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Rosiglitazone attenuates inflammation and CA3 neuronal loss following traumatic brain injury in rats
Biochemical and Biophysical Research Communications 472 (2016) 648e655
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Rosiglitazone attenuates inflammation and CA3 neuronal loss following traumatic brain injury in rats Hao Liu a, b, Marie E. Rose a, b, Sherman Culver a, c, d, Xiecheng Ma a, c, d, C. Edward Dixon a, c, d, Steven H. Graham a, b, * a
Geriatric Research Educational and Clinical Center, V.A. Pittsburgh Healthcare System, PA, USA Department of Neurology, University of Pittsburgh School of Medicine, PA, USA Department of Neurosurgery, University of Pittsburgh, PA 15216, USA d Department of Critical Care Medicine, University of Pittsburgh, PA 15216, USA b c
a r t i c l e i n f o
a b s t r a c t
Article history: Received 23 February 2016 Accepted 3 March 2016 Available online 3 March 2016
Rosiglitazone, a potent peroxisome proliferator-activated receptor (PPAR)-g agonist, has been shown to confer neuroprotective effects in stroke and spinal cord injury, but its role in the traumatic brain injury (TBI) is still controversial. Using a controlled cortical impact model in rats, the current study was designed to determine the effects of rosiglitazone treatment (6 mg/kg at 5 min, 6 h and 24 h post injury) upon inflammation and histological outcome at 21 d after TBI. In addition, the effects of rosiglitazone upon inflammatory cytokine transcription, vestibulomotor behavior and spatial memory function were determined at earlier time points (24 h, 1e5 d, 14e20 d post injury, respectively). Compared with the vehicle-treated group, rosiglitazone treatment suppressed production of TNFa at 24 h after TBI, attenuated activation of microglia/macrophages and increased survival of CA3 neurons but had no effect on lesion volume at 21 d after TBI. Rosiglitazone-treated animals had improved performance on beam balance testing, but there was no difference in spatial memory function as determined by Morris water maze. In summary, this study indicates that rosiglitazone treatment in the first 24 h after TBI has limited anti-inflammatory and neuroprotective effects in rat traumatic injury. Further study using an alternative dosage paradigm and more sensitive behavioral testing may be warranted. Published by Elsevier Inc.
Keywords: Traumatic brain injury PPARg Inflammation Rosiglitazone Behavioral Cell survival
1. Introduction Within minutes of TBI, numerous inflammatory mechanisms are triggered that lead to activation of microglia, migration of inflammatory macrophages into brain and expression of a host of cytokines including tumor necrosis factor (TNF)a, interleukins (IL), matrix metallopeptidases (MMP), and iNOS [1e3]. These mechanisms may exacerbate acute injury but may also be important in repair and recovery of function after TBI [4]. Alteration of this immune response to favor later beneficial effects of inflammation after TBI may be a valuable strategy to improve functional recovery. The nuclear PPARg receptor regulates transcription of a number of genes that promote the resolution of inflammation and healing
* Corresponding author. Geriatric Research Educational and Clinical Center, VA Pittsburgh Healthcare Center Research, Office Building #30, Mail Code 151 University Drive, Pittsburgh, PA 15240, USA. E-mail address: [email protected] (S.H. Graham). http://dx.doi.org/10.1016/j.bbrc.2016.03.003 0006-291X/Published by Elsevier Inc.
[5]. PPAR g expression is upregulated after TBI and may play a role in recovery after TBI [6,7]. A number of specific synthetic PPARg agonists have been developed that have potent effects upon lipid metabolism in adipocytes that makes them useful as anti-diabetic drugs. Rosiglitazone is an FDA-approved drug with few shortterm side effects making it an excellent candidate for rapid translation to clinical trials. It has been shown to have potent protective effects in models of stroke [5,8,9], neurodegenerative diseases [10,11] and spinal cord injury [12,13], but less is known about the effect in TBI. Previous studies have shown that rosiglitazone reduces neuroinflammation, lesion volume, and oxidative stress and apoptotic markers acutely in mouse models of TBI [6,14]. Some studies have shown short-term improvements in motor behavior, but longer term behavioral effects of rosiglitazone treatment have not been reported [14]. The current study aims to determine the effects of rosiglitazone treatment upon microglial/macrophage activation, and histological outcome at 21 d after TBI in rats. In addition, the effect of
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rosiglitazone treatment upon vestibulomotor behavior and spatial memory function was evaluated. The effect of rosiglitazone treatment upon acute cytokine transcription was also examined. 2. Materials and methods Animal studies were performed with the approval of the University of Pittsburgh Institutional Animal Care and Use Committee and conducted according to the National Institutes of Health Guide for the Care and Use of Laboratory Animals. Animals were housed in a temperature and humidity controlled environment with 12 h light cycles and free access to food and water. Chemicals: Unless otherwise noted, chemicals were purchased from Sigma Aldrich (St. Louis, MO). 2.1. Traumatic brain injury The controlled cortical impact model of TBI used in this study was performed as previously described [15,16]. Rats were anesthetized in 5% isoflurane in 60% nitrous oxide, balance oxygen, then endotracheally intubated, and maintained on a small-animal ventilator (Harvard Apparatus, Holliston, MA) at 1e2% isoflurane in the same carrier gas mixture. After craniectomy, the impactor tip (6 mm) was zeroed to the surface of the brain prior to injury (impact speed: 4 m/sec, injury depth: 2.7 mm, dwell time: 50 msec). Following TBI, anesthesia was discontinued, the incision was closed and the animals were ventilated with 100% oxygen until spontaneous return of respiration. Sham surgery was identical to that described above without trauma. Rats were administered 6 mg/kg rosiglitazone (Cayman Chemical, Ann Arbor, MI) diluted in DMSO and PBS (1:3) via intraperitoneal injection (IP) at 5 min, 6 h and 24 h post TBI or vehicle only (DMSO/PBS 1:3). n ¼ 15 per group. 2.2. Vestibular motor function Beam Balance and Beam Walking tests were performed post day injury 1e5 with training prior to injury on Day 0. Gross vestibulomotor function was assessed using a beam-balance task on days 0e5 post TBI [17]. The animal was placed on a suspended, narrow wooden beam (1.5 cm wide) 30 inches above a padded surface and latency on the beam was measured, up to 60 s. Three trials per animal per day were performed with a 30 s intertrial rest period. Finer components of vestibulomotor function and coordination were assessed using a modified beam-walking task. On the day prior to injury the rats were trained to escape a bright light and loud white noise by traversing a narrow wooden beam (2.5 100 cm), and entering a darkened goal box at the opposite end. Four pegs (3 mm diameter and 4 cm high) were equally spaced along the center of the beam to increase the difficulty of the task. Performance was assessed by measuring the latency to traverse the beam. If the rats did not cross the beam in 60 s or fell off, the light and noise were stopped and the rat was placed in the goal box. 2.3. Spatial learning Spatial learning was assessed by Morris water maze performance as previously described [18]. A large circular tank 180 cm in diameter and 45 cm high was filled with water maintained at 26 ± 1 C to a height of 30 cm containing a transparent circular platform (10 cm in diameter and 29 cm high) located in a fixed position in the tank 45 cm from the tank wall and 1 cm below the water surface. Extra-maze visual cues aid the rat in locating the escape platform. Animals underwent cognitive performance evaluation by placing them in the MWM on post-injury day (PID) 14 without prior training or exposure to the MWW, for five
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consecutive days (4 trials/day) with a hidden goal platform. Rats were randomly placed in the water against and facing the tank wall, and then released to swim freely about the tank to find the hidden platform, up to 120 s. If the rat was unable to locate the platform within the allotted time, it was manually directed to the platform. After a 4 min intertrial interval, the animal was returned to the maze for the next trial. On PID 19, the rats underwent a probe trial where the platform was removed from the maze and time spent in the “target quadrant” (the quadrant where the platform was previously located) was measured using a video-tracking system (AnyMaze, Stoelting, Inc. Wood Dale, IL) for 120 s. This was then compared with the time spent in the remaining three quadrants. A visible platform task was performed on PID 19e20 to evaluate nonspecific visual deficits. The platform was raised to 1 in. above the water level and the rats were released from each of four randomized starting positions to locate, swim to, and mount the visible platform. Latency in finding the platform was measured. 2.4. Cell survival measurement Rats were anesthetized with 4% isoflurane in N2O/O2 (2:1) and transcardially perfused with 200 mL heparinized saline and 200 mL 10% buffered formalin 21 days post TBI. Whole brains were embedded in paraffin and cut in 7 mm thick sections every 1 mm. Sections were stained with hematoxylin and eosin and hippocampi were examined using 20 brightfield magnification. Morphologically normal neurons (those with visible nucleoli) were counted in ipsilateral and contralateral CA1, CA3 and CA4 in slices located in the center of the contusion (3.60 mm from Bregma). Data are expressed as percent contralateral. 2.5. Assessment of lesion volume Brain lesion volumes were calculated by measuring ipsilateral and contralateral hemispheric areas at each slice then multiplying slice area X slice interval thickness and adding together all slices [18]. Ipsilateral lesion volumes are expressed as percent contralateral and are calculated as follows: (contralateral-ipsilateral)/ contralateral 100. 2.6. IBA-1 immunostaining assessment IBA-1 immunofluorescent staining was performed as previously described [19]. Brain sections taken at the center of the lesion were immunostained with anti-IBA-1 antibody (1:250, WAKO Chemicals, Richmond, VA) then incubated with AlexaFluor 488-conjugated secondary antibody (Life Technologies, Grand Island, NY). Brains sections were photographed using an Olympus BX51 microscope and Stereo Investigator software. IBA-1-positive cells were counted in 400 800 pixel fields located in CA1 and CA3 hippocampus, dorsal thalamus, and peri-contusional cortex using ImageJ software (U. S. National Institutes of Health, Bethesda, MD). Brain sections incubated without application of primary antibody served as controls. 2.7. RNA extraction and Quantitative real-time Polymerase chain reaction (qPCR) analysis A separate cohort of rats underwent TBI and drug administration as described above (n ¼ 6 per group) and were sacrificed 24 h after injury. Total brain tissue RNA and protein was extracted using a PARIS kit (Invitrogen, Grand Island, NY). Extracted RNA was treated with DNase to remove contaminating DNA. RNA concentrations were measured, and first-strand cDNA was synthesized using 5 mg of total RNA with a SuperScript III first-strand cDNA
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synthesis system (Invitrogen) primed with Oligo (dT), qPCR was performed using a Biosystems 7300 real time PCR System (Applied Biosystems, Foster City, CA). Duplex qPCR was performed to simultaneously amplify and measure gene expression levels of target genes TNFa, MMP-9, iNOS, or cyclooxygenase2 (COX2) and the internal control gene (GAPDH) in one reaction using a FAM dyelabeled Taqman gene expression assay for the target gene in combination with a primer-limited, VIC dye-labeled assay for GAPDH. After initial incubations at 50 C for 2 min 95 C for 10 min, the PCR amplification protocol consisted of 40 cycles of denaturing at 95 C for 15 s, and annealing and extension at 60 C for 60 s. The target gene expression levels were calculated after normalizing to GAPDH. 2.8. Statistical analysis Data are expressed as means ± SE and were analyzed using Student's T test. Behavioral data was analyzed using repeated measures ANOVA with Fisher LSD post hoc testing to calculate longitudinal differences between groups (SPSS, IBM Corp., Armonk, NY). Results were considered to be significant when p < 0.05.
A probe trial was run PID 19, where the platform was removed from the tank and percent dwell time in the target (platformcontaining) quadrant measured (Fig. 1D). There was a trend in improved target quadrant percent dwell time in rosiglitazonetreated vs. vehicle-treated trauma animals (28.21 ± 1.47% vs. 25.35 ± 2.06%) but this difference was not significant. 3.2. Rosiglitazone does not affect cortical contusion volume after TBI To examine whether treatment with the PPARg agonist, rosiglitazone, attenuates cortical contusion volume after TBI, sham or trauma surgery animals treated with rosiglitazone or vehicle were sacrificed 21 days post injury via brain perfusion fixation. After embedding in paraffin, serial sections were cut through the entire brain, stained with hematoxylin and eosin, and percent contralateral lesion volume was measured. In trauma groups, treatment with rosiglitazone did not decrease lesion volume as compared to the vehicle-treated group (14.34 ± 1.72% vs. 14.93 ± 1.87%, respectively, Fig. 2A). There was also no difference between sham rosiglitazone-treated and sham vehicle-treated ipsilateral brain volume (0.07 ± 0.86% vs. 0.80 ± 1.05%, respectively).
3. Results 3.1. The PPARg agonist rosiglitazone improves motor function after TBI To test the effect of rosiglitazone on motor function and spatial learning in rats after traumatic brain injury, rats received 6 mg/kg rosiglitazone or vehicle at 5 min, 6 h and 24 h post injury. Beam balance and beam walking latencies were measured on post injury days (PID) 1e5. Gross vestibulomotor function was assessed by placing rats on a suspended narrow wooden beam for 60 s. Latency on the beam was measured in three trials per day. Rats undergoing TBI and treatment with rosiglitazone had significantly higher 5 day beam balance latencies than rats treated with vehicle (55.7 ± 1.8 vs. 45.5 ± 4.3 s on PID 1, 53.3 ± 3.53 vs. 51.31 ± 3.67 s on PID 2, 54.12 ± 2.75 vs. 51.62 ± 4.27 s on PID 3, 58.87 ± 1.13 vs. 52.71 ± 3.31 s on PID 4, and 58.69 ± 1.31 vs. 58.64 ± 1.36 s on PID 5, respectively, Fig. 1A). There was no difference between rosiglitazone- and vehicle-treated groups in sham surgery animals. Finer vestibulomotor function was assessed by measuring latencies required to travel along a narrow beam to escape bright light and loud noise. Latencies were similar between rosiglitazoneand vehicle-treated groups (56.93 ± 1.97 vs. 49.91 ± 4.96 s on PID 1, 47.46 ± 4.92 vs. 48.62 ± 4.81 s on PID 2, 39.52 ± 6.16 vs. 35.93 ± 5.51sec on PID 3, 23.23 ± 5.95 vs. 25.52 ± 5.25 s on PID 4, and 10.25 ± 4.22 vs. 12.33 ± 3.63 s on PID 5, respectively). TBI and sham surgery groups had statistically different latencies regardless of treatment (p < 0.001, Fig. 1B). In both beam balance and beam walking tests, latencies were resolved by Day 5 and there were no measurable differences between any surgery or treatment groups. Morris Water Maze (MWM) performance was used to assess spatial learning deficits on PID 14e20 (Fig. 1C). On PID 14e18, rats were randomly placed in the maze using a hidden platform located 2 cm below the water surface. Latencies in trauma and sham surgery groups were significantly different (p < 0.02), regardless of treatment, but there was no difference between rosiglitazone- and vehicle-treated trauma groups. To evaluate nonspecific visual deficits, the platform was raised 2 cm above the water surface on PID 19e20 and latency to the platform measured. Again, there were significant, although modest, differences in latencies between sham and trauma surgery groups, regardless of treatment (P < 0.05), with no differences between rosiglitazone-treated and vehicle-treated trauma groups.
3.3. Rosiglitazone increases cell survival in CA3 region of brain hippocampus Neuronal protection by rosiglitazone was assessed in PID 21 brain sections taken at the center of the contusion. Neurons exhibiting normal morphology with visible nucleoli were counted in ipsilateral and contralateral CA1, CA3 and CA4 regions of hippocampus. Cell survival was significantly increased in the CA3 region of rosiglitazone-treated TBI surgery rats vs. vehicle-treated TBI surgery rats (69.54 ± 5.07% vs. 54.20 ± 5.08%, respectively) although there was no difference in CA1 and CA4 regions (Fig. 2B). All sham surgery animals retained approximately 100% cell survival in CA1, CA3 and CA4 regions, with either treatment. 3.4. Rosiglitazone prevents microglial inflammation after TBI PID 21 brain sections taken at the center of the contusion in trauma surgery rats and corresponding sections in sham-operated rats were immunostained with the microglial activation antibody, IBA-1 and fluorescent-conjugated secondary antibody. Positive cells were counted in ipsilateral and contralateral CA1, CA3, pericontusional cortex and dorsal thalamus regions (Fig. 3). Microglial activation was decreased in animals treated with rosiglitazone as compared to vehicle after trauma in CA1 (37.6 ± 7.4 vs. 84.4 ± 16.7 cells, respectively) and cortex (84.4 ± 16.7 vs. 127.0 ± 11.0 cells, respectively). Although there were trends in CA3 (92.6 ± 9.8 vs. 110.9 ± 12.8 cells) and thalamus (146.0 ± 11.9 vs. 163.0 ± 13.1 cells), these data did not reach statistical significance. There were no differences between regions in sham vehicle- and rosiglitazone-treated animals or between contralateral regions of trauma vehicle- and rosiglitazone-treated animals. Control brain slices incubated without primary antibody did not exhibit endogenous autofluorescence (data not shown). 3.5. Rosiglitazone attenuates TNFa gene expression after TBI A separate cohort of animals underwent trauma or sham surgery and was sacrificed at 24 h for real time PCR analysis of brain peri-contusional cortex. The anti-inflammatory cytokine TNFa mRNA was significantly decreased from 4.92 ± 0.27 to 3.59 ± 0.41 fold difference vs. sham contralateral vehicle-treated cortex in trauma animals treated with rosiglitazone as compared to vehicle
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Fig. 1. Motor function assessment in rats treated with rosiglitazone after TBI. Rats underwent sham or TBI surgery and treatment with vehicle (Veh, DMSO/PBS) or rosiglitazone (rosi). Beam Balance (A) and Beam Walking (B) latencies were measured prior to TBI (Pre) and Days 1e5 post TBI. Morris Water Maze (C) hidden and visible platform latencies were measured on days 14e18 and days 19e20, respectively. (D) Percent time in the target quadrant. n ¼ 10e15 per group. *P < 0.05 TBI Veh vs TBI rosiglitazone (repeated measures ANOVA with post hoc Fisher LSD). Data are means ± SE.
control (Fig. 4). However, there was no mRNA reduction in the matrix metallopeptidase gene, MMP9, the cytokine-inducible enzyme, iNOS, or in COX2, a gene whose expression is increased after inflammation. Contralateral gene expression was unchanged in all groups. 4. Discussion Many mechanisms or pathways are involved in secondary posttraumatic brain injury, such as inflammation, oxidative stress and neuronal cell apoptosis. Therefore, drugs which can simultaneously regulate multiple signal pathways may have an advantage in future trauma patient treatment [20]. PPARg is a ligand-activated transcription factor which regulates the transcription of many important genes involved in inflammation and oxidative stress [5,21,22]. Rosiglitazone, a PPARg agonist, has been shown to be neuroprotective in many acute neurological disease models, such as stroke, spinal injury and TBI [7,8,23,24]. This study evaluates the effects of rosiglitazone treatment in a rat model of TBI. The long-term effect of rosiglitazone upon spatial memory function, cell survival and lesion volume 21d after TBI was assessed. The major findings of the current study are 1) Rosiglitazone treatment attenuated trauma -induced TNFa mRNA expression but had no significant effect on cytokines MMP-9, iNOS or
COX2 at 24 h after TBI. 2) Rosiglitazone suppressed activation of microglia/macrophages at 21 d after TBI. 3) Rosiglitazone increased survival of CA3 neurons but had no effect on lesion volume at 21 d after TBI. 4) Rosiglitazone-treated animals had improved performance in beam balance testing compared with vehicle-treated control, but there was no difference in performance in Morris water maze testing of spatial memory function. Varying results have been reported in a rosiglitazone-treated mouse TBI model. Yi et al. found that rosiglitazone treatment decreased production of inflammatory cytokines including interleukin-6, ICAM and MCP1 at 24 h after TBI and decreased lesion volume at 7 d after injury in mice [6]. Microglial activation detected by GSI-B4 was suppressed at 48 h after TBI., Another report using a single dose of rosiglitazone administered at 30 min post injury did not result in protective effects upon lesion volume, cytokine production or acute motor function [25]. The current study in rats did not detect a reduction in lesion volume; however, lesion volume was determined at 21 d after injury when the effects of acute edema are less prevalent. In the current study, rosiglitazone-treated rats did not have significantly improved spatial memory function as compared to vehicle-treated controls. Although rosiglitazone treatment in the first 24 h resulted in suppression of microglial activation 21 d after injury, a longer duration of treatment may be needed to improve
Fig. 2. Effects of rosiglitazone on lesion volume and cell survival in rat brain after TBI. Vehicle (Veh)- or rosiglitazone (rosi)-treated rats were sacrificed 21 days post TBI and brains stained with hematoxylin and eosin. A. Lesion volume is presented as percent contralateral hemisphere. B. Upper: Representative photos of ipsilateral and contralateral CA1, CA3 and CA4 regions. Cells with normal morphology: yellow arrows; shrunken neurons: red arrows. Scale bar ¼ 25 mm. Lower: Cell survival presented as % contralateral cell counts. Data are means ± SE. *P < 0.05. n ¼ 10e15 per group.
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Fig. 3. Effects of rosiglitazone on microglial cell activation in rat brain after TBI. Vehicle (Veh)- or rosiglitazone (rosi)-treated rats were sacrificed 21 days post trauma, brains were immunostained with anti-IBA-1 antibody (green), and IBA-1 positive cells were counted in ipsilateral (ipsi) and contralateral (contra) CA1, CA3, cortex (CTX) and thalamus (Thal). Upper: representative photos of regional brain sections. Scale bar ¼ 50 mm. Lower: IBA-1-positive cells per region. *P < 0.05. n ¼ 10e15 per group. Data are means ± SE.
cognitive outcome. PPARg regulates a host of anti-inflammatory genes and genes with other functions that are unrelated to macrophage activation that could play a role in the recovery process after TBI [21]. Pioglitazone, a structurally related thiazolidinedione PPARg agonist, did improve spatial memory function in a rat TBI model [26]. Pioglitazone more readily crosses the blood brain barrier than rosiglitazone and also has PPARa activity, but is a less potent PPARg agonist than rosiglitazone [21,25]. Treatment with the PPARg antagonist T0070907 did not abolish the effects of pioglitazone upon cytokine production and lesion volume, suggesting that much of pioglitazone's effects may be mediated through a PPARg-independent mechanism such as PPARa [7,25]. Further studies are needed to delineate the roles of PPARg and
PPARa in recovery after TBI. In summary, rosiglitazone treatment in the first 24 h after TBI resulted in suppressed microglial activation and improved short term motor but not spatial memory function in rats. However, these protective effects were limited. Additional studies optimizing the rosiglitazone treatment window after TBI may be warranted.
Disclosure/conflict of interest The authors declare no conflict of interest. The contents do not represent the views of the Department of Veterans Affairs or the United States Government.
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Fig. 4. Effects of rosiglitazone on mRNA levels of inflammatory markers in rat brain cortex after TBI. Rats treated with vehicle (Veh) or rosiglitazone (rosi) were sacrificed 24 h after TBI and cortices dissected out. MMP9, COX2, TNFa and iNOS mRNA expression were measured using qPCR and are normalized to their respective contralateral sham Veh groups. n ¼ 6 per group. *P < 0.05. Data are means ± SE.
Acknowledgments This work was supported by the Veteran's Affairs Rehabilitation Research and Development Merit Review Program Award I01RX000310 (S.H.G.). Transparency document Transparency document related to this article can be found online at http://dx.doi.org/10.1016/j.bbrc.2016.03.003. References [1] P.M. Kochanek, T.C. Jackson, N.M. Ferguson, S.W. Carlson, D.W. Simon, E.C. Brockman, J. Ji, H. Bayir, S.M. Poloyac, A.K. Wagner, A.E. Kline, P.E. Empey, R.S. Clark, E.K. Jackson, C.E. Dixon, Emerging therapies in traumatic brain injury, Semin. Neurol. 35 (2015) 83e100. [2] D. Pearse, K. Jarnagin, Abating progressive tissue injury and preserving function after CNS trauma: The role of inflammation modulatory therapies, Curr. Opin. Investig. Drugs 11 (2010) 1207e1210. [3] A.F. Ramlackhansingh, D.J. Brooks, R.J. Greenwood, S.K. Bose, F.E. Turkheimer, K.M. Kinnunen, S. Gentleman, R.A. Heckemann, K. Gunanayagam, G. Gelosa, D.J. Sharp, Inflammation after trauma: microglial activation and traumatic brain injury, Ann. Neurol. 70 (2011) 374e383.
[4] P.F. Stahel, W.R. Smith, J. Bruchis, C.H. Rabb, Peroxisome proliferator-activated receptors: “key” regulators of neuroinflammation after traumatic brain injury, PPAR Res. 2008 (2008) 538141. [5] R. Bordet, T. Ouk, O. Petrault, P. Gele, S. Gautier, M. Laprais, D. Deplanque, P. Duriez, B. Staels, J.C. Fruchart, M. Bastide, PPAR: a new pharmacological target for neuroprotection in stroke and neurodegenerative diseases, Biochem. Soc. Trans. 34 (2006) 1341e1346. [6] J.H. Yi, S.W. Park, N. Brooks, B.T. Lang, R. Vemuganti, PPARgamma agonist rosiglitazone is neuroprotective after traumatic brain injury via antiinflammatory and anti-oxidative mechanisms, Brain Res. 1244 (2008) 164e172. [7] X.R. Chen, V.C. Besson, B. Palmier, Y. Garcia, M. Plotkine, C. Marchand-Leroux, Neurological recovery-promoting, anti-inflammatory, and anti-oxidative effects afforded by fenofibrate, a PPAR alpha agonist, in traumatic brain injury, J. Neurotrauma 24 (2007) 1119e1131. [8] M. Allahtavakoli, R. Moloudi, M.K. Arababadi, A. Shamsizadeh, K. Javanmardi, Delayed post ischemic treatment with Rosiglitazone attenuates infarct volume, neurological deficits and neutrophilia after embolic stroke in rat, Brain Res. 1271 (2009) 121e127. [9] C. Gu, Y. Wang, J. Li, J. Chen, F. Yan, C. Wu, G. Chen, Rosiglitazone attenuates early brain injury after experimental subarachnoid hemorrhage in rats, Brain Res. 1624 (2015) 199e207. [10] A.R. Carta, PPAR-gamma: therapeutic prospects in Parkinson's disease, Curr. Drug Targets 14 (2013) 743e751. [11] Y.C. Chen, J.S. Wu, H.D. Tsai, C.Y. Huang, J.J. Chen, G.Y. Sun, T.N. Lin, Peroxisome proliferator-activated receptor gamma (PPAR-gamma) and neurodegenerative disorders, Mol. Neurobiol. 46 (2012) 114e124.
H. Liu et al. / Biochemical and Biophysical Research Communications 472 (2016) 648e655 [12] E. Esposito, S. Cuzzocrea, Targeting the peroxisome proliferator-activated receptors (PPARs) in spinal cord injury, Expert Opin. Ther. Targets 15 (2011) 943e959. [13] Q.Q. Meng, X.J. Liang, P. Wang, X.P. Wang, J.W. Yang, Y.F. Wu, H.Y. Shen, Rosiglitazone enhances the proliferation of neural progenitor cells and inhibits inflammation response after spinal cord injury, Neurosci. Lett. 503 (2011) 191e195. [14] J. Yao, K. Zheng, X. Zhang, Rosiglitazone exerts neuroprotective effects via the suppression of neuronal autophagy and apoptosis in the cortex following traumatic brain injury, Mol. Med. Rep. 12 (5) (2015) 6591e6597. [15] C.E. Dixon, G.L. Clifton, J.W. Lighthall, A.A. Yaghmai, R.L. Hayes, A controlled cortical impact model of traumatic brain injury in the rat, J. Neurosci. Methods 39 (1991) 253e262. [16] S.S. Shin, E.R. Bray, C.E. Dixon, Effects of nicotine administration on striatal dopamine signaling after traumatic brain injury in rats, J. Neurotrauma 29 (2012) 843e850. [17] A.E. Kline, J. Yu, E. Horvath, D.W. Marion, C.E. Dixon, The selective 5-HT(1A) receptor agonist repinotan HCl attenuates histopathology and spatial learning deficits following traumatic brain injury in rats, Neuroscience 106 (2001) 547e555. [18] R. Tehranian, M.E. Rose, V. Vagni, R.P. Griffith, S. Wu, S. Maits, X. Zhang, R.S. Clark, C.E. Dixon, P.M. Kochanek, O. Bernard, S.H. Graham, Transgenic mice that overexpress the anti-apoptotic Bcl-2 protein have improved histological outcome but unchanged behavioral outcome after traumatic brain injury, Brain Res. 1101 (2006) 126e135. [19] H. Liu, M.E. Rose, T.M. Miller, W. Li, S.N. Shinde, A.M. Pickrell, S.M. Poloyac, S.H. Graham, R.W. Hickey, COX2-derived primary and cyclopentenone
[20] [21]
[22]
[23]
[24]
[25]
[26]
655
prostaglandins are increased after asphyxial cardiac arrest, Brain Res. 1519 (2013) 71e77. J. Culman, Y. Zhao, P. Gohlke, T. Herdegen, PPAR-gamma: therapeutic target for ischemic stroke, Trends Pharmacol. Sci. 28 (2007) 244e249. S. Mandrekar-Colucci, A. Sauerbeck, P.G. Popovich, D.M. McTigue, PPAR agonists as therapeutics for CNS trauma and neurological diseases, ASN Neuro 5 (2013) e00129. B.D. Semple, L.J. Noble-Haeusslein, Broad-spectrum neuroprotection against traumatic brain injury by agonism of peroxisome proliferator-activated receptors, Exp. Neurol. 229 (2011) 195e197. S.W. Park, J.H. Yi, G. Miranpuri, I. Satriotomo, K. Bowen, D.K. Resnick, R. Vemuganti, Thiazolidinedione class of peroxisome proliferator-activated receptor gamma agonists prevents neuronal damage, motor dysfunction, myelin loss, neuropathic pain, and inflammation after spinal cord injury in adult rats, J. Pharmacol. Exp. Ther. 320 (2007) 1002e1012. M. Sobrado, M.P. Pereira, I. Ballesteros, O. Hurtado, D. Fernandez-Lopez, J.M. Pradillo, J.R. Caso, J. Vivancos, F. Nombela, J. Serena, I. Lizasoain, M.A. Moro, Synthesis of lipoxin A4 by 5-lipoxygenase mediates PPARgammadependent, neuroprotective effects of rosiglitazone in experimental stroke, J. Neurosci. 29 (2009) 3875e3884. S.C. Thal, M. Heinemann, C. Luh, D. Pieter, C. Werner, K. Engelhard, Pioglitazone reduces secondary brain damage after experimental brain trauma by PPAR-gamma-independent mechanisms, J. Neurotrauma 28 (2011) 983e993. A. Sauerbeck, J. Gao, R. Readnower, M. Liu, J.R. Pauly, G. Bing, P.G. Sullivan, Pioglitazone attenuates mitochondrial dysfunction, cognitive impairment, cortical tissue loss, and inflammation following traumatic brain injury, Exp. Neurol. 227 (2011) 128e135.
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Rosiglitazone attenuates inflammation and CA3 neuronal loss following traumatic brain injury in rats Hao Liu a, b, Marie E. Rose a, b, Sherman Culver a, c, d, Xiecheng Ma a, c, d, C. Edward Dixon a, c, d, Steven H. Graham a, b, * a
Geriatric Research Educational and Clinical Center, V.A. Pittsburgh Healthcare System, PA, USA Department of Neurology, University of Pittsburgh School of Medicine, PA, USA Department of Neurosurgery, University of Pittsburgh, PA 15216, USA d Department of Critical Care Medicine, University of Pittsburgh, PA 15216, USA b c
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a b s t r a c t
Article history: Received 23 February 2016 Accepted 3 March 2016 Available online 3 March 2016
Rosiglitazone, a potent peroxisome proliferator-activated receptor (PPAR)-g agonist, has been shown to confer neuroprotective effects in stroke and spinal cord injury, but its role in the traumatic brain injury (TBI) is still controversial. Using a controlled cortical impact model in rats, the current study was designed to determine the effects of rosiglitazone treatment (6 mg/kg at 5 min, 6 h and 24 h post injury) upon inflammation and histological outcome at 21 d after TBI. In addition, the effects of rosiglitazone upon inflammatory cytokine transcription, vestibulomotor behavior and spatial memory function were determined at earlier time points (24 h, 1e5 d, 14e20 d post injury, respectively). Compared with the vehicle-treated group, rosiglitazone treatment suppressed production of TNFa at 24 h after TBI, attenuated activation of microglia/macrophages and increased survival of CA3 neurons but had no effect on lesion volume at 21 d after TBI. Rosiglitazone-treated animals had improved performance on beam balance testing, but there was no difference in spatial memory function as determined by Morris water maze. In summary, this study indicates that rosiglitazone treatment in the first 24 h after TBI has limited anti-inflammatory and neuroprotective effects in rat traumatic injury. Further study using an alternative dosage paradigm and more sensitive behavioral testing may be warranted. Published by Elsevier Inc.
Keywords: Traumatic brain injury PPARg Inflammation Rosiglitazone Behavioral Cell survival
1. Introduction Within minutes of TBI, numerous inflammatory mechanisms are triggered that lead to activation of microglia, migration of inflammatory macrophages into brain and expression of a host of cytokines including tumor necrosis factor (TNF)a, interleukins (IL), matrix metallopeptidases (MMP), and iNOS [1e3]. These mechanisms may exacerbate acute injury but may also be important in repair and recovery of function after TBI [4]. Alteration of this immune response to favor later beneficial effects of inflammation after TBI may be a valuable strategy to improve functional recovery. The nuclear PPARg receptor regulates transcription of a number of genes that promote the resolution of inflammation and healing
* Corresponding author. Geriatric Research Educational and Clinical Center, VA Pittsburgh Healthcare Center Research, Office Building #30, Mail Code 151 University Drive, Pittsburgh, PA 15240, USA. E-mail address: [email protected] (S.H. Graham). http://dx.doi.org/10.1016/j.bbrc.2016.03.003 0006-291X/Published by Elsevier Inc.
[5]. PPAR g expression is upregulated after TBI and may play a role in recovery after TBI [6,7]. A number of specific synthetic PPARg agonists have been developed that have potent effects upon lipid metabolism in adipocytes that makes them useful as anti-diabetic drugs. Rosiglitazone is an FDA-approved drug with few shortterm side effects making it an excellent candidate for rapid translation to clinical trials. It has been shown to have potent protective effects in models of stroke [5,8,9], neurodegenerative diseases [10,11] and spinal cord injury [12,13], but less is known about the effect in TBI. Previous studies have shown that rosiglitazone reduces neuroinflammation, lesion volume, and oxidative stress and apoptotic markers acutely in mouse models of TBI [6,14]. Some studies have shown short-term improvements in motor behavior, but longer term behavioral effects of rosiglitazone treatment have not been reported [14]. The current study aims to determine the effects of rosiglitazone treatment upon microglial/macrophage activation, and histological outcome at 21 d after TBI in rats. In addition, the effect of
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rosiglitazone treatment upon vestibulomotor behavior and spatial memory function was evaluated. The effect of rosiglitazone treatment upon acute cytokine transcription was also examined. 2. Materials and methods Animal studies were performed with the approval of the University of Pittsburgh Institutional Animal Care and Use Committee and conducted according to the National Institutes of Health Guide for the Care and Use of Laboratory Animals. Animals were housed in a temperature and humidity controlled environment with 12 h light cycles and free access to food and water. Chemicals: Unless otherwise noted, chemicals were purchased from Sigma Aldrich (St. Louis, MO). 2.1. Traumatic brain injury The controlled cortical impact model of TBI used in this study was performed as previously described [15,16]. Rats were anesthetized in 5% isoflurane in 60% nitrous oxide, balance oxygen, then endotracheally intubated, and maintained on a small-animal ventilator (Harvard Apparatus, Holliston, MA) at 1e2% isoflurane in the same carrier gas mixture. After craniectomy, the impactor tip (6 mm) was zeroed to the surface of the brain prior to injury (impact speed: 4 m/sec, injury depth: 2.7 mm, dwell time: 50 msec). Following TBI, anesthesia was discontinued, the incision was closed and the animals were ventilated with 100% oxygen until spontaneous return of respiration. Sham surgery was identical to that described above without trauma. Rats were administered 6 mg/kg rosiglitazone (Cayman Chemical, Ann Arbor, MI) diluted in DMSO and PBS (1:3) via intraperitoneal injection (IP) at 5 min, 6 h and 24 h post TBI or vehicle only (DMSO/PBS 1:3). n ¼ 15 per group. 2.2. Vestibular motor function Beam Balance and Beam Walking tests were performed post day injury 1e5 with training prior to injury on Day 0. Gross vestibulomotor function was assessed using a beam-balance task on days 0e5 post TBI [17]. The animal was placed on a suspended, narrow wooden beam (1.5 cm wide) 30 inches above a padded surface and latency on the beam was measured, up to 60 s. Three trials per animal per day were performed with a 30 s intertrial rest period. Finer components of vestibulomotor function and coordination were assessed using a modified beam-walking task. On the day prior to injury the rats were trained to escape a bright light and loud white noise by traversing a narrow wooden beam (2.5 100 cm), and entering a darkened goal box at the opposite end. Four pegs (3 mm diameter and 4 cm high) were equally spaced along the center of the beam to increase the difficulty of the task. Performance was assessed by measuring the latency to traverse the beam. If the rats did not cross the beam in 60 s or fell off, the light and noise were stopped and the rat was placed in the goal box. 2.3. Spatial learning Spatial learning was assessed by Morris water maze performance as previously described [18]. A large circular tank 180 cm in diameter and 45 cm high was filled with water maintained at 26 ± 1 C to a height of 30 cm containing a transparent circular platform (10 cm in diameter and 29 cm high) located in a fixed position in the tank 45 cm from the tank wall and 1 cm below the water surface. Extra-maze visual cues aid the rat in locating the escape platform. Animals underwent cognitive performance evaluation by placing them in the MWM on post-injury day (PID) 14 without prior training or exposure to the MWW, for five
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consecutive days (4 trials/day) with a hidden goal platform. Rats were randomly placed in the water against and facing the tank wall, and then released to swim freely about the tank to find the hidden platform, up to 120 s. If the rat was unable to locate the platform within the allotted time, it was manually directed to the platform. After a 4 min intertrial interval, the animal was returned to the maze for the next trial. On PID 19, the rats underwent a probe trial where the platform was removed from the maze and time spent in the “target quadrant” (the quadrant where the platform was previously located) was measured using a video-tracking system (AnyMaze, Stoelting, Inc. Wood Dale, IL) for 120 s. This was then compared with the time spent in the remaining three quadrants. A visible platform task was performed on PID 19e20 to evaluate nonspecific visual deficits. The platform was raised to 1 in. above the water level and the rats were released from each of four randomized starting positions to locate, swim to, and mount the visible platform. Latency in finding the platform was measured. 2.4. Cell survival measurement Rats were anesthetized with 4% isoflurane in N2O/O2 (2:1) and transcardially perfused with 200 mL heparinized saline and 200 mL 10% buffered formalin 21 days post TBI. Whole brains were embedded in paraffin and cut in 7 mm thick sections every 1 mm. Sections were stained with hematoxylin and eosin and hippocampi were examined using 20 brightfield magnification. Morphologically normal neurons (those with visible nucleoli) were counted in ipsilateral and contralateral CA1, CA3 and CA4 in slices located in the center of the contusion (3.60 mm from Bregma). Data are expressed as percent contralateral. 2.5. Assessment of lesion volume Brain lesion volumes were calculated by measuring ipsilateral and contralateral hemispheric areas at each slice then multiplying slice area X slice interval thickness and adding together all slices [18]. Ipsilateral lesion volumes are expressed as percent contralateral and are calculated as follows: (contralateral-ipsilateral)/ contralateral 100. 2.6. IBA-1 immunostaining assessment IBA-1 immunofluorescent staining was performed as previously described [19]. Brain sections taken at the center of the lesion were immunostained with anti-IBA-1 antibody (1:250, WAKO Chemicals, Richmond, VA) then incubated with AlexaFluor 488-conjugated secondary antibody (Life Technologies, Grand Island, NY). Brains sections were photographed using an Olympus BX51 microscope and Stereo Investigator software. IBA-1-positive cells were counted in 400 800 pixel fields located in CA1 and CA3 hippocampus, dorsal thalamus, and peri-contusional cortex using ImageJ software (U. S. National Institutes of Health, Bethesda, MD). Brain sections incubated without application of primary antibody served as controls. 2.7. RNA extraction and Quantitative real-time Polymerase chain reaction (qPCR) analysis A separate cohort of rats underwent TBI and drug administration as described above (n ¼ 6 per group) and were sacrificed 24 h after injury. Total brain tissue RNA and protein was extracted using a PARIS kit (Invitrogen, Grand Island, NY). Extracted RNA was treated with DNase to remove contaminating DNA. RNA concentrations were measured, and first-strand cDNA was synthesized using 5 mg of total RNA with a SuperScript III first-strand cDNA
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synthesis system (Invitrogen) primed with Oligo (dT), qPCR was performed using a Biosystems 7300 real time PCR System (Applied Biosystems, Foster City, CA). Duplex qPCR was performed to simultaneously amplify and measure gene expression levels of target genes TNFa, MMP-9, iNOS, or cyclooxygenase2 (COX2) and the internal control gene (GAPDH) in one reaction using a FAM dyelabeled Taqman gene expression assay for the target gene in combination with a primer-limited, VIC dye-labeled assay for GAPDH. After initial incubations at 50 C for 2 min 95 C for 10 min, the PCR amplification protocol consisted of 40 cycles of denaturing at 95 C for 15 s, and annealing and extension at 60 C for 60 s. The target gene expression levels were calculated after normalizing to GAPDH. 2.8. Statistical analysis Data are expressed as means ± SE and were analyzed using Student's T test. Behavioral data was analyzed using repeated measures ANOVA with Fisher LSD post hoc testing to calculate longitudinal differences between groups (SPSS, IBM Corp., Armonk, NY). Results were considered to be significant when p < 0.05.
A probe trial was run PID 19, where the platform was removed from the tank and percent dwell time in the target (platformcontaining) quadrant measured (Fig. 1D). There was a trend in improved target quadrant percent dwell time in rosiglitazonetreated vs. vehicle-treated trauma animals (28.21 ± 1.47% vs. 25.35 ± 2.06%) but this difference was not significant. 3.2. Rosiglitazone does not affect cortical contusion volume after TBI To examine whether treatment with the PPARg agonist, rosiglitazone, attenuates cortical contusion volume after TBI, sham or trauma surgery animals treated with rosiglitazone or vehicle were sacrificed 21 days post injury via brain perfusion fixation. After embedding in paraffin, serial sections were cut through the entire brain, stained with hematoxylin and eosin, and percent contralateral lesion volume was measured. In trauma groups, treatment with rosiglitazone did not decrease lesion volume as compared to the vehicle-treated group (14.34 ± 1.72% vs. 14.93 ± 1.87%, respectively, Fig. 2A). There was also no difference between sham rosiglitazone-treated and sham vehicle-treated ipsilateral brain volume (0.07 ± 0.86% vs. 0.80 ± 1.05%, respectively).
3. Results 3.1. The PPARg agonist rosiglitazone improves motor function after TBI To test the effect of rosiglitazone on motor function and spatial learning in rats after traumatic brain injury, rats received 6 mg/kg rosiglitazone or vehicle at 5 min, 6 h and 24 h post injury. Beam balance and beam walking latencies were measured on post injury days (PID) 1e5. Gross vestibulomotor function was assessed by placing rats on a suspended narrow wooden beam for 60 s. Latency on the beam was measured in three trials per day. Rats undergoing TBI and treatment with rosiglitazone had significantly higher 5 day beam balance latencies than rats treated with vehicle (55.7 ± 1.8 vs. 45.5 ± 4.3 s on PID 1, 53.3 ± 3.53 vs. 51.31 ± 3.67 s on PID 2, 54.12 ± 2.75 vs. 51.62 ± 4.27 s on PID 3, 58.87 ± 1.13 vs. 52.71 ± 3.31 s on PID 4, and 58.69 ± 1.31 vs. 58.64 ± 1.36 s on PID 5, respectively, Fig. 1A). There was no difference between rosiglitazone- and vehicle-treated groups in sham surgery animals. Finer vestibulomotor function was assessed by measuring latencies required to travel along a narrow beam to escape bright light and loud noise. Latencies were similar between rosiglitazoneand vehicle-treated groups (56.93 ± 1.97 vs. 49.91 ± 4.96 s on PID 1, 47.46 ± 4.92 vs. 48.62 ± 4.81 s on PID 2, 39.52 ± 6.16 vs. 35.93 ± 5.51sec on PID 3, 23.23 ± 5.95 vs. 25.52 ± 5.25 s on PID 4, and 10.25 ± 4.22 vs. 12.33 ± 3.63 s on PID 5, respectively). TBI and sham surgery groups had statistically different latencies regardless of treatment (p < 0.001, Fig. 1B). In both beam balance and beam walking tests, latencies were resolved by Day 5 and there were no measurable differences between any surgery or treatment groups. Morris Water Maze (MWM) performance was used to assess spatial learning deficits on PID 14e20 (Fig. 1C). On PID 14e18, rats were randomly placed in the maze using a hidden platform located 2 cm below the water surface. Latencies in trauma and sham surgery groups were significantly different (p < 0.02), regardless of treatment, but there was no difference between rosiglitazone- and vehicle-treated trauma groups. To evaluate nonspecific visual deficits, the platform was raised 2 cm above the water surface on PID 19e20 and latency to the platform measured. Again, there were significant, although modest, differences in latencies between sham and trauma surgery groups, regardless of treatment (P < 0.05), with no differences between rosiglitazone-treated and vehicle-treated trauma groups.
3.3. Rosiglitazone increases cell survival in CA3 region of brain hippocampus Neuronal protection by rosiglitazone was assessed in PID 21 brain sections taken at the center of the contusion. Neurons exhibiting normal morphology with visible nucleoli were counted in ipsilateral and contralateral CA1, CA3 and CA4 regions of hippocampus. Cell survival was significantly increased in the CA3 region of rosiglitazone-treated TBI surgery rats vs. vehicle-treated TBI surgery rats (69.54 ± 5.07% vs. 54.20 ± 5.08%, respectively) although there was no difference in CA1 and CA4 regions (Fig. 2B). All sham surgery animals retained approximately 100% cell survival in CA1, CA3 and CA4 regions, with either treatment. 3.4. Rosiglitazone prevents microglial inflammation after TBI PID 21 brain sections taken at the center of the contusion in trauma surgery rats and corresponding sections in sham-operated rats were immunostained with the microglial activation antibody, IBA-1 and fluorescent-conjugated secondary antibody. Positive cells were counted in ipsilateral and contralateral CA1, CA3, pericontusional cortex and dorsal thalamus regions (Fig. 3). Microglial activation was decreased in animals treated with rosiglitazone as compared to vehicle after trauma in CA1 (37.6 ± 7.4 vs. 84.4 ± 16.7 cells, respectively) and cortex (84.4 ± 16.7 vs. 127.0 ± 11.0 cells, respectively). Although there were trends in CA3 (92.6 ± 9.8 vs. 110.9 ± 12.8 cells) and thalamus (146.0 ± 11.9 vs. 163.0 ± 13.1 cells), these data did not reach statistical significance. There were no differences between regions in sham vehicle- and rosiglitazone-treated animals or between contralateral regions of trauma vehicle- and rosiglitazone-treated animals. Control brain slices incubated without primary antibody did not exhibit endogenous autofluorescence (data not shown). 3.5. Rosiglitazone attenuates TNFa gene expression after TBI A separate cohort of animals underwent trauma or sham surgery and was sacrificed at 24 h for real time PCR analysis of brain peri-contusional cortex. The anti-inflammatory cytokine TNFa mRNA was significantly decreased from 4.92 ± 0.27 to 3.59 ± 0.41 fold difference vs. sham contralateral vehicle-treated cortex in trauma animals treated with rosiglitazone as compared to vehicle
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Fig. 1. Motor function assessment in rats treated with rosiglitazone after TBI. Rats underwent sham or TBI surgery and treatment with vehicle (Veh, DMSO/PBS) or rosiglitazone (rosi). Beam Balance (A) and Beam Walking (B) latencies were measured prior to TBI (Pre) and Days 1e5 post TBI. Morris Water Maze (C) hidden and visible platform latencies were measured on days 14e18 and days 19e20, respectively. (D) Percent time in the target quadrant. n ¼ 10e15 per group. *P < 0.05 TBI Veh vs TBI rosiglitazone (repeated measures ANOVA with post hoc Fisher LSD). Data are means ± SE.
control (Fig. 4). However, there was no mRNA reduction in the matrix metallopeptidase gene, MMP9, the cytokine-inducible enzyme, iNOS, or in COX2, a gene whose expression is increased after inflammation. Contralateral gene expression was unchanged in all groups. 4. Discussion Many mechanisms or pathways are involved in secondary posttraumatic brain injury, such as inflammation, oxidative stress and neuronal cell apoptosis. Therefore, drugs which can simultaneously regulate multiple signal pathways may have an advantage in future trauma patient treatment [20]. PPARg is a ligand-activated transcription factor which regulates the transcription of many important genes involved in inflammation and oxidative stress [5,21,22]. Rosiglitazone, a PPARg agonist, has been shown to be neuroprotective in many acute neurological disease models, such as stroke, spinal injury and TBI [7,8,23,24]. This study evaluates the effects of rosiglitazone treatment in a rat model of TBI. The long-term effect of rosiglitazone upon spatial memory function, cell survival and lesion volume 21d after TBI was assessed. The major findings of the current study are 1) Rosiglitazone treatment attenuated trauma -induced TNFa mRNA expression but had no significant effect on cytokines MMP-9, iNOS or
COX2 at 24 h after TBI. 2) Rosiglitazone suppressed activation of microglia/macrophages at 21 d after TBI. 3) Rosiglitazone increased survival of CA3 neurons but had no effect on lesion volume at 21 d after TBI. 4) Rosiglitazone-treated animals had improved performance in beam balance testing compared with vehicle-treated control, but there was no difference in performance in Morris water maze testing of spatial memory function. Varying results have been reported in a rosiglitazone-treated mouse TBI model. Yi et al. found that rosiglitazone treatment decreased production of inflammatory cytokines including interleukin-6, ICAM and MCP1 at 24 h after TBI and decreased lesion volume at 7 d after injury in mice [6]. Microglial activation detected by GSI-B4 was suppressed at 48 h after TBI., Another report using a single dose of rosiglitazone administered at 30 min post injury did not result in protective effects upon lesion volume, cytokine production or acute motor function [25]. The current study in rats did not detect a reduction in lesion volume; however, lesion volume was determined at 21 d after injury when the effects of acute edema are less prevalent. In the current study, rosiglitazone-treated rats did not have significantly improved spatial memory function as compared to vehicle-treated controls. Although rosiglitazone treatment in the first 24 h resulted in suppression of microglial activation 21 d after injury, a longer duration of treatment may be needed to improve
Fig. 2. Effects of rosiglitazone on lesion volume and cell survival in rat brain after TBI. Vehicle (Veh)- or rosiglitazone (rosi)-treated rats were sacrificed 21 days post TBI and brains stained with hematoxylin and eosin. A. Lesion volume is presented as percent contralateral hemisphere. B. Upper: Representative photos of ipsilateral and contralateral CA1, CA3 and CA4 regions. Cells with normal morphology: yellow arrows; shrunken neurons: red arrows. Scale bar ¼ 25 mm. Lower: Cell survival presented as % contralateral cell counts. Data are means ± SE. *P < 0.05. n ¼ 10e15 per group.
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Fig. 3. Effects of rosiglitazone on microglial cell activation in rat brain after TBI. Vehicle (Veh)- or rosiglitazone (rosi)-treated rats were sacrificed 21 days post trauma, brains were immunostained with anti-IBA-1 antibody (green), and IBA-1 positive cells were counted in ipsilateral (ipsi) and contralateral (contra) CA1, CA3, cortex (CTX) and thalamus (Thal). Upper: representative photos of regional brain sections. Scale bar ¼ 50 mm. Lower: IBA-1-positive cells per region. *P < 0.05. n ¼ 10e15 per group. Data are means ± SE.
cognitive outcome. PPARg regulates a host of anti-inflammatory genes and genes with other functions that are unrelated to macrophage activation that could play a role in the recovery process after TBI [21]. Pioglitazone, a structurally related thiazolidinedione PPARg agonist, did improve spatial memory function in a rat TBI model [26]. Pioglitazone more readily crosses the blood brain barrier than rosiglitazone and also has PPARa activity, but is a less potent PPARg agonist than rosiglitazone [21,25]. Treatment with the PPARg antagonist T0070907 did not abolish the effects of pioglitazone upon cytokine production and lesion volume, suggesting that much of pioglitazone's effects may be mediated through a PPARg-independent mechanism such as PPARa [7,25]. Further studies are needed to delineate the roles of PPARg and
PPARa in recovery after TBI. In summary, rosiglitazone treatment in the first 24 h after TBI resulted in suppressed microglial activation and improved short term motor but not spatial memory function in rats. However, these protective effects were limited. Additional studies optimizing the rosiglitazone treatment window after TBI may be warranted.
Disclosure/conflict of interest The authors declare no conflict of interest. The contents do not represent the views of the Department of Veterans Affairs or the United States Government.
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Fig. 4. Effects of rosiglitazone on mRNA levels of inflammatory markers in rat brain cortex after TBI. Rats treated with vehicle (Veh) or rosiglitazone (rosi) were sacrificed 24 h after TBI and cortices dissected out. MMP9, COX2, TNFa and iNOS mRNA expression were measured using qPCR and are normalized to their respective contralateral sham Veh groups. n ¼ 6 per group. *P < 0.05. Data are means ± SE.
Acknowledgments This work was supported by the Veteran's Affairs Rehabilitation Research and Development Merit Review Program Award I01RX000310 (S.H.G.). Transparency document Transparency document related to this article can be found online at http://dx.doi.org/10.1016/j.bbrc.2016.03.003. References [1] P.M. Kochanek, T.C. Jackson, N.M. Ferguson, S.W. Carlson, D.W. Simon, E.C. Brockman, J. Ji, H. Bayir, S.M. Poloyac, A.K. Wagner, A.E. Kline, P.E. Empey, R.S. Clark, E.K. Jackson, C.E. Dixon, Emerging therapies in traumatic brain injury, Semin. Neurol. 35 (2015) 83e100. [2] D. Pearse, K. Jarnagin, Abating progressive tissue injury and preserving function after CNS trauma: The role of inflammation modulatory therapies, Curr. Opin. Investig. Drugs 11 (2010) 1207e1210. [3] A.F. Ramlackhansingh, D.J. Brooks, R.J. Greenwood, S.K. Bose, F.E. Turkheimer, K.M. Kinnunen, S. Gentleman, R.A. Heckemann, K. Gunanayagam, G. Gelosa, D.J. Sharp, Inflammation after trauma: microglial activation and traumatic brain injury, Ann. Neurol. 70 (2011) 374e383.
[4] P.F. Stahel, W.R. Smith, J. Bruchis, C.H. Rabb, Peroxisome proliferator-activated receptors: “key” regulators of neuroinflammation after traumatic brain injury, PPAR Res. 2008 (2008) 538141. [5] R. Bordet, T. Ouk, O. Petrault, P. Gele, S. Gautier, M. Laprais, D. Deplanque, P. Duriez, B. Staels, J.C. Fruchart, M. Bastide, PPAR: a new pharmacological target for neuroprotection in stroke and neurodegenerative diseases, Biochem. Soc. Trans. 34 (2006) 1341e1346. [6] J.H. Yi, S.W. Park, N. Brooks, B.T. Lang, R. Vemuganti, PPARgamma agonist rosiglitazone is neuroprotective after traumatic brain injury via antiinflammatory and anti-oxidative mechanisms, Brain Res. 1244 (2008) 164e172. [7] X.R. Chen, V.C. Besson, B. Palmier, Y. Garcia, M. Plotkine, C. Marchand-Leroux, Neurological recovery-promoting, anti-inflammatory, and anti-oxidative effects afforded by fenofibrate, a PPAR alpha agonist, in traumatic brain injury, J. Neurotrauma 24 (2007) 1119e1131. [8] M. Allahtavakoli, R. Moloudi, M.K. Arababadi, A. Shamsizadeh, K. Javanmardi, Delayed post ischemic treatment with Rosiglitazone attenuates infarct volume, neurological deficits and neutrophilia after embolic stroke in rat, Brain Res. 1271 (2009) 121e127. [9] C. Gu, Y. Wang, J. Li, J. Chen, F. Yan, C. Wu, G. Chen, Rosiglitazone attenuates early brain injury after experimental subarachnoid hemorrhage in rats, Brain Res. 1624 (2015) 199e207. [10] A.R. Carta, PPAR-gamma: therapeutic prospects in Parkinson's disease, Curr. Drug Targets 14 (2013) 743e751. [11] Y.C. Chen, J.S. Wu, H.D. Tsai, C.Y. Huang, J.J. Chen, G.Y. Sun, T.N. Lin, Peroxisome proliferator-activated receptor gamma (PPAR-gamma) and neurodegenerative disorders, Mol. Neurobiol. 46 (2012) 114e124.
H. Liu et al. / Biochemical and Biophysical Research Communications 472 (2016) 648e655 [12] E. Esposito, S. Cuzzocrea, Targeting the peroxisome proliferator-activated receptors (PPARs) in spinal cord injury, Expert Opin. Ther. Targets 15 (2011) 943e959. [13] Q.Q. Meng, X.J. Liang, P. Wang, X.P. Wang, J.W. Yang, Y.F. Wu, H.Y. Shen, Rosiglitazone enhances the proliferation of neural progenitor cells and inhibits inflammation response after spinal cord injury, Neurosci. Lett. 503 (2011) 191e195. [14] J. Yao, K. Zheng, X. Zhang, Rosiglitazone exerts neuroprotective effects via the suppression of neuronal autophagy and apoptosis in the cortex following traumatic brain injury, Mol. Med. Rep. 12 (5) (2015) 6591e6597. [15] C.E. Dixon, G.L. Clifton, J.W. Lighthall, A.A. Yaghmai, R.L. Hayes, A controlled cortical impact model of traumatic brain injury in the rat, J. Neurosci. Methods 39 (1991) 253e262. [16] S.S. Shin, E.R. Bray, C.E. Dixon, Effects of nicotine administration on striatal dopamine signaling after traumatic brain injury in rats, J. Neurotrauma 29 (2012) 843e850. [17] A.E. Kline, J. Yu, E. Horvath, D.W. Marion, C.E. Dixon, The selective 5-HT(1A) receptor agonist repinotan HCl attenuates histopathology and spatial learning deficits following traumatic brain injury in rats, Neuroscience 106 (2001) 547e555. [18] R. Tehranian, M.E. Rose, V. Vagni, R.P. Griffith, S. Wu, S. Maits, X. Zhang, R.S. Clark, C.E. Dixon, P.M. Kochanek, O. Bernard, S.H. Graham, Transgenic mice that overexpress the anti-apoptotic Bcl-2 protein have improved histological outcome but unchanged behavioral outcome after traumatic brain injury, Brain Res. 1101 (2006) 126e135. [19] H. Liu, M.E. Rose, T.M. Miller, W. Li, S.N. Shinde, A.M. Pickrell, S.M. Poloyac, S.H. Graham, R.W. Hickey, COX2-derived primary and cyclopentenone
[20] [21]
[22]
[23]
[24]
[25]
[26]
655
prostaglandins are increased after asphyxial cardiac arrest, Brain Res. 1519 (2013) 71e77. J. Culman, Y. Zhao, P. Gohlke, T. Herdegen, PPAR-gamma: therapeutic target for ischemic stroke, Trends Pharmacol. Sci. 28 (2007) 244e249. S. Mandrekar-Colucci, A. Sauerbeck, P.G. Popovich, D.M. McTigue, PPAR agonists as therapeutics for CNS trauma and neurological diseases, ASN Neuro 5 (2013) e00129. B.D. Semple, L.J. Noble-Haeusslein, Broad-spectrum neuroprotection against traumatic brain injury by agonism of peroxisome proliferator-activated receptors, Exp. Neurol. 229 (2011) 195e197. S.W. Park, J.H. Yi, G. Miranpuri, I. Satriotomo, K. Bowen, D.K. Resnick, R. Vemuganti, Thiazolidinedione class of peroxisome proliferator-activated receptor gamma agonists prevents neuronal damage, motor dysfunction, myelin loss, neuropathic pain, and inflammation after spinal cord injury in adult rats, J. Pharmacol. Exp. Ther. 320 (2007) 1002e1012. M. Sobrado, M.P. Pereira, I. Ballesteros, O. Hurtado, D. Fernandez-Lopez, J.M. Pradillo, J.R. Caso, J. Vivancos, F. Nombela, J. Serena, I. Lizasoain, M.A. Moro, Synthesis of lipoxin A4 by 5-lipoxygenase mediates PPARgammadependent, neuroprotective effects of rosiglitazone in experimental stroke, J. Neurosci. 29 (2009) 3875e3884. S.C. Thal, M. Heinemann, C. Luh, D. Pieter, C. Werner, K. Engelhard, Pioglitazone reduces secondary brain damage after experimental brain trauma by PPAR-gamma-independent mechanisms, J. Neurotrauma 28 (2011) 983e993. A. Sauerbeck, J. Gao, R. Readnower, M. Liu, J.R. Pauly, G. Bing, P.G. Sullivan, Pioglitazone attenuates mitochondrial dysfunction, cognitive impairment, cortical tissue loss, and inflammation following traumatic brain injury, Exp. Neurol. 227 (2011) 128e135.