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1 JOURNAL OF NEUROCHEMISTRY | 2010 | 115 | 921929 doi: 10.1111/j.1471-4159.2010.06969.x ,1 , ,1 , *Department of Surgery, Chi Mei Medical Center, Tainan, Taiwan Department of Neurosurgery, Taipei Medical University-Shuang-Ho Hospital, Taipei, Taiwan Graduate Institute of Clinical Medicine, Taipei Medical University, Taipei, Taiwan Department of Electrical Engineering, Southern Taiwan University, Tainan, Taiwan Department of Biotechnology, Southern Taiwan University, Tainan, Taiwan Abstract aUTP nick-end labeling and neuronal-specific nuclear protein Antagonism of tumor necrosis factor-alpha with etanercept has double-positive cells), glial apoptosis (e.g., increased numbers proved to be effective in the treatment of spinal cord injury and of terminal deoxynucleotidyl transferase aUTP nick-end centrally endotoxin-induced brain injury. However, etanercept labeling and glial fibrillary acidic protein double-positive cells), may offer promise as therapy for traumatic brain injury (TBI). In astrocytic (e.g., increased numbers of glial fibrillary acidic this study, anesthetized rats, immediately after the onset of protein positive cells) and microglial (e.g., increased numbers TBI, were divided into two major groups and given the vehicle of ionized calcium-binding adapter molecule 1-positive cells) solution (1 mL/kg of body weight) or etanercept (5 mg/kg of activation and activated inflammation (e.g., increased levels of body weight) intraperitoneally once per 12 h for consecutive tumor necrosis factor-alpha, interleukin-1b and interleukin-6) 3 days. Etanercept caused attenuation of TBI-induced cere- were all significantly reduced by etanercept treatment. These bral ischemia (e.g., increased cellular levels of glutamate and findings suggest that etanercept may improve outcomes of TBI lactate-to-pyruvate ratio), damage (e.g., increased cellular by penetrating into the cerebrospinal fluid in rats. levels of glycerol) and contusion and motor and cognitive Keywords: apoptosis, etanercept, inflammation, traumatic function deficits. TBI-induced neuronal apoptosis (e.g., brain injury, tumor necrosis factor-alpha. increased numbers of terminal deoxynucleotidyl transferase J. Neurochem. (2010) 115, 921929. Traumatic brain injury (TBI), the leading cause of morbidity when administered systemically at the dosage approved for and mortality in young adults and children, is a major public its licensed indications ( 50 mg/week in humans), would health problem globally. TBI is associated with microglial not be expected to achieve therapeutic levels in the CSF and astrocytic activation as well as the release of proinam- because of its high molecular weight (Francis et al. 2004). matory cytokines (Lucas et al. 2006). The most studied proinammatory cytokines related to development of cere- bral edema, breakdown of the blood-brain barrier, and Received June 21, 2010; revised manuscript received August 4, 2010; secondary neuronal injury are interleukin (IL)-1, tumor accepted August 17, 2010. Address correspondence and reprint requests to Ching-Ping Chang, necrosis factor (TNF)-a and IL-6 (Hailer et al. 2005). Using Department of Biotechnology, Southern Taiwan University, Tainan, anti-inammatory agents for therapy of TBI to reduce blood- Taiwan. E-mail: [email protected] 1 brain barrier dysfunction, intracranial neutrophil inltration These authors contributed equally to this study. and neuronal cell death and to improve neurological outcome Abbreviations used: FPI, uid percussion injury; GFAP, glial brillary is a promising treatment (Stahel et al. 2000). acidic protein; Iba1, ionized calcium-binding adapter molecule 1; IL, interleukin; NeuN, neuronal-specic nuclear protein; PBS, phosphate Etanercept is a recombinant dimer of human TNF-a buffer saline; TBI, traumatic brain injury; TNF-a, tumor necrosis factor- receptor proteins fused and bound to human IgG 1. It is a alpha; TTC, triphenyltetrazolium chloride; TUNEL, terminal deoxynu- TNF antagonist with anti-inammatory effects. Etanercept, cleotidyl transferase dUTP nick-end labeling. 2010 The Authors Journal of Neurochemistry 2010 International Society for Neurochemistry, J. Neurochem. (2010) 115, 921929 921

2 922 | C.-C. Chio et al. More recent studies showed that etanercept was able to once per 12 h for consecutive 3 days (n = 8); (ii) TBI + etanercept intervene in the brain inammatory response and to protect group this group was the same as the TBI + saline group, with the brain and spinal cord from secondary damage caused by exception that etanercept at the dose of 5 mg/kg was administered inltrating leukocytes (Campbell et al. 2007; Genovese et al. intraperitoneally once per 12 h for consecutive 3 days (n = 8); (iii) Sham + saline group rats were subjected to the surgical 2006). This raised the possibility that experimental TBI could procedures as the above groups, with the exception that the FPI be affected by etanercept therapy. was not applied (n = 8); and (iv) Sham + etanercept group this To deal with the question, current experiments were group was the same as group 3, with exception that etanercept at the conducted to assess the motor and cognitive function, dose of 5 mg/kg was administered intraperitoneally once per 12 h cerebral ischemia and damage, neuronal and glial apoptosis, for consecutive 3 days (n = 8). gliosis, and expression of TNF-a, IL-1b and IL-6 in TBI- affected rats treated with or without etanercept. Experimental procedures In Experiment 1, an i.p. dose of etanercept (5 mg/kg) or saline (1 mL/kg) was randomly administered immediately after TBI, and Experimental procedures their effects on core temperature, mean arterial pressure, heart rate, and the hippocampus level of glutamate, glycerol, and the lactate- Animals to-pyruvate ratio were assessed after TBI for 120 min. ENBRELTM Male SpragueDawley rats (weight, 249 11 g) were obtained (etanercept) (Wyeth Pharmaceuticals, New Lane, Havant, Hamp- from the Animal Resource Center of the National Science Council shire, UK) was reconstituted with normal saline according to the of Republic of China (Taipei, Taiwan). The animals were housed manufacturers instructions. four in a group at an ambient temperature of 22 1C with a 12-h In Experiment 2, an i.p. dose of etanercept (5 mg/kg) or saline light-dark cycle. Pelleted rat chow and tap water were available was randomly administered immediately after TBI once per 12 h for ad libitum. All protocols were approved by the Animal Ethics consecutive 3 days, and their effect on the maximal angle animals Committee of the Chi Mei Medical Center (Tainan, Taiwan) to could cling to an inclined plane as well as passive avoidance minimize discomfort in the animals during surgery and in the performance was assessed 4 days after TBI. recovery period. In Experiment 3, an i.p. dose of etanercept (5 mg/kg) or saline was randomly administered immediately after TBI once per 12 h for Surgery consecutive 3 days, and their effect on cerebral contusion zone was Animals were anesthetized with sodium pentobarbital (25 mg/kg, assessed 4 days after TBI. An i.p. dose of 5 mg/kg of etanercept was intraperitoneally; Sigma Chemical, St. Louis, MO, USA) and a found to be effective for treating spinal cord injury in rats (Genovese mixture containing ketamine (4.4 mg/kg, intramuscularly; Nanku- et al. 2006). Therefore, the dosage of etanercept was adopted in the ang Pharmaceutical, Tainan, Taiwan), atropine (0.02633 mg/kg, present experiments. intramuscularly; Sintong Chemical, Ind., Taoyuan, Taiwan) and In Experiment 4, an i.p. dose of etanercept (5 mg/kg) or saline xylazine (6.77 mg/kg, intramuscularly; Bayer, Leverkusen, was randomly administered immediately after TBI once per 12 h for Germany). Both the femoral artery and vein on the right side consecutive 3 days, and their effect on the extent of neuronal and were cannulated with PE50 polyethylene tubing for monitoring glial apoptosis as well as gliosis was assessed 4 days after TBI. blood pressure and analyzing blood gas. After cannulation, the In Experiment 5, an i.p. dose of etanercept (5 mg/kg) or saline wound was sutured and animals were turned prone. The animals was randomly administered immediately after TBI once per 12 h for were placed in a stereotaxic frame, and the scalp was incised consecutive 3 days, and their effect on the relative levels of selected sagittally. The animals were subjected to a lateral uid percussion rat cytokines in serum was assessed 4 days after TBI. injury (FPI) (McIntosh et al. 1987). After the scalp was incised, a 4.8-mm circular craniotomy was performed midway between Monitoring cellular ischemia and damage markers in hippocampus lambda and bregma 3.0 mm to the right of the central suture. A The animals head was mounted in a stereotoxic apparatus (David modied Luer-lock connector (trauma cannula), 2.6 mm inner Kopf Instruments, Tujunga, CA, USA) with the nose bar positioned diameter, was secured into the craniotomy with cyanoacrylate 3.3 mm below the horizontal line. After a midline incision in the adhesive and dental acrylic. A moderate FPI (2.2 atm) was skull, a dialysis probe (4 mm in length CMA/2; Carnegie Medicine, produced by rapidly injecting a small volume of saline into the Stockholm, Sweden) was inserted. The microdialysis probe was closed cranial cavity with a uid percussion device (VCU stereotaxically and obliquely (anterior 4.3 mm) implanted into the Biochemical Engineering, Richmond, VA, USA). The animal right hippocampus (or dentate gyrus) according to the atlas and was removed from the device, the acrylic removed, and the coordinates of Paxinos and Watson (1982): P, 8 mm; R, 3 mm; H, incision sutured. Each injured and sham-injured animal for the FPI 5 mm. According to the methods described previously (Chio et al. model was closely evaluated immediately after FPI for behavioral 2007; Kuo et al. 2007), the micro-dialysis probe was perfused at recovery. Mean arterial pressure, heart rate and core temperature 2.0 lL/min and the dialysates were sampled in microvials. The were continuously monitored during 60 min after FPI. dialysates were collected every 20 min in a CMA/140 fraction collector (Carnegie Medicine). Aliquots of dialysates (5 lL) were Experimental groups injected onto a CMA 600 microdialysis analyzer (Carnegie Rats were randomly allocated into the following groups: (i) Medicine) for measurement of lactate, glycerol, pyruvate and TBI + saline group rats were subjected to TBI plus an glutamate. intraperitoneal (i.p.) dose of normal saline (mL/kg body weight) 2010 The Authors Journal of Neurochemistry 2010 International Society for Neurochemistry, J. Neurochem. (2010) 115, 921929

3 Etanercept in a traumatic brain injury | 923 Inclined plane then treated with a TUNEL reaction mixture (terminal deoxynu- The inclined plane was used to measure limb strength. The animals cleotidyl transferase nucleotide; Roche, Mannheim, Germany) at were placed, facing right and then left, perpendicular to the slope of 37C for 1 h, and then the sections were washed with distilled a 20 20-cm ruffer ribbed surface of an inclined plane starting at an water. They were then re-incubated in an anti-uorescein antibody- angle of 55o (Chang et al. 2008). The angle was increased or cojugated with horseradish peroxidase at 26C for 30 min, decreased in 5C increments to determine the maximal angle an rewashed, and then visualized using the avidin-biotin-peroxidase animal could hold to the plane. The data for each day were the mean complex technique and 0.05% 3, 3-diamino-benzidine tetrachloride of left and right side maximal angles. All behavioral tests were (Sigma Chemical) as a chromogen. The numbers of TUNEL- examined and independently scored by two observers who were positive cells were pathologist-counted in 30 elds/sections (200 unaware of prior treatment. These scores were averaged to arrive at magnication). The blinding was performed for the pathology one score for each animal for the behavioral session. grading of the results. Passive avoidance performance Cytokine assay This passive avoidance task is a one trial fear-motivated avoidance The concentrations of TNF-a, IL-1b, IL-6 and IL-10 in the serum task in which the rat learns to refrain from stepping through a door were determined using double-antibody sandwich enzyme-linked to an apparently safer but previously punished dark compartment. immunosorbent assay (R&D systems, Minneapolis, MN, USA) The latency to refrain from crossing into the punished compartment according to the manufacturers instructions. serves as an index of the ability to avoid, and allows memory to be assessed. The apparatus (Shuttlebox-Passive Avoidance, Accuscan Immunofluorescence staining Instrument, Inc., Columbus, Ohio, USA) consists of a rectangular Adjacent 50-lm sections, corresponding to coronal coordinates chamber divided into two compartments. One compartment is 0.200.70 mm anterior to the bregma, were incubated in 2 mol/L lighted by an overhead stimulus light and the other is black. The two HCl for 30 min, rinsed in 0.1 mol/L boric acid (pH 8.5) for 3 min compartments are separated by an automatic guillotine door and at 26C, and then incubated with primary antibodies in PBS each has a grid oor placed through which a foot shock can be containing 0.5% normal bovine serum at 4C overnight. The delivered. On test day (24 h after training), the rat is returned to the following primary antibodies were used in the present study: mouse lighted compartment, facing away from the dark compartment. After anti-glial brillary acidic protein (GFAP for astrocytes), mouse anti- 5 s, the guillotine door is lifted. When the rat enters the dark ionized calcium-binding adapter molecule 1 (Iba1; for microglia) compartment with four paws, the guillotine is closed, and the and mouse anti-neuronal-specic nuclear protein (NeuN for latency to enter the dark compartment is recorded (from the time the neurones). The sections were then detected with Alexa-Flour 568 door is lifted). The rat is removed and returned to the homecage. goat anti-mouse (IgG) antibody. The number of labeled cells was calculated in a coronal sections from each rat and expressed as the Cerebral contusion assay mean number of cells per section. For negative control sections, all The triphenyltetrazolium chloride (TTC) staining procedures fol- procedures were performed in the same manner without the primary lowed those described elsewhere (Wang et al. 1997). All the animals antibodies. were killed 4 days after TBI. Under deep anesthesia (sodium pentobarbital, 100 mg/kg, intraperitoneally), the animals were per- Statistical analysis fused intracardially with saline. The brain tissue was then removed, The data are presented as mean SD. The repeated measures immersed in cold saline for 5 min, and sliced into 2.0-mm sections analysis of variance was conducted to test the treatment-by-time with a tissue slice. The brain slices were incubated in 2% TTC interactions and the effect of treatment over time on each score. The dissolved in phosphate buffer saline (PBS) for 30 min at 37C and Duncans multiple range test was used for post hoc multiple then transferred to 5% formaldehyde solution for xation. The volume comparisons among means. p < 0.05 was considered evidence of of infarction, as revealed by negative TTC stains indicating statistical signicance. dehydrogenase-decient tissue, was measured in each slice and summed using computerized planimetry (Image-Pro Plus 5.0 soft- ware, Media Cybernetics, Inc., Bethesda, MD, USA). The volume of Results infarction was calculated as 2 mm (thickness of the slice) [sum of the infarction area in all brain slices (mm2)] (Wang et al. 1997). Etanercept did not affect physiological parameters changes during TBI Terminal deoxynucleotidyl-transferase-mediated and dUTP-biotin The physiological responses (including arterial blood pres- nick end-labeling assay sure, heart rate and core temperature) observed for (TBI plus The terminal deoxynucleotidyl transferase dUTP nick-end labeling saline)-treated group were insignicantly different from (TBI (TUNEL) assay was performed using the same brain tissues used in plus etanercept)-treated group. (The data are not shown here). histologic verication. The color was developed using 3,3-diamino- benzidine tetra chloride. The sections were xylene-and ethanol- Etanercept attenuated increased hippocampal levels of treated for parafn removal and for dehydration. They were then washed with PBS and incubated in 3% H2O2 solution for 20 min. cellular hypoxia and damage markers during TBI The sections were treated with 5 lg/mL proteinase k for 2 min at The values for hippocampal levels of glutamate, lactate- 26C and rewashed with PBS (0.1 M, pH 7.4). The sections were to-pyruvate ratio and glycerol in (TBI + saline)-treated rats 2010 The Authors Journal of Neurochemistry 2010 International Society for Neurochemistry, J. Neurochem. (2010) 115, 921929

4 924 | C.-C. Chio et al. were all signicantly higher at 20120 min after the start of icantly attenuated the TBI-induced alteration in microdialysis TBI than they were for the sham-operated controls (Fig. 1). concentrations of cellular ischemia (e.g., glutamate and Resuscitation with etanercept immediately after TBI signif- lactate-to-pyruvate ratio) and damage (e.g., glycerol) markers in hippocampus (Chio et al. 2007; Kuo et al. 2007; Chen 18 * * et al. 2009). * 16 * * 14 * * Etanercept improved motor and cognitive function during Glutamate (mol/L) 12 TBI At 4 days after TBI, behavioral tests revealed that the (TBI 10 plus saline)-treated rats had signicantly lower performance 8 in both motor and cognitive function tests than they were for + 6 sham-operated controls (Fig. 2). The TBI-induced motor and 4 + + + 2 + + 0 2 80 60 40 20 0 20 40 60 80 100 120 140 180 * * * * 160 * 140 + * * Glycerol (mol/L) 120 + 100 + 80 + 60 + 40 + 20 0 80 60 40 20 0 20 40 60 80 100 120 140 80 * * * 70 Lactate/pyruvate ratio 60 * * 50 * 40 + + 30 + 20 10 0 80 60 40 20 0 20 40 60 80 100 120 140 Time (min) Fig. 1 Etanercept decreased the TBI-induced increased levels of cellular ischemia and damage markers in hippocampus. The (TBI + Fig. 2 Etanercept decreased the TBI-induced motor (a) and cognitive saline)-treated group (s; n = 8) showed a significant increase of hip- (b) dysfunction. The (TBI + saline)-treated group (h; n = 8) showed a pocampal levels of glutamate, glycerol and lactate/pyruvate ratio at significant decrease of maximal angle but a significant increase of 20120 min after TBI (*p < 0.01) compared with the sham controls (d; latency to avoid at 3 days after TBI (*p < 0.01) compared with the n = 8). The (FPI + etanercept)-treated group (.; n = 8) had a signifi- sham controls ( ; n = 8). The (TBI + etanercept)-treated group cant decrease (+p < 0.01) of glutamate, glycerol and lactate/pyruvate (h; n = 8) had a significant increase (+p < 0.01) of maximal angle but ratio in hippocampus compared with the (TBI + saline)-treated group a significant decrease (+p < 0.01) of latency to avoid compared with (s; n = 8). TBI, trauma brain injury. the (TBI + saline)-treated group (h; n = 8). TBI, traumatic brain injury. 2010 The Authors Journal of Neurochemistry 2010 International Society for Neurochemistry, J. Neurochem. (2010) 115, 921929

5 Etanercept in a traumatic brain injury | 925 cognitive dysfunction could be signicantly reduced by etanercept treatment. Etanercept decreased contusion during TBI The TTC-stained sections at 4 days after TBI showed a signicant decrease in the contusion area of the etanercept- treated TBI group (Fig. 3). Both the cortical and hippo- campal contusion areas were distinctly smaller in the etanercept-treated rats than in saline-treated TBI ones. Etanercept attenuated systemic inflammation during TBI The serum levels of TNF-a, IL-1b and IL-6 decreased and IL-10 increased, respectively, in the etanercept-treated TBI (a) Fig. 4 Etanercept attenuated TBI-induced activated inflammation. The (TBI + saline)-treated group (h; n = 8) showed a significant increase of serum TNF-a, IL-1b and IL-6 at 3 days after TBI (*p < 0.01) compared with the sham controls (h; n = 8). The (TBI + etanercept)-treated group (h; n = 8) had a significant decrease of TNF-a, IL-1b and IL-6 (p < 0.01) compared with the (TBI + saline)- treated group. In addition, the (TBI + etanercept)-treated group had a significant increase of IL-10 at 3 days after TBI (p < 0.05) compared with the (TBI + saline)-treated group. Each column and bar denoted mean SD of eight animals per group (p < 0.05). (b) group (p < 0.05; Fig. 4) compared with the (TBI + saline)- treated group at 4 days after TBI. Etanercept decreased neuronal and glial apoptosis during TBI Both the TUNEL-stained and NeuN-stained sections at 4 days after TBI revealed a signicant decrease of the numbers of TUNEL-NeuN double positive cells in the ischemic brain of the Etanercept-treated TBI group (Fig. 5). In addition, both the TUNEL-stained and GFAP-stained sections at 7 days after TBI revealed a signicant decrease in the numbers of TUNEL-GFAP double positive cells in the ischemic regions of the etanercept-treated TBI group (Fig. 6). Etanercept decreased TBI-induced gliosis in core region during TBI The GFAP-stained sections at 4 days after TBI revealed a Fig. 3 Etanercept attenuated the TBI-induced contusion area. The signicant decrease of the numbers of GFAP-positive cells in (TBI + saline)-treated group (s;n = 8) showed a significant increase the contusion region of the etanercept-treated group com- of contusion volume at 3 days after TBI (*p < 0.01) compared with the pared with the (TBI + saline)-treated group (Fig. 7). In sham controls (d; n = 8). The (TBI + etanercept)-treated group (.; particular, the GFAP-positive cells in the contusion area of n = 8) had a significant decrease (+p < 0.01) of contusion volume the vehicle-treated group became swollen, which could be compared with the (TBI + saline)-treated group (a). TBI, traumatic brain. (b) Representative triphenyltetrazolium chloride staining of brain reduced by etanercept therapy. In addition, Iba1-stained slices for a sham control (i; d), a (TBI + saline)-treated rat (ii; s) and a sections at 4 days after TBI showed a signicant decrease of (TBI + etanercept)-treated rat (iii; .). Both cortical and hippocampal the numbers of Iba1-positive cells of the etanercept-treated areas were affected in the injured side after TBI. In particular, the TBI group compared with the (TBI + saline)-treated group dentate gyrus was involved. (Fig. 8). 2010 The Authors Journal of Neurochemistry 2010 International Society for Neurochemistry, J. Neurochem. (2010) 115, 921929

6 926 | C.-C. Chio et al. GFAP TUNEL Merged (a) (a) (b) (b) (c) (c) 50 m 50 m Fig. 5 Etanercept decreased TBI-induced neuronal apoptosis in Fig. 6 Etanercept decreased TBI-induced astrocytic apoptosis in ischemic brain regions. The TBI plus saline-treated group showed a ischemic brain. The TBI plus saline-treated group showed a significant significant increase of the numbers of NeuN-TUNEL double positive increase of the numbers of GFAP-TUNEL double positive cells in the cells in the ischemic brain 3 days after TBI compared with the sham ischemic cortex 3 days after TBI compared with the sham controls controls (*p < 0.01). The TBI plus Etanercept-treated group had a (*p < 0.01). The TBI plus Etanercept-treated group had a significant significant decrease of the numbers of NeuN-TUNEL double positive decrease (+p < 0.01) of the numbers of GFAP-TUNEL-positive cells cells compared with the TBI plus saline-treated group (p < 0.01). compared with the TBI plus saline-treated group. Each column and bar Each column and bar denoted mean SD of eight animals per group denoted mean SD of eight animals per group. Top panels depicted (c). Representative panel (a, b and c) stainings at 3 days after TBI, representative stainings at 3 days after TBI respectively for a sham respectively, for a sham control, a (TBI + saline)-treated rat, and a control (a), a (TBI + saline)-treated rat (b) and a (TBI + etanercept)- (TBI + etanercept)-treated rat. NeuN, neuronal-specific nuclear pro- treated rat (c). GFAP, glial fibrillary acidic protein; TUNEL, terminal tein; TUNEL, terminal deoxynucleotidyl transferase dUTP-end label- deoxynucleotidyl transferase dUTP-nick-end labeling; TBI, traumatic ing; TBI, traumatic brain injury. brain injury. et al. 1994). The experimental dose is therefore more than 70 Discussion times higher than the usual human therapeutic dose, which Antagonism of TNF-a with etanercept has proved to be one might expect to result in a higher dose of etanercept effective in the treatment of acute spinal cord injury delivered into the CSF, as there is experimental data that (Genovese et al. 2006) and centrally recombinant interleu- suggests that 0.10.5% of the dose of a systemically kin-1 or endotoxin-induced brain injury (Campbell et al. administered large molecule will reach the CSF (Banks 2007). The present results further showed that systemic 2004). Therefore, administration of a dose of etanercept 70 delivery of etanercept signicantly improved outcomes of times larger than the therapeutic dose in humans might result TBI in rats. It should be stressed that the etanercept doses in signicant penetration of etanercept into the CSF; used in the current experimental set-up, namely 5 mg/kg i.p. particularly in an experimental setting such as that involving per 12 h for three consecutive days (total dose of 30 mg/kg) TBI, in which the blood-cerebrospinal uid barrier might be are far higher than the normal subcutaneous dose used for damaged. Nevertheless, the present study is valuable and rheumatoid arthritis (about 0.4 mg/kg per 3 days) (Elliott provides scientic data that supports a potential therapeutic 2010 The Authors Journal of Neurochemistry 2010 International Society for Neurochemistry, J. Neurochem. (2010) 115, 921929

7 Etanercept in a traumatic brain injury | 927 (a) (b) (c) (a) (b) (c) 50 m Fig. 7 Etanercept decreased TBI-induced astrocyte gliosis in ische- Fig. 8 Etanercept decreased TBI-induced microglia gliosis in ische- mic brain. The TBI plus saline-treated group showed a significant in- mic brain. The TBI plus saline-treated group (b) showed a significant crease of the numbers of GFAP-positive cells in the ischemic core increase of the numbers of Iba1-positive cells in the ischemic core region 3 days after TBI compared with the sham controls (*p < 0.05). region 3 days after TBI compared with the sham controls (a) The TBI plus Etanercept-treated group had a significant decrease (*p < 0.05). The TBI + etanercept-treated group (c) had a significant (+p < 0.05) of the numbers of GFAP-positive cells compared with the decrease (+p < 0.05) of the numbers of Iba1-positive cells compared TBI plus saline-treated group. Each column and bar denoted with the TBI plus saline-treated group (b). Each column and bar mean SD of eight animals per group. Top panels depicted the GFAP denoted mean SD of eight animals per group. Top panels depicted stainings at 3 days after TBI for a sham control (a), a (TBI + saline)- the Iba1-stainings for a sham control (a), a (TBI + saline)-treated rat treated rat (b) and a (TBI + etanercept)-treated rat (c). The GFAP- (b) and a (TBI + etanercept)-treated rat (c). positive cells became swollen in TBI rats with saline therapy. role of etanercept in TBI. However, methods of administra- nist-soluble TNF-a receptor fusion protein at 15 min tion that may enhance delivery of etanercept into the CSF before and 1 h after TBI, improved performance in a (such as perispinal administration) (Tobinick 2010) may be series of standard motor tasks after injury (Knoblach et al. necessary to provide adequate and selective delivery of 1999). etanercept to the CSF for therapeutic purposes. Our data are In the current results, the TBI-induced overproduction of supported by several previous reports. For example, IL-1b, TNF-a and IL-6 in serum was signicantly reduced substantial basic science and clinical evidence suggests that by etanercept. In contrast, etanercept therapy signicantly excess TNF-a is centrally involved in the pathogenesis of increased the serum levels of IL-10 during TBI in rats. In Alzheimers disease (Tobinick and Gross 2008). Etanercept, fact, IL-10 had important anti-inammatory properties a biologic antagonist of TNF-a, delivered by perispinal through inhibiting the production of the pro-inammatory administration over a period of 6 months, has been reported cytokines TNF-a, IL-1b and IL-6, while upgrading the to induce rapid cognitive improvement in a patient with late- expression of IL-1 receptor antagonist (Cartmell et al. 2003). onset Alzheimers disease. A well characterized mouse Elevated plasma IL-10 levels had been reported in animals model where a systemic injection of interleukin-1b during with lipopolysaccharide injection (Wang et al. 2001) and in the rst ve postnatal days (inammatory insult) is combined patients with sepsis (van der Poll et al. 1997). In addition, with an intracerebral injection of the glutamatergic analogue systemic delivery of recombinant IL-10 protected mice form ibotenate (excitotoxic insult) at postnatal day 5 was used lethal endotoxaemia via reducing TNF-a release (Marchant (Aden et al. 2010). It was found that TNF-a blockade by et al. 1994). Neutralization of endogenously produced IL-10 etanercept given after the combined inammatory and resulted in an increased production of several proinamma- excitotoxic insult reduced brain damage by 50%. Using a tory cytokines and enhanced mortality in endotoxaemic mice rat model, it was found that systemic administration of (Standiford et al. 1995). IL-10-knockout mice had also been etanercept prior to IL-1b-microinjection into the anterior shown to have an increased likelihood of inammatory hypothalamus inhibited certain IL-1b-mediated sickness bowel disease (Rennick et al. 1997), a higher sepsis-induced behavior, such as the depression of open-eld activity and mortality rate (Berg et al. 1995) and an exacerbated and reduced glucose consumption (Jiang et al. 2008). Intracere- prolonged endotoxin-induced fever (Leon et al. 1999). broventricular administration of a selective TNF-a antago- Collectively, these data implied that etanercept may have 2010 The Authors Journal of Neurochemistry 2010 International Society for Neurochemistry, J. Neurochem. (2010) 115, 921929

8 928 | C.-C. Chio et al. improved outcomes of TBI in rats by inhibiting overproduc- In summary, the following behavioral, biochemical and tion of pro-inammatory cytokines. histopathologic characteristics in the contusion brain were Our data are conrmed by many previous results. Imme- noted after TBI: (i) motor and cognitive dysfunction; (ii) diate anti-TNF-a (etanercept) therapy enhanced the rate of cerebral ischemia and damage (evidenced by increased axonal regeneration after nerve injury in rats (Kato et al. levels of cellular glutamate, lactate-to-pyruvate ratio and 2010). TNF-a inhibited neurite outgrowth of cultured dorsal glycerol) (Chio et al. 2007; Kuo et al. 2007; Chen et al. root ganglia (Larsson et al. 2005) and hippocampal (Neu- 2009); (iii) cerebral contusion; (i.v.) systemic inammation mann et al. 2002) neurons. An estrogen sulfate ameliorated (evidence by increased levels of serum IL-1b, TNF-a and systemic inammation during TBI by decreasing TNF-a but IL-6); (v) neuronal and glial apoptosis; and (vi) gliosis of increasing IL-10 production in serum (Chen et al. 2009). In both astrocytes and microglia. All these destructive addition, indomethacin enhanced neurogenesis after experi- characteristics were favorably inuenced by etanercept mental stroke (Hoehn et al. 2005) and reduced intracranial therapy during TBI. In particular, etanercept may improve hypertension in patients of TBI (Rosmussen 2005). Endo- the outcomes of TBI by penetrating into the cerebrospinal toxin-induced inammation impaired neurogenesis (Ekdahl uid. et al. 2003), whereas blockade of inammation restored neurogenesis in adult brain following ischemic insult (Hoehn Acknowledgements et al. 2005). IL-10 had direct neuronal effects with important implications for neuroprotection (Zhou et al. 2009). This study was funded in part by the National Science Council of In addition to inammation, glial scar formation was a the Republic of China (Grant Nos. NSC 98-2314-B-218 -001 -MY2 signicant blockade to actual functional neuronal regener- and NSC 99-2314-B-384 -004 -MY3, and DOH99-TD-B-111-003, ation. An excellent recent review (Silver and Miller 2004) Center of Excellence for Clinical Trial and Research in Neuro- science. had proposed that to overcome the inhibitory environment of the glial scar might allow long-distance functional regener- ation after TBI. Indeed, as demonstrated in the current References results, the TBI-induced gliosis of both astrocytes and Aden U., Favrais G., Plaisant F., Winerdal M., Felderhoff-Mueser U., microglia were signicantly reduced by etanercept therapy. Lampa J., Lelievre V. and Gressens P. (2010) Systemic inam- Apparently, etanercept might enhance actual functional mation sensitizes the neonatal brain to excitotoxicity through a neuronal regeneration via inhibiting glial scar formation pro-/anti-inammatory imbalance: key role of TNFalpha pathway during TBI. and protection by etanercept. Brain Behav. Immun. 24, 747758. Banks W. A. (2004) Are the extracellular [correction of extracelluar] It has been well documented that the lactate/pyruvate ratio pathways a conduit for the delivery of therapeutics to the brain? and glutamate are well-known markers of cellular ischemia, Curr. Pharm. Des. 10, 13651370. whereas glycerol is a marker of how severely cells are Berg D. J., Kuhn R., Rajewsky K., Muller W., Menon S., Davidson N., affected by the ongoing pathology (Chou et al. 2003). Grunig G. and Rennick D. (1995) Interleukin-10 is a central reg- Excessive concentrations of glutamate have been noted in ulator of the response to LPS in murine models of endotoxic shock and the Shwartzman reaction but not endotoxin tolerance. J. Clin. ischemic brain tissue (Bullock et al. 1995; Nilsson et al. Invest. 96, 23392347. 1996). Our previous study (Chio et al. 2007) showed that the Bullock R., Zauner A., Woodward J. and Young H. F. (1995) Massive animals with TBI had higher values of extracellular levels of persistent release of excitatory amino acids following human glutamate, lactate-to-pyruvate ratio, and glycerol in ischemic occlusive stroke. Stroke 26, 21872189. cortex and intracranial pressure, but lower values of cerebral Campbell S. J., Jiang Y., Davis A. E., Farrands R., Holbrook J., Leppert D. and Anthony D. C. (2007) Immunomodulatory effects of eta- perfusion pressure. In the present study, we further demon- nercept in a model of brain injury act through attenuation of the strated that etanercept therapy signicantly attenuated the acute-phase response. J. Neurochem. 103, 22452255. TBI-induced increased cerebral ischemia and injury markers Cartmell T., Ball C., Bristow A. F., Mitchell D. and Poole S. (2003) in the ischemic cortex as early as 20 min post-injection. Endogenous interleukin-10 is required for the defervescence of Etanercept might cause attenuation of the TBI-induced fever evoked by local lipopolysaccharide-induced and Staphylo- coccus aureus-induced inammation in rats. J. Physiol. 549, cortical ischemia and damage via reducing intracranial 653664. hypertension and cerebral hypoperfusion that occurred Chang M. W., Young M. S. and Lin M. T. (2008) An inclined plane during the early phase of TBI. It should be stated that our system with microcontroller to determine limb motor function of experimental results, using doses 70 times larger than the laboratory animals. J. Neurosci. Methods 168, 186194. usual human therapeutic dose, cannot properly be extrapo- Chen S. H., Chang C. Y., Chang H. K., Chen W. C., Lin M. T., Wang J. J., Chen J. C. and Chang F. M. (2009) Premarin stimulates lated to infer that etanercept administered systemically at its estrogen receptor-alpha to protect against traumatic brain injury in usual therapeutic dose can be used to treat TBI in humans. In male rats. Crit. Care Med. 37, 30973106. addition, levels of etanercept in the CSF following systemic Chio C. C., Kuo J. R., Hsiao S. H., Chang C. P. and Lin M. T. (2007) administration of etanercept should be measured in future Effect of brain cooling on brain ischemia and damage markers after studies. uid percussion brain injury in rats. Shock 28, 284290. 2010 The Authors Journal of Neurochemistry 2010 International Society for Neurochemistry, J. Neurochem. (2010) 115, 921929

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