrontal grid; observation under a high-power optical field (×40) was used where necessary for clarification of cellular pathology. To obtain standardized results from all animals, evaluations of pathology were performed on sections at the level of hippocampi (between −1 and −2 mm anteroposteriorly), which included traumatic area. GFAP-positive astrocytes were counted in the hemisphere ipsilateral to the trauma site, and data are presented as positive cells per square millimeter. In addition, the surface covered by the processes of these cells (GFAP-positive small squares in the prefrontal grid per the total number of squares, i.e., 100) was evaluated as an indicator of their activity level and is presented as the percentage of the section area studied. Lectin-positive microglia and macrophages were counted within the ipsilateral hemisphere, and data are presented as positive cells per square millimeter. Cells positive for active caspase-3 were counted in adjacent sections within the trauma area, penumbral tissue, and the corresponding hippocampus on d 3 following injury, and data are presented as positive cells per square millimeter. Accordingly, on d 21 postinjury, caspase-3-positive cells were counted in the area of traumatic scar borders and the surrounding tissue (extended by one prefrontal grid from the scar borders).

Finally, neurodegeneration analysis was performed by counting FJB-positive cells and processes in the area of trauma and hippocampus in accordance with a slightly modified method, as previously reported by our group (25). Briefly, FJB-positive cells were quantified by capturing images from 1 to 3 visual fields (depending on the size of each area) onto an Intel Pentium computer using a digital camera (Nikon DS-5Mc-L1) mounted on the microscope. Images of the respective areas were captured at a final magnification of ×480. In each image, a 305- × 122-μm rectangle with 40 subdivisions (3.05×3.05 μm each) was positioned over the captured area. For all images, a square subdivision was counted if it contained ≥1 labeled element, defined as a labeled cell body and/or process, and data were presented as FJB-positive square subdivisions per square millimeter.

Measurement of lesion volume

Mice (n=7/group) were sacrificed 16 h following ITF2357 or vehicle injection (40 h postinjury). Brains were removed and coronally sectioned at 2-mm thickness. The sections were immersed in 2% 2,3,5-triphenyltetrazolium chloride (TTC; Sigma Chemical Corp.) in saline for 1 h at 37°C and fixed in 4% formaldehyde. Images of all sections were captured using a digital camera (Nikon Coolpix 990) and analyzed using ImageJ software (public domain program developed at the National Institutes of Health, Bethesda, MD, USA). Lesion areas were corrected for edema via division by the area of the contralateral hemisphere. Values were then multiplied by the slice thickness and integrated for calculating total lesion volume as described previously (26).

Western immunoblotting

Ipsilateral frontal cortical segments (n=4/group) were taken 6 or 24 h following the injection of either vehicle or ITF2357 (i.e., 30 or 48 h postinjury) and homogenized (16). Following sonication on ice for 45 s and centrifugation at 12,000 rpm, protein concentrations in the supernatants were determined using the Bradford method (Bio-Rad Laboratories, Munich, Germany). Protein (40 μg) was separated on 4–20% gradient gels (Bio-Rad Laboratories) and subsequently transferred onto nitrocellulose membranes (0.2 μm; Scleicher and Schuell, Dassel, Germany). Blots were probed overnight at 4°C with primary antibodies against acetylated histone H3 (Ac-H3; 1:20000; Upstate Biotechnology-Millipore Corporation, Billerica, MA, USA), heat-shock protein 70 kDa (HSP70; 1:1000; Cell Signaling Technology, Danvers, MA, USA), phospho-Akt (Ser-473; p-Akt; 1:1000; R&D Systems) and p53 (1:20; a gift from Prof. Y. Haupt, Hebrew University). All primary antibody incubations were followed by an appropriate horseradish peroxidase-conjugated secondary antibody (Jackson Immunoresearch, Soham, UK). Reactive bands were visualized using an enhanced chemiluminescence system (Biological Industries, Beit Ha’Emek, Israel). All blots were probed with anti-β-actin (β-actin; 1:1000; Cell Signaling Technology) to control for protein loading. The films were scanned and subsequently analyzed using Tina software (Raytest, Straubenhardt, Germany). Data are presented as percentages of control, as no difference was observed between sham groups. For p53, optical density values relative to β-actin are shown, since the level of this factor in sham animals was nearly zero.

Statistical analysis

Data are presented as means ± se. Statistical analysis was conducted using a commercial statistics package (GraphPad Prism 4; Graph Pad Software, San Diego, CA, USA; or SPSS 11.5; SPSS Inc., Chicago, IL, USA). Data were subjected to ANOVA followed by Bonferroni post hoc multiple comparison tests. Nonparametric data (NSS values) were analyzed using the Kruskal-Wallis rank test. Following the Kruskal-Wallis test, the Dunn’s test was used for investigating pair-wise comparisons, which takes into account multiple comparisons. Student’s t tests or Mann-Whitney U tests were used for 2-group comparisons as appropriate (normality tested with Kolmogorov-Smirnov test). Significance level was set at P ≤ 0.05 in all cases.

RESULTS

Post-TBI administration of ITF2357 enhances neurobehavioral recovery

Functional recovery was assessed by serial ΔNSS measurements of respective mouse groups (n≥9/group). NSS values at 1 h after injury were similar in all groups and confirmed comparable and moderate severity of injury (pretreatment: NSS 1 h=6.2±0.2 and 6.4±0.2 in vehicle and ITF2357-treated mice, respectively; 1 h postinjury: NSS 1 h=7.0±0.4 and 6.6±0.3 in vehicle and ITF2357 groups, respectively; 24 h postinjury: NSS 1 h=6.6±0.2 in both groups). As shown in Fig. 1, ITF2357 administered 24 h following injury induced a prolonged significant improvement in functional recovery, namely increased ΔNSS. ITF2357, given in accordance with either of the three examined treatment protocols (pretreatment, 1 h, and 24 h postinjury), induced a consistent tendency toward improved recovery; however, whereas pretreatment elicited a nonsignificant tendency at both 3 and 6 d post-CHI (Fig. 1A), administration of ITF2357 at 1 h after traumatic impact (Fig. 1B) led to a transient borderline effect evident at 3 d postinjury (P=0.08) and improved recovery over time, as indicated by a statistically significant improvement evident in the ITF2357 group by 3 d postinjury (P≤0.05 vs. 24 h in the same group) but not in control mice (significance reached only at 6 d postinjury). The effects observed in mice treated with early administration protocols were again observed in the group receiving ITF2357 at 24 h following CHI (Fig. 1C). ITF2357 treatment at this latter time point induced a significant and sustained beneficial effect indicated by increased ΔNSS 6 d after trauma (0.7±0.1 vs. 1.6±0.2 in the vehicle control and ITF2357 groups, respectively; P≤0.01) and at 10 (1.3±0.1 vs. 2.1±0.3; P≤0.05) and 14 d (1.4±0.2 vs. 2.1±0.2; P≤0.01). The beneficial effect subsequently diminished by 21 d postinjury. In light of the pronounced improvement following the administration of ITF2357 24 h postinjury and the potential clinical implications of this belated administration, subsequent experiments were conducted using the 24-h postinjury treatment protocol.

Figure 1.

Postinjury administration of ITF2357 improves neurobehavioral recovery after CHI. ITF2357 was administered either 30 min prior to injury (A) or as postinjury treatment given at 1 h or 24 h after impact (B, C). Neurobehavioral status was assessed using the Neurological Severity Score (NSS), such that an increase in ΔNSS reflects faster recovery. Whereas pretreatment with ITF2357 only tended to improve recovery, a significant beneficial effect was observed in mice administered with the agent after injury as compared to matched vehicle-treated controls. Following administration of ITF2357 at 1 h postinjury, recovery over time was accelerated (B). When the compound was administered at 24 h after injury (C), functional benefit was evident from 6 d after trauma and up to 2 wk postinjury (n≥9 mice/group). Values are means ± se. *P ≤ 0.05, **P ≤ 0.01; Kruskal-Wallis rank test with Dunn’s post hoc test.

ITF2357 reduces TBI-induced neurodegeneration and lesion volume

Neurodegeneration was evaluated using FJB staining of brain sections taken from ITF2357- or vehicle-treated mice (n=4/group). As shown in Fig. 2, FJB-positive cells were mainly located within the dentate gyrus. ITF2357 significantly reduced the number of degenerating cells on d 3 as well as d 21 after injury, as compared to control (14±2 vs. 2±1 FJB-positive square subdivisions/mm² in vehicle and ITF2357 groups on d 3, respectively; and 12±3 vs. 0±0 for vehicle and ITF2357 groups on d 21, respectively; P≤0.001 between groups at both time points). TTC staining was used for measuring lesion volume at 16 h following ITF2357 administration (40 h postinjury). ITF2357 reduced ipsilateral lesion size as compared to vehicle controls (Fig. 3; 17.7±1.0 vs. 13.8±0.76% lesion volume in vehicle- and ITF2357-treated groups, respectively; 22% reduction relative to control; P≤0.01).

Figure 2.

FluoroJade-B histochemistry. Fluoro-Jade B (FJB)-positive cells and processes (arrows) within the hippocampus of the injured hemisphere of vehicle-treated (A, C) or ITF2357-treated (B, D) mice on d 3 (A, B) and d 21 (C, D) postinjury. Data are means ± se (FJB-positive square subdivisions/mm²); n = 4 mice/group. ***P ≤ 0.001 Mann-Whitney U test.

Figure 3.

ITF2357 reduces TBI-induced lesion size. Lesion volume was evaluated 40 h postinjury (16 h after vehicle or ITF2357 injection) using TTC staining. A) Representative stains. B) Lesion volume was reduced in ITF2357-treated mice as compared to vehicle controls. Values are means ± se; n = 7 mice/group. **P ≤ 0.01; Student’s t test.

Effect of ITF2357 on H3 acetylation, HSP70, p-Akt, and p53

We next evaluated the levels of acetylated histone H3 protein to confirm the availability and expected activity of ITF2357 within the injured hemisphere. Acetylated H3 was evaluated at 6 and 24 h following ITF2357 or vehicle injection (30 or 48 h postinjury, respectively; n=4/group), time points at which the effects of other HDAC inhibitors have been reported (15, 16). As shown in Fig. 4A, Ac-H3 levels decreased following injury in vehicle-treated mice, whereas this reduction was less pronounced in ITF2357-treated animals, resulting in preservation of higher levels of acetylated histone at 6 h after injection (Fig. 4A; 71±3 vs. 33±6% reduction in vehicle and ITF2357 groups, respectively; P≤0.01 between the groups at 6 h after injection).

Figure 4.

Effects of ITF2357 on levels of AC-H3 (A), HSP70 (B), and p53 (C). ITF2357 was given 24 h after injury, and mice were sacrificed 6 or 24 h following injection (i.e., 30 or 48 h postinjury, respectively). Protein levels within ipsilateral frontal cortical segments were evaluated using Western immunoblotting. Optical density values relative to β-actin within the same sample were obtained using TINA software. Data are means ± se; n = 4 mice/group. *P ≤ 0.05, **P ≤ 0.01; ANOVA with Bonferroni post hoc test.

The levels of HSP70, p-Akt, and p53 were evaluated in the same mice at both time points. These factors were examined, as changes in their expression have previously been shown to be associated with neuroprotective effects of other HDAC inhibitors in the setting of brain ischemia (15, 16) and in light of the established role of HSP70 and p-Akt in neuroprotection against ischemic or traumatic brain injury (27,28,29,30,31,32). At 6 h after injection, injury was found to induce a significant decrease in HSP70 protein levels in vehicle-treated mice. This reduction was attenuated in the group treated with ITF2357 (Fig. 4B; 45±11 vs. 26±7% decrease at 6 h after injection in vehicle and ITF2357 groups, respectively; P≤0.01 vs. sham in vehicle only). This effect was transient, as similar HSP70 levels were evident in both groups by 24 h after injection. p-Akt levels were only slightly affected by either injury or treatment. A similar trend, namely attenuation of postinjury reduction by ITF2357, was observed but did not reach statistical significance (data not shown). In contrast, the levels of p53 robustly increased following injury. The postinjury increase in p53 levels was more pronounced in mice treated with ITF2357, as reflected by higher protein content vs. vehicle controls at 24 h after injection (Fig. 4C; 0.61±0.14 vs. 1.18±0.14; P≤0.01).

ITF2357 reduces the number and activation of astrocytes within the injured hemisphere and decreases microgliosis at the hippocampus following TBI

Given reports on potent anti-inflammatory properties of ITF2357 (24, 33), the effects of treatment on gliosis, a hallmark of the central nervous system inflammatory response, were examined. Three and 21 d postinjury, there were significantly fewer astrocytes within the injured hemisphere in mice treated with ITF2357 (P≤0.001 on d 3 and P≤0.01 on d 21 post-trauma; n=4/group; Fig. 5) as compared to controls (150±7 vs. 105±5 GFAP-positive cells/mm² in vehicle- and ITF2357-treated groups on d 3 postinjury; 60±4 vs. 48±3 on d 21, respectively). In addition, these cells occupied significantly less tissue surface in the ITF2357-treated group compared to vehicle-treated mice 3 d after trauma (85.8±2.5 vs. 75.9±2.1% covered surface in vehicle and ITF2357 groups, respectively; P≤0.01). However, 21 d after injury, there was no difference in the tissue surfaces between the groups (58.2±3.1 vs. 52.8±2.6% covered surface in vehicle- and ITF2357-treated groups, respectively; P>0.05). Therefore, astrocytes were equally activated in both groups of mice after 21 d, although these cells were present at lower numbers in the case of ITF2357-treated animals.

Figure 5.

GFAP histochemistry. GFAP immunohistochemistry is shown at the striatum of the injured hemisphere in vehicle-treated (A, C) or ITF2357-treated (B, D) animals on d 3 (A, B) and 21 (C, D) following traumatic injury. Numbers of activated astrocytes and the area covered by their processes were reduced in ITF2357-treated mice on d 3 after trauma. On d 21 postinjury, astrocytes were present at lower numbers in animals given ITF2357 as compared to controls, but surface areas did not differ between groups. Data are means ± se; n = 4 mice/group. **P ≤ 0.01, ***P ≤ 0.001; Mann-Whitney U test.

ITF2357 did not affect the total lectin-positive cellular reaction (microglia and infiltrating macrophages) within the injured hemisphere at either time point (192±9 vs. 200±9 and 62±7 vs. 67±7 lectin-positive cells/mm² in control and ITF2357 groups on d 3 and 21 postinjury, respectively; n=4/group; P>0.05, not shown). However, our subanalysis indicated that ITF2357-treated animals exhibited significantly reduced microglial/macrophage immunoreactivity in the underlying hippocampus. As shown in Fig. 6, this difference was evident at 3 d postinjury (P≤0.001) and persisted on d 21 after trauma, though to a lesser extent (P≤0.05).

Figure 6.

Lectin immunoreactivity. Activated microglia/macrophages within the parahippocampal area of vehicle-treated (A, C) and ITF2357-treated (B, D) animals at 3 d (A, B) and 21 d (C, D) after traumatic injury. Data are presented as means ± se; n = 4 mice/group. *P ≤ 0.05, ***P ≤ 0.001; Mann-Whitney U test.

ITF2357 enhances active caspase-3 immunoreactivity at the area of trauma

Study of active caspase-3-positive cells (Fig. 7A, B) revealed that ITF2357 induced a 2-fold increase (P≤0.001) in the amount of these cells within the traumatic area (core and penumbra) as compared to vehicle controls on d 3 after injury (18±2 vs. 39±5 positive cells/mm² in vehicle- and ITF2357-treated groups, respectively; n=4/group). Immunoreactivity was not found within hippocampal areas. In both groups, labeled cells were mainly macrophages or activated microglia, as indicated by coexpression of active caspase-3 and Mac-3 immunoreactivity (Fig. 7C, D). On d 21 post-trauma, cells positive for active caspase-3 were only evident in a restricted area, with very few cells observed in any other brain region. At this time point, caspase-3-positive cells, if present, were only found at the borders of the traumatic scar and were mainly macrophage-like round cells. No differences were found between the two groups at this time point (13±7 and 12±10 positive cells/mm² in vehicle- and ITF2357-treated groups, respectively; n=4/group; P>0.05, not shown). No caspase-3-positive cells were found in areas other than the trauma site at either time point.

Figure 7.

Caspase-3 labeling. Active caspase-3 immunohistochemistry at the area of injury in vehicle-treated (A) and ITF2357-treated (B) animals on d 3 post-trauma. Double immunohistochemistry for active caspase-3 (apoptosis; red fluorescence) and MAC-3 (macrophages/activated microglia; green fluorescence) at the area of trauma in vehicle-treagted (C) and ITF2357-treated (D) animals. Coexpression of both markers was identified using confocal microscopy, suggesting that apoptotic cells were macrophages/activated microglia (yellow cells). Data are means ± se; n = 4 mice/group. ***P ≤ 0.001; Mann-Whitney U test.

DISCUSSION

Several studies have shown the involvement of a disruption of acetylation homeostasis in multiple neurodegenerative conditions. Neurodegeneration has been suggested to be associated with relative overdeacetylation, stemming from a global decrease in HAT activity (2, 5). Consequent to this line of evidence, the ability of HDAC inhibiting compounds to ameliorate damage associated with disorders such as Alzheimer’s disease, intracerebral hemorrhage, neuronal oxidative stress, dentatorubral-pallidoluysian atrophy, amyotrophic lateral sclerosis, and Huntington’s disease was demonstrated (7, 9, 34,35,36,37,38,39,40,41,42). The present study indicates that HDAC inhibitor ITF2357 is beneficial in the setting of CHI, demonstrating for the first time the ability of postinjury HDAC inhibitor treatment to enhance overall functional, neurobehavioral outcome after brain trauma. Of particular significance is the finding of a sustainable benefit when a single dose of ITF2357 is given as late as 24 h following injury. As opposed to immediate post-trauma administration, the interpretation of which is greatly hindered by lack of clinical application, our data may be relevant to the treatment of TBI patients since many arrive late to seek medical attention. Furthermore, our observation that a single dose of ITF2357 24h after injury is sufficient to induce functional and sustained improvement for over a week establishes a prolonged timeframe for treatment and warrants further study of later administration or multiple dosing with this clinically approved drug in the setting of brain injury.

Similar to findings obtained in stroke models, showing the ability of HDAC inhibitors such as valproic acid, sodium butyrate, trichostatin A (TSA), and SAHA to decrease brain lesion volume (15, 16), we observed that ITF2357 decreases lesion volume after CHI. This would indicate that post-TBI administration of the compound not only facilitated improved functional recovery but also reduced tissue damage within hours following injection. The ameliorating effect of ITF2357 at tissue level was further corroborated by its ability to reduce the extent of neurodegeneration.

The neuroprotective properties of HDAC inhibitors have been associated with their ability to restore adequate acetylation levels (34, 37, 43). Particularly, a decrease in histone H3 acetylation following brain injury was demonstrated, alongside beneficial effects brought about by correction of the latter using VPA, sodium butyrate (SB), SAHA, or 4-dimethylamino-N- [5-(2-mercaptoacetylamino)pentyl] benzamide (DMA-PB). Consistent with these reports, we found that CHI induces a pronounced decrease in H3 acetylation in vehicle-treated mice but significantly less so in mice treated with ITF2357. Higher levels of acetylated H3 were evident within the injured ipsilateral cortex of mice 6 h after administration of ITF2357, confirming the availability and expected activity of the compound within the injured hemisphere.

Concurrently, treatment also attenuated the decrease in the levels of HSP70 after injury but only slightly affected p-Akt levels. We examined these factors since both have been shown to peak within hours following ischemic or traumatic brain injury and to be associated with neuroprotective and antiapoptotic effects (27,28,29,30,31,32). Furthermore, postischemic administration of HDAC inhibitors, including VPA, SB, trichostatin A (TSA), and SAHA, has been reported to induce increased expression of HSP70 alongside neuroprotection (15, 16). SB and TSA were shown to also prevent ischemia-induced reduction in p-Akt levels, evident in vehicle-treated animals 24 h after injury induction (16). However, it should be noted in this context that the effects of HDAC inhibitors on both HSP70 and p-Akt were shown to be dose and agent dependent. Specifically, Faraco et al. (15) demonstrated that the extent of the effect of SAHA on HSP70 levels varies with different administered doses and did not observe any effect of this agent on p-Akt levels, as was later shown by Kim et al. (16), when applying SB or TSA. In the current investigation, we evaluated the levels of these factors at later times after injury (30 and 48 h) than in these previous reports, since ITF2357 was given at 24 h post-trauma. Decreased levels of HSP70 and to some extent p-Akt were found in vehicle-treated mice at 30 h after injury (6 h after injection). This is conceivable given the presence of prominent apoptotic processes in the injured hemisphere within this time frame, as described previously (44, 45). As the injury-induced decrease in HSP70 was somewhat attenuated by ITF2357 and given its well-established neuroprotective properties and association with HDAC-inhibitor-induced neuroprotection, a role for maintenance of higher levels of this factor in the benefits afforded by ITF2357 is plausible, yet the moderate and transient nature of the observed effect should be considered when weighing its potential contribution. p-Akt levels were only slightly affected by treatment, suggesting that the effect of ITF2357 on this factor is similar to that of SAHA and is not likely to comprise a predominant mechanism in providing functional benefit.

At low nanomolar concentrations, ITF2357 is a potent anti-inflammatory agent in vitro and in vivo (24, 33, 46). In addition, in a phase II clinical trial in children with active systemic onset juvenile idiopathic arthritis, a daily dose of ITF2357 given for the duration of 12 wk was well tolerated, exhibited no organ toxicity, and achieved a significant (P<0.01) reduction in the systemic feature score and number of painful joins (20). HDAC inhibitors were also shown to convey anti-inflammatory effects in rodent brain injury models, namely reduced cyclooxygenase 2 levels and decreased density of phagocytic microglia (16, 17). Our current observations demonstrate a reduction in lectin-positive microglia/macrophage accumulation within the ipsilateral hippocampus following postinjury ITF2357 administration. Zhang et al. (17) reported no effect of the HDAC inhibitor DMA-PB on resting and activated microglia, whereas phagocytic microglia was reduced within the CA2/CA3 hippocampal region in the treated group following TBI. Consistent with the latter may be considered our observation that microglia/macrophages at the area of trauma are apoptotic following ITF2357 treatment, leading to accelerated clearance of these cells from the injured tissue. We further expand previous findings, as we show that ITF2357 not only reduces microglial accumulation following injury but also decreases the number of astrocytes and their level of activation. Moreover, our data provide new insight into mechanisms that may underlie the evident reduction in microglia numbers, as we found that the reduced levels of these inflammatory cells at 3 d following injury are accompanied by higher abundance of active caspase-3-positive microglial/macrophage cells in the trauma core and penumbra of ITF2357-treated mice. Hence, ITF2357-induced functional neuroprotection is preceded by enhanced microglial apoptosis. Reduced numbers of astroglia and microglia within the hippocampus are an additional indication of attenuated degeneration in this area with ITF2357 treatment. It remains unclear whether the neuroprotection observed within the hippocampus is attributed to a direct effect on hippocampal cells and/or is associated with reduced cytokine activity. However, a reduction in the number of functional (nonapoptotic) microglia/macrophages at the area of trauma may reduce glial proliferation within the underlying ipsilateral hippocampus. One possible mechanism would be ITF2357 inhibition of chemokines, as has been reported in models of inflammatory bowel disease (47, 48). Because ITF2357 enhances the clearance of activated microglia/macrophages from the trauma penumbra via apoptosis, such a mechanism would also result in decreased gliosis.

Certainly, the induction of tumor cell death by HDAC inhibitors, including ITF2357 is the basis for their clinical use in human cancer patients (49, 50). Acetylation of p53 was proposed as a mechanism for tumor cell death by TSA, SAHA, and ITF2357 in human hepatoma cells (51). In that report, cell death was correlated with the acetylation of p53 and formation of nuclear immunocomplexes containing acetylated p53 in conjunction with acetylated forms of histones and HAT proteins. In the experiments reported here, we observed a robust increase in the levels of total p53 within the trauma area following ITF2357 administration. Given the presence of large numbers of glial cells within this anatomical area following injury, the potential involvement of p53 in microglial/macrophage apoptosis with subsequent neuroprotection warrants further study and may account for the beneficial effects of post-trauma administration of ITF2357.

The need for developing new therapeutics for TBI treatment and the current lack of specific therapy for this indication underscore the importance of identification and characterization of novel neuroprotective compounds. The present findings contribute an essential link to the assessment of HDAC inhibitors as potential novel agents for TBI, as we establish neurobehavioral value for such therapy and demonstrate that functional and molecular benefits can be achieved when treatment is given as late as 24 h after impact. We chose to focus on ITF2357 because the compound has an established favorable safety and therapeutic profile in children with systemic onset juvenile idiopathic arthritis. The present experimental findings, demonstrating benefits in a clinically relevant model of CHI, support further consideration of ITF2357 for treatment of TBI.