Retracted: Administration of MS‐275 Improves Cognitive Performance and Reduces Cell Death Following Traumatic Brain Injury in Rats

Summary

Aims

The MS‐275 is a selective inhibitor of class I histone deacetylases (HDACs), which has been reported as a potential strategy in some central nervous system diseases associated with neurodegeneration and disturbed learning. However, its role in traumatic brain injury is not well defined. In this study, we examined the behavioral–cognitive performance as well as histology outcome in adult rats to evaluate whether postinjury administration of MS‐275 (15 and 45 mg/kg) would provide neuroprotection benefits and ameliorate cognitive deficits following fluid percussion injury.

Methods

Traumatic brain injury (˜2.15 ATMs) was produced using a fluid percussion device with the lateral orientation. MS‐275 was administered (15 and 45 mg/kg) systemically once daily for 7 days starting at 30 min after lateral fluid percussion TBI. Acquisition of spatial learning and memory retention was assessed using the Morris water maze (MWM) on days 10–14 after TBI. Brain tissues were collected and stained with Fluoro‐Jade B histofluorescence (for degenerating neurons) at 24 h after injury and cresyl violet (for long‐term neuronal survival) on day 14 postinjury.

Results

Behavioral outcome after TBI revealed MS‐275 treatment groups, at all doses examined, performed significantly better in the Morris Water Maze (P < 0.001). Acute histology analysis demonstrated that 45 mg/kg MS‐275 significantly reduced the number of degenerating neurons in the ipsilateral CA2–3 hippocampus at 24 h postinjury (P = 0.007). There was a trend for MS‐275 to increase the survival of neurons in the CA2–3 hippocampus on 14 days after TBI (P = 0.164).

Conclusion

Our present data highlight the fact that MS‐275 may provide neuroprotective effect and improve cognitive performance after TBI. We concluded that MS‐275 is a potential novel treatment and will have an ameliorative effect on some of the pathological features associated with TBI.

Introduction

Traumatic brain injury (TBI) is a major public health issue and the leading cause of death and disability worldwide. Despite considerable progresses were made in the field of research and treatment of TBI, it is still a serious and complex clinical problem that affects approximately 1.7 million people each year in the United States 1, 2. TBI produces a broad spectrum of symptoms and disabilities, and the pathological consequences of TBI can be variable including diffuse axonal injury, ischemia, hemorrhages, cell death, brain swelling, and inflammation 3. Although the prevalence and increased recognition of TBI and the mechanisms of TBI‐induced brain injury are still far from understood, no effective treatments have been applied yet.

The acetylation of histones plays an important role in remodeling chromatin structure and regulating gene expression. Histone acetylation involves the reversible actions of the histone acetyltransferases (HAT) and histone deacetylases (HDACs). Histone acetylation is associated with more transcriptional activity, while histone deacetylation is associated with preventing transcription and repression of gene expression 4, 5. Therefore, the action of the HDAC inhibitors is to promote the posttranslational acetylation status of histones by modulating the condensation status of chromatin to control gene expression 6. In addition, HDAC inhibitors may also exert antiinflammatory effects through acetylation of nonhistone proteins 7. Much of the research has focused on HDAC inhibitors, where several agents have demonstrated in vitro and in vivo their neuroprotective effects in models of central nervous system disorders including ischemic stroke 8, 9, Huntington's disease 10, 11, and memory deficits or disturbed learning 12, 13, 14. In the previously published experiments 15, showed HDAC6 inhibitor DMA‐PB reduced the number of degenerating neurons in the ipsilateral CA2–3 hippocampus 24 h after TBI. Together, these data support the hypothesis that HDAC inhibitors may play a neuroprotective role both in acute neurological diseases and in chronic degenerative neurological diseases. This effect may be responsible for their potential therapeutic benefits for some pathological features associated with TBI.

A previous research experiment in moderate pediatric TBI model has revealed a decrease in histone H3 acetylation and methylation in hippocampus CA1–3 region during the first hours to days postinjury 16. A novel HDAC inhibitor, pyridin‐3‐ylmethyl N‐[[4‐[(2‐aminophenyl) carbamoyl] phenyl] methyl] carbamate (MS‐275), was reported as a potent, long‐lasting brain region‐selective inhibitor of class I HDACs 17. In vivo studies showed that MS‐275 demonstrated therapeutic benefits in various types of malignancies, as well as promising antiinflammatory activities 18, 19. Recent study has noted that MS‐275, as a potent brain region‐selective HDAC inhibitor, exhibited the blood–brain barrier penetration and can be effective in increasing brain Ac‐H3 levels in rodent brain with a maximal effective dose of 120 μmol/kg (~45 mg/kg) and did not have significant toxicity 17, 20. Here, we will investigate the neuroprotective potential of postinjury administration of MS‐275 (15 mg/kg and 45 mg/kg) on improving cognitive deficits as well as reduction in TBI‐induced acute neuronal degeneration and longer‐term neuronal cell loss in clinically relevant animal TBI models. The employed dosing intervals and injection mode in this study were chosen daily for 7 days with i.p. injection. Results were assessed using histology techniques and behavior analysis to evaluate the therapeutic efficacy of MS‐275 for TBI.

Materials and Methods

Animals

Adult male Sprague‐Dawley rats (N = 78) weighing 300 ± 20 g were used in this study. Animals were housed in individual cages at a controlled room temperature (22°C) and humidity‐controlled (50% relative) animal facility with a 12‐h light/dark cycle. Food and water were readily available and accessible. Animals were acclimated to the animal facility at least 7 days prior to TBI surgery.

Experimental Design

Investigators were blind to the injury and treatment for all experiments included in this study. MS‐275 was purchased from Sigma‐Aldrich, Inc. (St. Louis, MO, USA) and dissolved in DMSO. Two doses (15 mg/kg and 45 mg/kg) or an equal volume of DMSO was injected intraperitoneally (i.p.) at a volume of 1 mL/kg. Animals were randomly subjected to four groups (sham + vehicle, n = 12; TBI + vehicle, n = 20; TBI + MS‐275 [15 mg/kg], n = 20; TBI + MS‐275 [45 mg/kg], n = 20, respectively). Surgery and moderate brain injury were produced on day 0.

Experiment 1: To evaluate the effect of MS‐275 on TBI‐induced acute neuronal degeneration at 24 h postinjury, three groups of animals were subjected to TBI for 30 min followed later by one of two doses of MS‐275 (15 and 45 mg/kg) or an equal volume of DMSO vehicle (n = 8, each group). A fourth group of sham–TBI animals (n = 6) was administered an equal volume of DMSO 30 min after sham–TBI. Animals were euthanized at 24 h after surgery, and brains were removed and stained with Fluoro‐Jade B histofluorescence to analyze acute neuronal degeneration.

Experiment 2: To evaluate the effect of MS‐275 on TBI‐induced cognitive deficits and progressive neuronal death, four groups of animals (n = 12/TBI group; n = 6 Sham–TBI) were administered either one of two doses of MS‐275 (15 and 45 mg/kg) or an equal volume of DMSO vehicle once daily for 7 days starting at 30 min after lateral fluid percussion TBI or Sham–TBI. A fifth group of sham‐injury animals (n = 6) were given MS‐275 (45 mg/kg) at the same dosing interval. All groups were tested on the Morris water maze (MWM) on postinjury days 10–14. Animals were euthanized, and brains were collected for staining with cresyl violet to analyze neuronal survival following the final MWM trial.

Surgical Procedure

Rats were initially anesthetized with 4% isoflurane in a 2:1 nitrous oxide/oxygen mixture, intubated, and mechanically normoventilated with a rodent volume ventilator (Harvard Apparatus model 683, Holliston, MA, USA) with anesthesia reduced to 2% for maintenance. Depth of anesthesia was continuously monitored throughout the duration of surgery. Rats were mounted in a stereotaxic frame, a scalp incision was made along the midline, and a 4.8‐mm‐diameter craniotomy was performed with a trephine on the right parietal bone (centered at 4.5 mm posterior from bregma and 3.0 mm right lateral to the sagittal suture). A rigid plastic injury tube (modified Luer‐loc needle hub, 2.6 mm inside diameter) was secured over the exposed intact dura with cyanoacrylate adhesive. Two skull screws (2.1 mm diameter, 6.0 mm length) were placed into burr holes, 1 mm rostral to bregma and 1 mm caudal to lambda. The assembly was secured to the skull with cranioplastic cement (Plastics One, Roanoke, VA, USA). Rectal temperature was continuously monitored and maintained within normal ranges (37 ± 0.5°C) throughout the surgical procedure by a feedback temperature controller pad (CWE model TC‐1000, Ardmore, PA, USA). Brain temperature was also measured (Physitemp model TH‐5, Clifton, NJ, USA) by insertion of a needle temperature probe (Physitemp model MT‐29/2) between the skull and temporalis muscle pre‐ and post‐TBI.

Induction of Traumatic Brain Injury

Lateral fluid percussion (LFP) TBI was induced using a fluid percussion device (VCU Biomedical Engineering, Richmond, VA, USA) 21 using the lateral orientation 22. The device consists of a Plexiglas cylindrical reservoir filled with isotonic saline. One end of the reservoir has a Plexiglas piston mounted on O‐rings, and the opposite end has a transducer housing with a 2.6‐mm‐inside‐diameter male Luer‐loc opening. Upon injury, the rat was disconnected from the ventilator and the injury tube connected to the fluid percussion cylinder. TBI was induced by the descent of a pendulum striking the piston, which injects a small volume of saline epidural into the closed cranial cavity, producing a brief displacement and deformation of neural tissue. The pressure pulse was measured in atmospheres (ATM) by an extracranial transducer (model EPN‐0300A*‐100A; Entran Devices, Inc., Fairfield, NJ, USA) and recorded on a digital storage oscilloscope (model TDS 1002; Tektronix Inc., Beaverton, OR, USA). Moderate TBI is considered to be within the range of 2.12–2.18 ATM. Immediately after TBI, the rat was reventilated with a 2:1 nitrous oxide/oxygen mixture in the absence of isoflurane. The plastic injury tube and skull screws were removed, and the scalp incision was closed with 4.0 braided silk sutures. As soon as spontaneous breathing was observed, the animal was disconnected from the ventilator and transferred to a warm cage for recovery. The righting reflex was assessed by placing the rat in a supine position at regular intervals (~30 seconds) to test the rat's ability to spontaneously recover to a prone position. The duration of suppression of the righting reflex was used as an additional indicator of traumatic unconsciousness and injury severity. The TBI–sham control group underwent all surgical and anesthesia procedures described above, except no fluid pulse was delivered to the brain.

Postoperative Care and Observation

Each postinjury animal was housed in individual cage containing the information of animal ID and date of surgery and remained on the heating pad (30–32°C) in the vivarium until it recovered baseline weight (~3–4 days). The postoperative record sheet for each animal was filled and placed under each animal cage. Wound was observed and cleaned before stitch removal at day 7 after the operation. A new food tray soaked with water was placed inside a cage, and body weight and percent body weight loss were recorded each day up until prior to perfusion. The teeth of the rats that lost weight more than 10 g each day will be checked and administrated 6 cc Ringers to maintain adequate hydration until they started to gain weight. The animal had to be euthanized when it lost greater than 20% of pre‐injury body weight or appeared moribund.

Behavioral Analysis

On days 10–14 post‐TBI, spatial learning and memory were tested by using the Morris water maze (MWM) behavior paradigm. The maze is a large circular pool (220 cm diameter by 60 cm high). A transparent Plexiglas escape platform (12.8 cm diameter by 20 cm high) was placed 2 cm below the water (24–28°C). Rats were randomly placed in one of four directions (N, E, S, W) facing the wall first and were required to find the escape platform. A total of four trials per day were performed across five consecutive days. Rats were allowed a maximum of 120 seconds to find the escape platform. In the event a rat failed to find a platform within the allotted time limit, an experimenter helped guide the rat to the platform. All rats remained on the platform for 30 seconds before being removed from the maze. On the fifth training day, a probe test was run by removing the escape platform. Rats were allowed 60 seconds to navigate to an area containing the removed hidden platform. In addition, visual acuity was assessed by allowing the animals to locate a black flag mounted on a raised platform (1 cm above water). The latency and distance to find the platform were tracked with a video tracking system (Poly‐Track Video Tracking System version 2.1, San Diego Inst., San Diego, CA, USA).

Tissue Collection and Sectioning

Rats were deeply anesthetized with 4% isoflurane and given a dose of pentobarbital (100 mg/kg, i.p.). The rats were perfused transcardially with 100 mL of 0.1 M sodium phosphate buffer (PB; pH = 7.4), followed by 200 mL of 4% paraformaldehyde (PFA; pH = 7.4). Brains were removed and stored overnight in 4% paraformaldehyde at 4°C. Tissues were cryoprotected in 10% sucrose for 1 day followed by 2–3 days in a 30% sucrose solution and then frozen on powdered dry ice. Coronal sections (−2.12 to −4.80 mm bregma; [45 μm] were cut with a sliding microtome [American Optical, Model 860]). Systematic random sampling techniques were used for selecting tissue sections for mounting and staining. Every fifth section for a total of seven sections per brain was sampled starting at a section randomly determined from the first through fifth rostral‐most sections. The tissue sections were then mounted onto gelatin‐coated slides using a 1:1 distilled water/0.1 M PB mixture and allowed to dry.

Histology

Acute Neuronal Degeneration (Experiment 1)

Fluoro‐Jade‐B, as a histofluorescent marker, is commonly used to label degenerating neurons in the CA2–3 region of the ipsilateral hippocampus at 24 h after injury.

Tissue sections were mounted on 1% gelatin‐coated slides and air‐dried overnight. The slide‐mounted tissue sections were subsequently immersed in 100% alcohol (5 min), 70% alcohol (5 min), 50% alcohol (3 min), dH₂O (2 min), 0.006% potassium permanganate (15 min shaking) and dH₂O (1 min). The sections were then incubated in 0.001% Fluoro‐Jade B staining solution in 0.1% acetic acid for 30 min at room temperature, rinsed again in dH₂O (1 min×3) and air‐dried. Finally, the sections were immersed in xylene and mounted on glass slides with DPX mounting medium. Sections stained with Fluoro‐Jade B were examined under UV light with a FITC fluorescence filter cube (Nikon B‐2A, Tokyo, Japan).

Long‐Term Neuronal Cells Survival (Experiment 2)

Cresyl violet (CV) staining is an effective and reliable stain used for long‐term neuronal survival analysis associated with TBI. Tissue sections were dehydrated and defatted by immersion in 70% (2 min ×1), 95% (2 min ×2), 100% (2 min ×2) ethanol at room temperature and in xylene for 16 min. They were then placed sequentially in 100% (2 min ×2), 95% (2 min ×2), and 70% (2 min ×1) ethanol, followed by 30 seconds in dH₂O. Slides were stained in 0.1% cresyl violet acetate for 6 min then rinsed for 15 seconds in dH₂O (2×), 4 min in 95% alcohol with acetic acid, and 30 seconds each in 95% (2×) and 100% (2×) ethanol and xylene (5 min ×2). The slides were covered with Permount (Fisher Scientific, Hampton, NH).

Anatomical Regions of Interest and Stereological Cell Counts

The region of interest for measurement of surviving neurons encompasses the stratum pyramidale of the hippocampus CA2–3 bounded on one end by its entry into the dentate gyrus at the lateral tips of the dorsal and ventral blades of the granule cells and at the other end by the narrowing of the stratum pyramidale at the boundary of the CA2 to CA1. Cell counting was made with the aid of a microscope (Nikon E600, Nikon) connected to a motorized stage (Bioprecision2, Ludl Electronic Products, Inc., Hawthorne, NY, USA) and computer software (Stereo Investigator^TM 8.0, Microbrightfield, Inc., Williston, VT, USA). The region of interest for CA2–3 pyramidal neurons was traced under 4X, and neuronal counting and identification were quantified with a 100 × oil objective. The criterion for selection and quantification of neurons was a morphologically distinct cell body with at least one clearly identifiable dendrite.

Statistical Analysis

The data in the text and graphs were expressed as mean ± standard error of the mean (SEM). The data in the Table were expressed as mean ± standard deviation. Data analysis was performed using SPSS satistical software (Version 17.0, SPSS Inc., Chicago, IL, USA), which adheres to a general linear model. Alpha level for type I error was set at 0.05 for rejecting null hypothesis. Differences between body weights, injury magnitudes, righting times, and temperature measurements were analyzed using separate one‐way analysis of variance (ANOVA) between groups. Behavioral measurement for MWM latency was analyzed using repeated‐measures ANOVA with assessment days as the repeated variable within subjects followed by Dunnett's post hoc analysis. The numbers of either acute degenerating neurons or long‐term surviving neurons in the CA2–3 of the ipsilateral hippocampus were analyzed using one‐way ANOVA followed by Dunnett's post hoc analysis.

Results

There were no significant differences between groups in mean injury magnitude, initial mean body weight, and rectal or temporalis muscle temperature values during surgery and post‐TBI. As expected, the mean righting time of sham–TBI group was significantly less compared with all TBI groups (P < 0.05) (Table 1). Although there was no significant difference in percent mortality following TBI in each group, a total of 9 animals died or had to be out of study (TBI + vehicle, n = 3; TBI + MS‐275 [15 mg/kg], n = 2; TBI + MS‐275 [45 mg/kg], n = 4, respectively) after TBI for the following reasons: fluid leak during induction of TBI (TBI + vehicle, n = 1; TBI + MS‐275 [15 mg/kg], n = 1); appeared moribund after injury (TBI + vehicle, n = 2; TBI + MS‐275 [45 mg/kg], n = 1); and weight loss greater than 20% post‐TBI (TBI + MS‐275 [15 mg/kg], n = 1; TBI + MS‐275 [45 mg/kg], n = 3).

Table 1.

Groups and injury parameters in experiments (Value are mean ± SD)

Treatment groups	Sample (n)	Body weight (g)		Injury magnitude (ATM)	Righting time (seconds)	Rectal temp (°C)		Temporalis temp (°C)
		Pre‐TBI	Pre‐WMW			Pre‐TBI	Post‐TBI	Pre‐TBI	Post‐TBI
Experiment 1: Acute histology study
Sham–TBI, Vehicle	6	305 ± 12			183 ± 24a	37.2 ± 0.4		36.2 ± 0.2
TBI, Vehicle	8	315 ± 13		2.13 ± 0.03	517 ± 68	37.3 ± 0.4	37.1 ± 0.3	36.3 ± 0.3	36.1 ± 0.3
TBI, MS‐275 15 mg/kg	8	307 ± 9		2.14 ± 0.02	525 ± 49	37.0 ± 0.2	37.0 ± 0.4	36.2 ± 0.2	36.1 ± 0.1
TBI, MS‐275 45 mg/kg	8	309 ± 11		2.13 ± 0.02	509 ± 66	37.2 ± 0.2	37.0 ± 0.3	36.2 ± 0.3	35.9 ± 0.4
Experiment 2: MWM and Long‐term histology study
Sham, Vehicle	6	307 ± 7	328 ± 12		176 ± 17a	37.3 ± 0.2		36.0 ± 0.4
Sham, MS‐275 45 mg/kg	6	304 ± 9	331 ± 14		180 ± 19a	37.0 ± 0.4		36.1 ± 0.2
TBI, Vehicle	9	308 ± 10	297 ± 13	2.13 ± 0.02	496 ± 60	37.1 ± 0.3	37.1 ± 0.4	36.1 ± 0.2	36.0 ± 0.2
TBI, MS‐275 15 mg/kg	10	309 ± 11	296 ± 16	2.15 ± 0.01	507 ± 43	36.9 ± 0.4	37.0 ± 0.3	36.1 ± 0.4	35.9 ± 0.4
TBI, MS‐275 45 mg/kg	8	312 ± 14	294 ± 21	2.13 ± 0.03	512 ± 71	37.2 ± 0.3	36.9 ± 0.4	36.2 ± 0.1	36.0 ± 0.3

^a P < 0.05 was considered statistically significant as compared to all TBI groups.

Acute Neuronal Degeneration (Experiment 1)

Degenerating neurons were observed in the hippocampus of the ipsilateral CA2–3 region with Fluoro‐Jade B at 24 h postinjury using stereological techniques. Positive degenerating neurons were quantified as brightly fluoresced, and somas appeared large with extensive dendritic arborization into the stratum radiatum. Gross visual inspection at 2× objective indicated that moderate lateral fluid percussion injury produced significant numbers of FJ‐B‐positive cells in the injured hippocampus region compared with sham–TBI. For the group of TBI + vehicle, FJ‐B staining showed a more noticeable and extent cell injury than we observed with other two MS‐275‐treated groups (Figure 1A–D). Analysis of cell quantification at 40 × objective revealed the number of Fluoro‐Jade‐positive ipsilateral degenerating neurons was significantly different among the vehicle‐ and drug‐treated groups [F _(2,21) = 10.12, P < 0.05]. Administration of MS‐275 at 45 mg/kg significantly reduced the number of degenerating neurons in the CA2–3 region as compared to vehicle‐treated control group [t ₍₁₄₎ = 3.82, P = 0.007] (Figure 1E). There was no Fluoro‐Jade B‐positive neuron that was detected in the contralateral and dorsal hippocampus in any groups at 24 h after postinjury (data not shown).

Figure 1.

Fluoro‐Jade B (FJ‐B) histofluorescence of degenerating neurons stained at 24 h. Coronal sections (−3.6 mm Bregma) of the dorsal hippocampal CA2–3 region: (A) There were no detectable F‐J B‐positive neurons in the sham group. (B) A single moderate TBI injury produced robust FJ‐B‐positive neurons in the ipsilateral (right) CA2–3 region. Treatment with MS‐275 at dose of 15 mg/kg (C) or 45 mg/kg (D) produced scattered FJ‐B‐positive cells than vehicle control group. (E) Quantification of degenerating neurons in the hippocampus using stereological techniques. Administration of MS‐275 at 45 mg/kg significantly reduced the number of ipsilateral degenerating neurons in the hippocampus CA2–3 region at 24 h after TBI. Values are means ± SEM. *P < 0.05 compared with vehicle control. Calibration bars: A–D = 100 μm.

Behavior Test and Long‐Term Neuronal Survival (Experiment 2)

Change of Body Weight Postinjury

There was no significant difference in initial mean body weight on the day of TBI in all groups (Table 1). Rats following moderate brain injury had significant more body weight loss over the 14 days after TBI compared with sham‐injury groups [F _(4,33) = 57.34, P < 0.001]. There was no difference in the rate of weight changing for sham‐injury animals with or without MS‐275 treatment (P>0.05). Rats of all TBI groups lost weight rapidly for the first 4 days after injury and can recover to pre‐injury body weight afterward. No significant difference was found in the changing of body weight over continuous 14 days postinjury in all TBI groups [F _(2,24) = 0.149, P = 0.862]; however, there was a slight trend that higher dose (45 mg/kg) of MS‐275 treatment produced more weight loss. Mean body weights (±SEM) on day 14 post‐TBI were 297.2 ± 13.4 g, 296.4 ± 15.7 g, and 294.1 ± 20.7 g for the group of TBI + vehicle, TBI + MS‐275(15 mg/kg), and TBI + MS‐275(45 mg/kg), respectively (Figure 2).

Figure 2.

Body weight loss/growth curves over 2 weeks after lateral fluid percussion TBI or sham. TBI produced significantly greater weight loss compared with sham–TBI. There was no significant difference between TBI groups regardless of treatment. All TBI groups had rapid body weight loss for the first 4 days as well as nearly identical growth rates after TBIs. Values are means ± SEM. Values are means ± SEM. *P < 0.001 repeated‐measures ANOVA.

Morris Water Maze assessment

Traumatic brain injury distinctly affected the acquisition MWM performance and produced obviously cognitive deficits when compared with the sham–TBI group. Our data indicated that vehicle‐treated group had a significant cognitive deficits as compared to both MS‐275 treatment groups over all test days [F _(2,24) = 25.39, P < 0.001]. The MS‐275‐treated group also had significantly shorter latency to find the hidden platform on the last day in the maze (day 14) than the vehicle control group [t ₍₁₇₎ = 3.66, P = 0.004 (15 mg/kg); t ₍₁₅₎ = 3.07, P = 0.002 (45 mg/kg)] (Figure 3A). Additionally, performance over the 5 days of testing was not significantly different between two sham‐injury groups regardless of MS‐275 treatment [F _(1,10) = 0.024, P = 0.871] (Figure 3B). The swim speed (sham + vehicle = 30.3 ± 2.2 cm/seconds; sham + MS‐275(45 mg/kg) = 29.9 ± 1.9 cm/seconds; TBI + vehicle = 28.6 ± 3.0 cm/seconds; TBI + MS‐275(15 mg/kg) = 29.4 ± 1.3 cm/seconds; TBI + MS‐275(45 mg/kg) = 28.7 ± 2.4 cm/seconds, respectively) revealed no difference between groups [F _(4,34) = 0.157, P > 0.05].

Figure 3.

Acquisition of spatial learning performance and memory retention on the MWM over days 10–14 after TBI: (A) Sham–TBI group had the best cognitive performance with the shortest latency to find the platform. Both of the MS‐275 treatment groups had better overall performance and significantly shorter latencies to find the hidden platform on last day of testing than the vehicle group (repeated‐measures ANOVA).Values are means ± SEM. *P < 0.05 compared with the vehicle control on day 14. (B) Performance over the 5 days of testing was not significantly different between two sham‐injury groups regardless of MS‐275 treatment. (C) The Sham–TBI group spent significantly more time in the target quadrant (Quad 1) compared with the vehicle‐treated group. The dashed line represents chance performance (15 seconds). Values are means ± SEM. *P < 0.05 compared with the sham group.

Following the final maze trail on the last day (day 14), we removed the platform, and spatial memory retention measured with probe trial revealed that the two sham–TBI groups spent significantly longer time in the target quadrant compared with the vehicle control group (P < 0.05). The MS‐275‐treated groups showed better preference than vehicle‐treated group, and there was no difference between two doses of MS‐275‐treated groups in time spent in the platform quadrant (MS‐275 15 mg/kg, 20.6 ± 2.3 seconds vs. MS‐275 45 mg/kg, 19.9 ± 1.7 seconds) (Figure 3C). Additionally, the visual acuity test indicated no difference between groups (sham–TBI = 7.2 ± 1.1 seconds; sham + MS‐275 [45 mg/kg] = 7.2 ± 1.8 cm/seconds; TBI + vehicle = 7.7 ± 1.5 cm/seconds; TBI + MS‐275 [15 mg/kg] = 7.7 ± 1.1 cm/seconds; TBI + MS‐275 [45 mg/kg] = 7.0 ± 1.4 cm/seconds, respectively) suggesting the animals in all groups had a normal visual processing and the difference in the acquisition cognitive performance was not due to impaired vision.

Histology Measures

Long‐term surviving neurons stained with cresyl violet were counted in the ipsilateral CA2–3 region of hippocampus using stereological techniques on day 14 after TBI (Figure 4A–D). Analysis of the mean numbers of neuronal cells revealed that TBI significantly produced progressive neuronal cell death in the ipsilateral CA2–3 region, and all TBI groups had significantly fewer survival neurons compared with the sham–TBI group [F _(4,33)=47.64, P < 0.05]. There was no significant difference in numbers of surviving CA2–3 pyramidal neurons for any TBI group regardless of MS‐275 treatment [F _(2,24)=1.955, P = 0.164]. There was a trend, however, for increasing the total numbers of surviving neurons in both the MS‐275 treatment groups compared with vehicle control group at day 14 postinjury (Figure 4E). Moreover, there was no positive relationship between the dose of MS‐275 treatment and the numbers of survival neurons (MS‐275 15 mg/kg, 82,693 ± 1804 vs. MS‐275 45 mg/kg, 83,501 ± 2071).

Figure 4.

Stereological quantification of cresyl violet‐stained neurons in the ipsilateral CA2–3 region of hippocampus at 14 days postinjury. (A) Gross pathology of ipsilateral parietal cortex. (B) Cresyl violet staining of ipsilateral hippocampus (Sham‐injury, 4X). (C) Representative section from the ipsilateral hippocampus CA2 region (Sham‐injury, 100X oil). (D) Representative section from the ipsilateral hippocampus CA3 region (Sham‐injury, 100X oil). (E) TBI significantly produced neuronal death in the ipsilateral CA2–3 region with or without MS‐275 treatment (P < 0.05). Values are means ± SEM. *P < 0.05 compared with the sham–TBI group. Calibration bars: B = 500 μm; C, D = 20 μm.

Discussion

In this study, we evaluated the neuroprotective potential of a novel class I HDAC inhibitor, MS‐275, on behavioral performance and histological outcome of acute neuronal degeneration as well as long‐term neuronal survival in ipsilateral hippocampus following a moderate lateral fluid percussion TBI in rats. Two doses (15 and 45 mg/kg) or an equal volume of vehicle was delivered systemically (i.p.) each daily for 7 days starting at 30 min after TBI. Our data demonstrated that moderate TBI produced obviously learning and memory deficits and resulted in a significant increase in the number of acute neuronal degeneration and progressive neuronal death with or without MS‐275 treatment. The MS‐275 treatment, however, at all doses examined reduced acute neuronal degeneration with the higher dose (45 mg/kg) producing significant reduction in neuronal degeneration in the CA2–3 region at 24 h after TBI compared with vehicle control group. Results of experiment 2 indicated that MS‐275 treatment exhibited significantly better overall as well as terminal cognitive performance over the 5 days of MWM training. Administration of MS‐275 also showed a trend to increase numbers of surviving neurons in the ipsilateral hippocampus at 14 days postinjury. Group difference in behavioral and histological outcome was not likely due to difference in the amount of force transmitted to the brain as all TBI groups received equivalent injury magnitudes and had no difference in righting times. These data highlighted the hypothesis that MS‐275 could be a potential therapeutic approach for some pathological features following TBI.

Traumatic brain injury is a complex pathological process characterized by acute excitatory cell death occurring within hours after injury and delayed apoptotic cell death occurring days to weeks after injury 23. There are heterogeneous mechanisms responsible for cellular death or dysfunction after TBI involving glutamate neurotoxicity, inflammation, production of free radicals, calcium overload, and many more 24. The two primary mechanisms for the inflammatory cascade are releasing of upstream proinflammatory cytokines 25 and activation of microglia after TBI 26, which can exacerbate other pathophysiological processes associated with injury 27. Considerable previous studies have reported that HDAC inhibitors exerted neuroprotective effects by preventing microglia activation and antineuroinflammatory effects in some central nervous system (CNS) diseases 9, 15, 28, 29, 30. Meanwhile, it has been generally accepted that change in epigenetic gene expression, which is related to histone posttranslational modification, has an equally prominent role in brain cognitive function after TBI 31, and a series of HDAC inhibitors have showed functional benefits to attenuate cognitive deficits in some models of cognitive dysfunction 32, 33.

The primary stage of TBI is characterized by acute neuronal degeneration occurring within hours after injury following some bimolecular and cellular changes due to direct damage and distortion of brain tissue associated with initial injury forces 23. Fluoro‐Jade is an anionic fluorochrome used as a marker for acute degenerating neurons resulting from experimental TBI 34, 35, 36, 37. It has high specific affinity for neurons undergoing degeneration and is able to selectively stain them in their entirety, including cell bodies, distal dendrites, axons, and terminals 38, 39. As much, this effect is not affected by administration of membrane‐permeabilizing drugs 40. The 24 h after TBI is the optimal time point capable to detect maximal degenerating neurons based on previous experimental TBI researches that examined neuronal degeneration from hours to days postinjury. Consistent with previous studies 36, 37, 38, 41, 42, the present findings showed a notable increase in the number of FJ‐B‐positive neurons in the ipsilateral hippocampus CA2–3 region at 24 h following TBI compared with the sham group. Inhibition of HDAC activity with MS‐275 showed a dose response with the most effective dose (45 mg/kg) providing significant reduction in the numbers of degenerating neurons when compared with vehicle control group indicating that administration of MS‐275 specifically attenuated the TBI‐induced reduction in neuronal degeneration assessed at 24 h postinjury and had potential therapeutic benefits for some pathological process related to acute excitatory cell death following lateral fluid percussion TBI by selectively modulating histone acetylation in brain.

Changes in body weight, which is characteristic of TBI in rats, could be due to many different influences including appetite, food intake, bowel excretion, and side effect of drug treatment 43, 44. The body weight is an indicator of general health of the animal. Data in our study clearly indicated that all animals of TBI groups lost similar and considerable body weight over the first 4 days after TBI and had almost identical rates of weight gain over days 4–16 postinjury period, suggesting the employed doses and dosing intervals (each daily) of MS‐275 treatment in the present study did not significantly affect the postinjury body weight with unwanted systemic side effects.

The Morris water maze is a widely used task in behavioral neuroscience to analyze the psychological processes and mechanisms of spatial learning and memory function 45, 46, 47. The results of the present study were not influenced by differences in swim speed or visual processing between groups, indicating that motor swim deficits and visual acuity were not confounding factors in performance between groups. The present analysis revealed sham–TBI resulted in very little or no measurable cognitive deficit, performance over the last 3 days of testing was not significantly different between the sham control and MS‐275 treated groups. However, only the animals in vehicle‐treated group showed a significant poorer cognitive deficit over all 5 days of testing. Using repeated‐measures ANOVA indicated there was a trend for the MS‐275‐treated group to have a steeper slope of learning over days 10–14 after injury, and it showed a significant shorter latency to find the hidden platform on the last day of test (day 14) compared with the vehicle control group. Additionally, animals treated with MS‐275 spent more time in the target quadrant after the final MWM acquisition trials. Taken together, our data revealed that MS‐275 treatment in the present study significantly attenuated cognitive deficits and improved spatial memory retention compared with the vehicle‐treated groups in experimental lateral fluid percussion TBI model in rats.

Progressive neuronal death in the hippocampal CA2–3 region following brain injury has been documented as a hallmark of head injury in humans as well as in experimental animal TBI models 48, 49, 50. Long‐term neuronal death was detected in the ipsilateral hippocampus CA2–3 at 14 days after TBI on a subset of cresyl violet‐stained brains using stereological counting technique. Quantification of surviving neurons revealed a trend for increased numbers of surviving neurons in the MS‐275‐treated group compared with the vehicle‐treated group. The modest effects on long‐term neuronal survival indicated that cognitive outcomes may be more related to neuron function rather than neuron survival. Additionally, it may be related to the fact that there are some different members of histone deacetylases including HDAC6 and HDAC10 15, 51 in the brain except for class I HDACs, which may not be regulated by the selective inhibitor of class I HDAC, MS‐275, employed in the present study. Previous experimental TBI studies have demonstrated behavior improvements, but lack of benefits on long‐term survival neurons 38, 52, are in accordance with our present results that MS‐275 attenuated cognitive deficits without a significant reduction in dying neurons following TBI. The trend for reduced long‐term neuronal loss coupled with the significant neuroprotective effects on MWM performance as well as the TBI‐induced reduction in acute neurodegeneration after treatment with MS‐275 also suggested a certain link between these pathophysiological processes associated with TBI.

Conclusion

Our present study demonstrated significant reduced acute neurodegeneration and improvement in MWM cognitive performance and a trend to increase long‐term survival of neuronal cell following the treatment with the novel selective class I HDAC inhibitor MS‐275 compared with vehicle control group after lateral fluid percussion TBI in rats. These data suggested that treatment with MS‐275 may have potential neuroprotective benefits on some of the pathological features following TBI. Further works are required to understand the underlying specific mechanisms and relationship between histone acetylation, acute neurodegeneration, and neurobehavioral performance following TBI.

Conflict of Interest

The authors declare no conflict of interest.