- Email: [email protected]
Suppression of Ischemia-Induced Hippocampal Pyramidal Neuron Death by Hyaluronan Tetrasaccharide through Inhibition of Toll-Like Receptor 2 Signaling Pathway
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62
The American Journal of Pathology, Vol. 186, No. 8, August 2016
ajp.amjpathol.org
Suppression of Ischemia-Induced Hippocampal Pyramidal Neuron Death by Hyaluronan Tetrasaccharide through Inhibition of Toll-Like Receptor 2 Signaling Pathway Q35
Q2
Takehiko Sunabori,*y Masato Koike,* Akira Asari,z Yoji Oonuki,x and Yasuo Uchiyamay From the Departments of Cell Biology and Neuroscience* and Cellular and Molecular Neuropathology,y Juntendo University Graduate School of Medicine, Bunkyo-Ku, Tokyo, Japan; the Department of Biomedical Engineering (ND20),z Lerner Research Institute, Cleveland Clinic Foundation, Cleveland, Ohio; and Glycoscience Laboratories Inc.,x Tokyo, Japan Accepted for publication March 28, 2016.
Q5
Q6
Address correspondence to Yasuo Uchiyama, M.D., Ph.D., Department of Cellular and Molecular Neuropathology, Juntendo University Graduate School of Medicine, 2-1-1 Hongo, Bunkyo-Ku, Tokyo 1138421, Japan. E-mail: y-uchi@ juntendo.ac.jp.
Toll-like receptors (TLRs) are one of the main contributors that induce inflammation under tissue injury and infection. Because excessive inflammation can aggravate disease states, it is important to control inflammation at a moderate level. Herein, we show that hyaluronan (HA) oligomer, HA tetrasaccharide (HA4), could suppress the expression of proinflammatory cytokine IL-1b when stimulated with both TLR2- and TLR4-specific agonists in primary hippocampal neurons. To understand the effect of HA4 against ischemic insult, we performed hypoxic-ischemic (H/I) brain injury against neonatal mice. HA4 treatment significantly prevented hippocampal pyramidal cell death even 7 days after H/I injury, compared with the control mice. Although TLR2 and TLR4 are known as receptors for HA and also act as a receptor for inducing inflammation, only TLR2-deficient mice showed tolerance against H/I injury. Moreover, HA4 administration suppressed gliosis by inhibiting the activation of NF-kB, the downstream target of TLR2, which led to the suppression of IL-1b expression. Taken together, our data suggest that the neuroprotective effect of HA4 relies on antagonizing the TLR2/NF-kB pathway to reduce inflammation through suppressing the expression of proinflammatory cytokines after neonatal H/I brain injury. (Am J Pathol 2016, 186: 1e9; http://dx.doi.org/10.1016/j.ajpath.2016.03.016)
Hypoxic-ischemic (H/I) brain injury in the perinatal period is known to link to subsequent neurological complications, such as seizure, cerebral palsy, mental retardation, and epilepsy.1 As recognized in neonates, symptomatic perinatal stroke occurs in one in 4000 live births.2 Morphological, biochemical, and genetic approaches have revealed that both apoptotic and nonapoptotic neuronal death takes place in the pyramidal layer of the hippocampus after H/I injury.3e5 Oxidative stress and glutamate excitotoxicity are suspected to be major executors that lead to cell death in the initial stage.1 However, reducing inflammation could prevent the progression of damage in the following step.6 An understanding of the molecular pathways underlying neuronal cell death induced by neonatal H/I brain injury could pave the way for new therapeutic approaches.
Toll-like receptors (TLRs) are the key receptors that initiate innate immune responses by recognizing specific molecular structures of pathogens known as pathogenassociated molecular patterns.7 However, TLRs are also stimulated by endogenous intracellular molecules released by activated or necrotic cells and extracellular matrix molecules that are up-regulated on injury or degraded after tissue damage, namely damage-associated molecular Supported by Japan Society for the Promotion of Science Kakenhi grants Q3 23390041 and 23659102 (Y.U.) and grants 23790239 and 26860139 (T.S.); the Scientific Research on Innovative Areas from the Ministry of Education, Science, Sports and Culture grants 23111004 and 23110517 (Y.U.); the MEXT-supported Program for the Strategic Research Foundation at Private Universities grant S1101009 (Y.U. and T.S.); and the Juntendo University Project Research grant-in-aid 2317 (T.S.). Q4 Disclosures: Y.O. is employed by Glycoscience Laboratories Inc.
Copyright ª 2016 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.ajpath.2016.03.016
FLA 5.4.0 DTD AJPA2352_proof 18 June 2016 7:53 am EO: AJP15_0460
63 64 65 66 67 68 69 70 71 72 73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 91 92 93 94 95 96 97 98 99 100 101 102 103 104 105 106 107 108 109 110 111 112 113 114 115 116 117 118 119 120 121 122 123 124
125 126 127 128 129 130 131 132 133 134 135 136 137 138 139 140 141 142 143 144 145 146 147 148 149 150 151 152 153 154 155 156 157 158 159 160 161 162 163 164 165 166 167 168 169 170 171 172 173 174 175 176 177 178 179 180 181 182 183 184 185 186
Sunabori et al patterns (DAMPs), even in the absence of infection.8,9 Accumulation of DAMPs initiates positive feedback loops of inflammation that increases tissue damage after impairment. Therefore, regulating the TLR signaling pathway is important for managing pathological conditions. Although hyaluronan (HA) is known as one of the DAMPs that stimulates TLR2 and TLR4,10,11 interestingly, the activity of HA depends on size. In general, high-molecularweight-HA (HMW-HA; 6 105 to 1 106 Da) is antiangiogenic, anti-inflammatory, and immunosuppressive, whereas low-molecular-weight-HA (LMW-HA; <5 104 Da) is inflammatory, immunostimulatory, and angiogenic.12 However, the definition of LMW-HA is ambiguous and little is known about small HA oligomers. Herein, we revealed the effect of a small HA oligomer, HA tetrasaccharide (HA4; 820.24 Da) against neonatal H/I brain injury. Several recent articles reported that HA4 treatment has improvement in a symptom after spinal cord injury13 or experimental autoimmune encephalomyelitis.14 However, little is known about the underlying molecular mechanisms. To further elucidate the effects of HA4 after neonatal H/I brain injury, we focused on the relationship between TLRs and the induction of inflammation.
Materials and Methods Animals
Q7 Q8 Q9
The procedures involving animal care, surgery, and sample preparation were approved by the Animal Experimental Committee of Juntendo University (Tokyo, Japan) and performed in accordance with the Guide for the Care and Use of Laboratory Animals.15 Neonatal C57BL/6J mice were obtained from Japan SLC (Shizuoka, Japan), and the C57BL/6 background TLR2 and TLR4 knockout (KO) mice were purchased from Oriental BioService (Kyoto, Japan). Mice were subsequently housed under specific pathogen-free conditions with a 12-hour light-dark cycle at Juntendo University.
Preparation of Hyaluronan Tetrasaccharide Preparation of HA4 was as follows. Hyaluronan (Shiseido, Tokyo, Japan) was digested in 0.3 mol/L methanesulfonic Q10 acid at 80 C for several hours (18 to 20 hours). The digest was diluted in water and then adjusted to a pH between 5 and 7 with sodium hydroxide before being loaded onto a column containing Dowex 1 4 anion exchange resin (Wako Chemical, Osaka, Japan; 200 to 400 mesh). The Q11 1 4 column was washed with an aqueous 40 mmol/L sodium chloride solution, and HA4 was eluted with a linear gradient consisting of 40 to 100 mmol/L sodium chloride. The Dowex 1 4 purified HA4 was adjusted to between pH 4 and pH 5 with HCl, diluted with water, and loaded onto a column containing BioRad High Q (anion exchange) resin (Bio-Rad Laboratories, Hercules, CA). The High Q column Q12 was washed with water, and HA4 was eluted with 0.5 mol/L sodium chloride. The pH of the eluted HA4 solution was adjusted to pH approximately 5 with HCl, concentrated by rotary evaporation, and then desalted by passing it through a BioGel P2 size exclusion column (Bio-Rad Laboratories) using water as the eluant. The desalted HA4 was purified with a Dowex 50Wx4 column (Wako Chemical) in the acid form. This purified HA4 was passed through ultrafilter membranes with a molecular weight cutoff of 10,000 Da (Merck Millipore, Darmstadt, Germany), concentrated, and then dried to a Q13 powder in a rotary evaporator. The purity of HA4 was determined by high-performance liquid chromatography.
Hyaluronan Administration HA4 was administered i.p. to P6 mouse pups 12 hours before, and just before, H/I injury at P7 with a concentration of 10 mg/kg body weight. Saline administration was performed against littermates or age-matched mice as a control.
Sample Preparation for Histochemical and Morphological Analyses
Hypoxic-Ischemic Injury Neonatal H/I brain injury was induced in mice on post-natal day 7 (P7), essentially according to the Rice-Vannucci model16 with minor modifications.17 After the mice were deeply anesthetized with isoflurane (2%), the left common carotid artery was dissected and ligated with silk sutures (6/0), followed by a recovery for 1 hour at 37 C. They were then placed in chambers maintained at 37 C with a flow purged with nitrogen gas to control oxygen concentration to 8% for 25 minutes. After hypoxic exposure, the pups were returned to their dams and were allowed to recover for 1, 3, 6, 12, and 24 hours, and 7 days. At each stage, the brains were processed for molecular, biochemical, and morphological analyses. This procedure resulted in brain injury in the ipsilateral hemisphere, consisting of cerebral infarction mainly in the hippocampus.17e19 Pups with severe damage in the cortex
2
were eliminated from the experiment. Control littermates were neither operated on nor subjected to hypoxia.
After H/I injury, mice were deeply anesthetized and fixed by cardiac perfusion with 4% paraformaldehyde buffered with 0.1 mol/L phosphate buffer (pH 7.2) containing 4% sucrose. Brain tissues were quickly removed from the mice and further immersed in the same fixative overnight at 4 C. Samples processed for paraffin embedding were cut into sections (4-mm thick) with a semimotorized rotary microtome (RM2245; Leica, Wetzlar, Germany) and placed on Q14 silane-coated glass slides. For routine histological studies, paraffin sections were stained with hematoxylin and eosin.
Immunoblot Analysis Each tissue was independently homogenized in RIPA buffer [50 mmol/L Tris-HCl (pH 7.6) with 150 mmol/L
ajp.amjpathol.org
-
The American Journal of Pathology
FLA 5.4.0 DTD AJPA2352_proof 18 June 2016 7:53 am EO: AJP15_0460
187 188 189 190 191 192 193 194 195 196 197 198 199 200 201 202 203 204 205 206 207 208 209 210 211 212 213 214 215 216 217 218 219 220 221 222 223 224 225 226 227 228 229 230 231 232 233 234 235 236 237 238 239 240 241 242 243 244 245 246 247 248
249 250 251 252 253 254 255 256 257 258 259 260 261 262 263 264 265 266 267 268 269 270 271 272 273 274 275 276 277 278 279 280 281 282 283 284 285 286 287 288 289 290 291 292 293 294 295 296 297 298 299 300 301 302 303 304 305 306 307 308 309 310
HA4 Protects Neuron from H/I Q1
Q15
Q16
Q17
NaCl, 1% NP-40, 0.5% sodium deoxycholate, and 0.1% SDS] containing a protease inhibitor-cocktail (Nacalai, Kyoto, Japan) and a phosphatase inhibitor-cocktail (Nacalai). After centrifugation at 20,000 g for 10 minutes at 4 C, the protein concentrations in the supernatants were determined using the BCA protein assay system (Pierce, Rockford, IL), and were used for immunoblotting. The lysates were eluted by boiling in loading buffer, separated by SDS-PAGE, and transferred to an Immobilon P-membrane (Millipore). The blots were probed with antieNF-kB p65 antibodies (1:1000 dilution; Cell Signaling Technology, Danvers, MA) and antiephospho-NF-kB p65 (Ser536) antibodies (1:500; Cell Signaling Technology).
Primary Cultures of Hippocampal Neurons Primary hippocampal neurons were prepared for culturing, as previously described.20 Briefly, the hippocampi were dissected and dissociated from 18-day-old fetal mice. Dissociated hippocampal neurons were plated on poly-Lornithine (Sigma) coated 6-well plates with 2% B27 (Invi- Q27 trogen, San Diego, CA) and 2 mmol/L L-glutamine (Nacalai) Q28 in Neurobasal medium (Invitrogen). Two days later, 10 mmol/L cytosine arabinoside (Nacalai) was added to inhibit nonneuronal growth. After 14 days, hippocampal neurons were stimulated with 1 mg/mL Pam3CSK4 (Invivogen) and 1 mg/mL LPS-SM Ultrapure (Invivogen) in the presence (50 ng/mL, 5 mg/mL) or absence of HA4 for 6 hours on day 14.
Immunohistochemical Analysis Q18
Q19 Q20
Q21 Q22
Q23
Deparaffinized sections were incubated overnight at 4 C with anti-NeuN antibodies (1:200; Merck Millipore), anti-Iba1 antibodies (1:200; Wako), anti-Mac2 antibodies (1:200; Cedarlane, Burlington, NC), and antieglial fibrillary acidic protein antibodies (1:200; Sigma, St. Louis, MO). Sections were further reacted with peroxidase-conjugated secondary antibodies at room temperature for 2 hours and washed three more times. To visualize the sections, they were reacted with peroxidase, using 0.0125% 3,30 -diaminobenzidine tetrahydrochloride and 0.002% H2O2 in 0.05 mol/L Tris-HCl buffer (pH 7.6) for 5 minutes. For immunofluorescence staining antimouse, anti-rabbit IgG Alexa Fluor 488 antibodies (Thermo Fisher Scientific, Rockford, IL), and anti-rat Cy3 antibodies (Jackson ImmunoResearch Laboratories, West Grove, PA) were used as secondary antibodies. Immunostained sections were observed using a BX50 (Olympus, Tokyo, Japan) and confocal laser scanning microscope FV1000 (Olympus).
qPCR and RT-PCR
Q24 Q25
Q26
For real-time quantitative PCR (qPCR) and RT-PCR analyses, total RNA was prepared using RNeasy (Qiagen, Hilden, Germany) and cDNA was synthesized using a PrimeScript master mix (Takara, Shiga, Japan), according to the manufacturer’s recommendations. The expression level of IL-1b for qPCR was analyzed using a Thunderbird SYBR qPCR mix (Toyobo, Osaka, Japan) on thermal cycler dice (Takara). The primer pairs were as follows: IL-1b, 50 -ACCTGTGGCCTTGGGCCTCAAA-30 (forward) and 50 -TTGGGATCCACACTCTCCAGCTGC-30 (reverse); b-actin, 50 -GGTGGGCCGCCCTAGGCACCA-30 (forward) and 50 -TTGGCCTTAGGGTTCAGGGGG-30 (reverse). The expressions of TLR2, TLR4, and b-actin were analyzed using KOD-plus (Toyobo). The primer pairs were as follows: TLR2, 50 -TGGTGTCTGGAGTCTGCTGTG-30 (forward) and 50 -CGCTCCGTACGAAGTTCTCAG-30 (reverse); TLR4, 50 -CAGTGGTCAGTGTGATTGTGG-30 (forward) and 50 -TTCCTGGATGATGTTGGCAGC-30 (reverse); and b-actin, 50 -GGTGGGCCGCCCTAGGCACCA-30 (forward) and 50 -TTGGCCTTAGGGTTCAGGGGG-30 (reverse).
The American Journal of Pathology
-
TUNEL Staining Terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick end labeling (TUNEL) staining was applied to deparaffinized sections as previously described.21 Briefly, sections were incubated with 100 U/mL TdT (Promega, Madison, WI) and 10 nmol/L biotinylated 16-20 -dUTP Q29 (Roche Diagnostics, Basel, Switzerland) in TdT buffer (100 Q30 mmol/L sodium cacodylate, pH 7.0, 1 mmol/L cobalt chloride, 50 mg/mL gelatin) in a humid atmosphere at 37 C for 1 hour, followed by further incubation with peroxidaseconjugated streptavidin (Vector Laboratories, Burlingame, CA) for 1 hour at room temperature. Staining for peroxidase Q31 was performed as stated above.
Cell Counting The number of TUNEL-positive cells was counted in the whole pyramidal regions of the hippocampus, located in the same portion (bregma: -1.82 mm of adult C57BL/J mice), according to the previous report.17
Neuropathological Evaluation H/I injury and neuroprotection were evaluated by the number of TUNEL-positive (dead) cells in the pyramidal layers of the hippocampus 24 hours after H/I injury using brain sections corresponding to the same portion of the hippocampus as mentioned above. The damaged area in the hippocampal pyramidal layers was also examined 7 days after H/I injury, in which time clear-cut shrinkage occurred in the ipsilateral hippocampus of the control mice, resulting in a loss of ipsilateral hippocampal neurons. To quantitate these damaged areas after neonatal H/I, the areas of lesioned and unlesioned hippocampi were measured and compared within the same section corresponding to the same portions mentioned above, using ImageJ software version 1.38 (NIH, Bethesda, MD). The Q32 area loss of the lesioned ipsilateral hippocampus was calculated as a percentage of the intact contralateral hippocampus, and was determined for each animal.
ajp.amjpathol.org
FLA 5.4.0 DTD AJPA2352_proof 18 June 2016 7:53 am EO: AJP15_0460
3
311 312 313 314 315 316 317 318 319 320 321 322 323 324 325 326 327 328 329 330 331 332 333 334 335 336 337 338 339 340 341 342 343 344 345 346 347 348 349 350 351 352 353 354 355 356 357 358 359 360 361 362 363 364 365 366 367 368 369 370 371 372
Sunabori et al 373 Statistical Analysis 374 375 All quantified data were expressed as the means SEM. 376 Differences between two mean values were evaluated using 377 an unpaired Student’s t-test and two-way analysis of vari378 ance post-hoc tests that were considered statistically sig379 nificant when the P value was <0.05. 380 381 382 Results 383 384 HA4 Antagonizes Both TLR2 and TLR4 Signaling 385 Pathway in Primary Hippocampal Neurons 386 387 The hippocampus is the brain region that is most vulnerable 388 to cerebral ischemic insult,18,19 and Toll-like receptors 389 (TLRs) are known to be involved in neuronal cell death after 390 ischemia.22e25 To determine the effect of a small HA 391 oligomer, HA tetrasaccharide (HA4; 820.24 Da), we 392 focused on the TLR signaling pathway for three reasons: i) 393 TLR2 and TLR4 are known as the receptors for HA10,11; ii) 394 stimulation of TLRs would activate NF-kB and induce 395 proinflammatory cytokines7; and iii) primary cortical neu396 397 rons generated from TLR2-KO and TLR4-KO mice are 398 resistant to glucose deprivation.22 Therefore, we first 399 explored whether HA4 could participate in the TLR/NF-kB 400 signaling pathway in primary hippocampal neurons. To 401 confirm the expressions of TLR2 and TLR4 in primary 402 hippocampal neurons, we performed reverse transcriptase403 PCR. Primary hippocampal neurons cultured for 14 days 404 in vitro expressed both TLR2 and TLR4 as well as the 405 microglial cell line BV2 (Figure 1A). Thus, we stimulated ½F1 406 TLR2 and TLR4 with specific agonists, Pam3CSK4 and 407 LPS, respectively, and observed the expression of the 408 409 proinflammatory cytokine IL-1b by qPCR in the presence 410 and absence of HA4. The increased expression of IL-1b by 411 Pam3CSK4 stimulation (2.7-fold) and LPS stimulation (2.8412 fold) was suppressed in a dose-dependent manner by HA4 413 administration (50 ng/mL, 5 mg/mL of HA4, respectively), 414 whereas no change was observed when HA4 was adminis415 tered alone (Figure 1B). These data suggest that HA4 can 416 play an antagonistic role against TLR2 and TLR4 in the 417 hippocampal neuron in vitro. 418 419 420 HA4 Administration Protects Hippocampal Pyramidal 421 Neurons from Neonatal H/I Injury 422 423 To elucidate the effect that HA4 administration exerts 424 against cerebral ischemic insult in vivo, we next examined 425 the damage in the pyramidal regions of the hippocampus in 426 neonatal mouse brains after H/I injury. HA4 (10 mg/kg 427 body weight) was administered i.p., 12 hours, and imme428 diately before H/I injury, and physiological saline was 429 injected as a control. The degree of damage in the hippo430 431 campal pyramidal cells, quantified as the number of 432 TUNEL-positive nuclei, was extremely reduced in mice 433 treated with HA4, compared with those treated with saline 434
4
Figure 1
HA4 antagonizes both TLR2 and TLR4 signaling pathway in primary hippocampal neurons. A: Expression of TLR2 and TLR4 mRNA was confirmed by reverse transcriptase-PCR in lysates from BV2 cells (lane 1; a microglial cell line), primary hippocampal neurons (lane 2), TLR2-KO mouse brain (lane 3), and TLR4-KO mouse brain (lane 4). B: Quantification of expression levels of IL-1b mRNA in primary hippocampal neurons cultured in the presence of Pam3CSK4 and lipopolysaccharide (LPS) with 50 ng/mL and 5 mg/mL of HA4 or without HA4, as measured by real-time quantitative PCR. Data are expressed as means SEM (B). n Z 4 independent experiments (B). *P < 0.05.
Q34
24 hours after H/I injury (Figure 2, AeD). The number of ½F2 TUNEL-positive nuclei within the pyramidal layer of mouse brains (Figure 2, B and D) that were administered with HA4 was significantly decreased (179 19) to fewer than half the number of those treated with saline (377 51) (Figure 2E). We farther evaluated the number of TUNELpositive nuclei, in the CA1 and CA3 region of the hippocampal pyramidal cell layer. The number of TUNELpositive nuclei within CA1 region (Figure 2F) significantly decreased by HA4 administration (44 10) compared with saline (214 30). However, although slight reduction of the number of TUNEL-positive nuclei was also observed, no significance was detected between HA4 (17 6) and saline (61 24) administration in the CA3 region (Figure 2F). These data suggest that HA4 administration can protect hippocampal pyramidal cells after neonatal H/I brain injury in a region-specific manner.
Pathological Alterations Are Not Detected in the Hippocampal Pyramidal Neurons of a HA4-Administered Neonatal Hippocampus 7 Days after H/I Injury Delayed neuronal death reportedly occurs in the pyramidal layers of the hippocampus within 7 days after transient brain ischemia, and damaged areas are replaced by glial cells.19
ajp.amjpathol.org
-
The American Journal of Pathology
FLA 5.4.0 DTD AJPA2352_proof 18 June 2016 7:53 am EO: AJP15_0460
435 436 437 438 439 440 441 442 443 444 445 446 447 448 449 450 451 452 453 454 455 456 457 458 459 460 461 462 463 464 465 466 467 468 469 470 471 472 473 474 475 476 477 478 479 480 481 482 483 484 485 486 487 488 489 490 491 492 493 494 495 496
print & web 4C=FPO
Figure 2
HA4 administration protects hippocampal pyramidal cells from neonatal H/I injury. Representative demonstration of the ipsilateral hippocampi of mice administered saline (A and B) and HA4 (C and D), respectively, stained with hematoxylin and eosin (H&E; A and C) and for terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL; B and D) 24 hours after H/I injury. E: Quantitative analysis of the number of TUNELpositive cells in the pyramidal layer of an identical portion (bregma: -1.82 mm of adult C57BL/J mice) of the hippocampus, administered saline and HA4, 24 hours after H/I injury. F: Quantitative analysis of the number of TUNEL-positive cells in the CA1 and CA3 region of an identical portion (bregma: -1.82 mm of adult C57BL/J mice) of the hippocampus, administered saline and HA4, 24 hours after H/I injury. Data are expressed as means SEM (E and F). n Z 13 and 14 mice per group (E); n Z 8 and 6 mice per group (F). **P < 0.01, ***P < 0.001. Scale bar Z 200 mm (AeD).
print & web 4C=FPO
497 498 499 500 501 502 503 504 505 506 507 508 509 510 511 512 513 514 515 516 517 518 519 520 521 522 523 524 525 526 527 528 529 530 531 532 533 534 535 536 537 538 539 540 541 542 543 544 545 546 547 548 549 550 551 552 553 554 555 556 557 558
HA4 Protects Neuron from H/I 559 control littermate mice 7 days after H/I injury. Loss of 560 NeuN-positive neurons with their neurites that was elimi561 nated by microglial cells contributed to the areal reduction 562 of pyramidal layers of the control mouse hippocampus 563 (Figure 3, C and D). The total area of the ipsilateral hip- ½F3 564 pocampus was significantly diminished, compared with the 565 contralateral hippocampus (Figure 3, A and B). Meanwhile, 566 in the HA4-administered mice, the remaining pyramidal 567 neurons were largely TUNEL-negative (data not shown), 568 569 and staining for hematoxylin and eosin, and NeuN showed 570 those neurons to be intact (Figure 3, AeF). The ipsilateral 571 hippocampal area in the HA4-administered mice was only 572 slightly diminished to 96% 9% compared with the 573 contralateral hippocampi (Figure 3, B and G). However, in 574 the physiological saline-injected littermate controls, the 575 ipsilateral hippocampal area was reduced to nearly half the 576 contralateral hippocampal area 7 days after H/I injury 577 (53% 7%) (Figure 3, A and G). These data indicate that 578 the prevention of hippocampal pyramidal cell death by HA4 579 administration is sustained at least until 7 days after H/I 580 581 injury.
582 583 584 The Neuroprotective Effect of HA4 Largely Relies on the 585 Inhibition of TLR2, but Not TLR4 in Neonatal H/I Injury 586 587 HA4 antagonized both TLR2 and TLR4 in primary hippo588 campal neurons in vitro (Figure 1) and protected hippo589 campal pyramidal cells after H/I injury in vivo (Figures 2 590 and 3). However, the major target of the neuroprotective 591 role of HA4 after neonatal H/I injury remained unclear. To 592 confirm the relationship between TLR2, TLR4, and HA4 593 To exclude the possibility that the administration of HA4 in vivo, we used TLR2-KO and TLR4-KO mice and applied 594 simply delayed the onset of pyramidal cell death, we further them to H/I injury. When saline was administered, the 595 analyzed the degree of damage in the neurons of the degree of damage in the hippocampal pyramidal cells was 596 extremely reduced in TLR2-KO mice (Figure 4C), compared ½F4 597 hippocampal pyramidal layers in HA4-administered and 598 599 600 601 602 603 604 605 606 607 608 609 610 611 612 613 614 615 Figure 3 Prevention of neuronal loss in the hippocampus by HA4 administration is further sustained up to 7 days after H/I injury. Representative 616 demonstration of the hippocampi of mice administered saline (A, C, and E) or HA4 (B, D, and F) stained with hematoxylin and eosin (H&E; A and B) and anti617 NeuN antibodies (CeF) 7 days after H/I injury. G: Evaluation of the area of ipsilateral (Ipsi) against contralateral (Contra) hippocampi of HA4-treated and 618 littermate control mouse brains of an identical portion (bregma: -1.82 mm of adult C57BL/J mice) of the hippocampus 7 days after H/I injury. Data are 619 expressed as means SEM (G). n Z 9 and 7 mice per group (G). **P < 0.01. Scale bars: 400 mm (AeD); 200 mm (E and F). 620
The American Journal of Pathology
-
ajp.amjpathol.org
FLA 5.4.0 DTD AJPA2352_proof 18 June 2016 7:53 am EO: AJP15_0460
5
together, these data indicate that the major target of HA4 after neonatal H/I brain injury was TLR2 but not TLR4.
HA4 Administration Inhibits the Expression of Proinflammatory Cytokine IL-1b through Inactivating NF-kB after H/I Injury
print & web 4C=FPO
621 622 623 624 625 626 627 628 629 630 631 632 633 634 635 636 637 638 639 640 641 642 643 644 645 646 647 648 649 650 651 652 653 654 655 656 657 658 659 660 661 662 663 664 665 666 667 668 669 670 671 672 673 674 675 676 677 678 679 680 681 682
Sunabori et al
Figure 4 TLR2 is the major target for the neuroprotective effect of HA4 after H/I injury in vivo. Representative demonstration of the ipsilateral hippocampus of wild-type (A and B), TLR2-KO (CeF), and TLR4-KO (GeJ) mice administered saline (AeD, G, and H) or HA4 (E, F, I, and J), and obtained 24 hours after H/I injury. A, C, E, G, and I: Hematoyxlin and eosin (H&E) staining. B, D, F, H, and J: Terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) staining. K: Quantification of the number of TUNELpositive cells in the pyramidal layer of an identical portion (bregma: -1.82 mm of adult C57BL/J mice) of the hippocampus of wild-type, TLR2-KO, and TLR4-KO mice administered saline or HA4 and obtained 24 hours after H/I injury. Data are expressed as means SEM (K). n Z 6 or 7 mice per group (K). *P < 0.05, **P < 0.01. Scale bar Z 200 mm (AeJ).
with both TLR4-KO (Figure 4G) and wild-type mice (Figure 4A) 24 hours after H/I injury, which was consistent with previous reports.23,24 Moreover, the improvement after administration of HA4 was observed only in TLR4-KO mice, but not in TLR2-KO mice (Figure 4, E and I). The TUNEL-positive cells within the ipsilateral hippocampal pyramidal layer (Figure 4, B, D, F, H, and J) were significantly decreased in TLR2-KO mice (130 16), compared with wild-type mice (280 19), and no additional improvement was observed after the administration of HA4 (113 24) (Figure 4K). In contrast, the TUNEL-positive cells in TLR4-KO mice (268 26) showed comparable level to the control mice, whereas the TUNEL positivity was significantly suppressed and the insult was improved by HA4 administration (194 15) (Figure 4K). Taken
6
H/I injury induces an inflammatory reaction in the neonatal brain, and the early proinflammatory phase is thought to deteriorate pathological states.6 Inhibition of proinflammatory cytokines, such as IL-1b26 and IL-18,27 and the activator of IL-1 and IL-18, caspase-1,28 reduces H/I brain injury to a moderate degree. Therefore, we evaluated the expression level of IL-1b in the ipsilateral hippocampus with or without HA4 administration and compared with TLR2-KO mice by qPCR. The expression level of IL-1b increased within an hour and peaked 6 hours after H/I injury in the control mice (Figure 5A). However, HA4 adminis- ½F5 tration, as well as TLR2-KO mice, significantly inhibited IL-1b expression 3 and 6 hours after H/I injury (Figure 5A). To further confirm the molecular mechanism regulating the expression of IL-1b, we next verified the activity of the NF-kB/Rel family. The Ser536 residue of p65/RelA, a member of the NF-kB/Rel family, is phosphorylated when NF-kB is activated and translocates to the nucleus to regulate gene expression including IL-1b.29 Western blot analyses revealed that the activation of NF-kB in the ipsilateral hippocampi, represented as an expression ratio of phospho-p65/RelA (Ser536) per pan-p65/RelA, increased 5.2-fold within an hour in control mice when compared with sham-operated mice, whereas the activation was significantly suppressed to 1.4-fold by HA4 administration and 0.98-fold in TLR2-KO mice (Figure 5, B and C). These data indicate that HA4 administration can inhibit the expression of proinflammatory cytokine IL-1b in vivo, in a closely related manner to TLR2-KO mice, by inactivating NF-kB in the early stages after H/I Injury.
HA4 Administration Inhibits Gliosis after H/I Injury Although the expression of IL-1b mRNA was suppressed by HA4 administration (Figure 5), the activation (cleavage for the secretion form) of IL-1b, which causes inflammation, was undetectable by both Western blot analyses and enzyme-linked immunosorbent assay analyses after H/I brain injury. Therefore, we focused on gliosis as the hallmark of inflammation. Gliosis consists of the activation of astrocytes and microglial cells, stimulated by proinflammatory cytokines, including IL-1b. The activation of microglial cells and astrocytes was investigated by evaluating the expression of Mac2 (for reactive microglial cells), Iba1 (for pan-microglial cells), and glial fibrillary acidic protein (for reactive astrocytes) 24 hours after H/I injury (Figure 6). Interestingly, the Iba1-positive microglial cells ½F6 slightly increased the number and many coexpressed Mac2 in the control hippocampus (Figure 6A), whereas HA4
ajp.amjpathol.org
-
The American Journal of Pathology
FLA 5.4.0 DTD AJPA2352_proof 18 June 2016 7:53 am EO: AJP15_0460
683 684 685 686 687 688 689 690 691 692 693 694 695 696 697 698 699 700 701 702 703 704 705 706 707 708 709 710 711 712 713 714 715 716 717 718 719 720 721 722 723 724 725 726 727 728 729 730 731 732 733 734 735 736 737 738 739 740 741 742 743 744
suppressed the emergence of reactive astrocytes (Figure 6, C and D). Taken together, these data indicate that HA4 Q33 administration protects neuronal cell death through suppressing inflammation via inhibiting the expression of proinflammatory cytokine IL-1b.
Discussion Inflammation is induced by both tissue injury and infection for facilitating wound healing and stimulating innate immunity. However, excessive inflammation can also aggravate symptoms. Although lines of evidence from KO animals have revealed that TLRs are one of the main contributors not only to pathogen-induced inflammation, but also to injury-induced inflammation in various organs,30 the strategies to attenuate inflammation after tissue damage have been limited. Herein, we demonstrated the following five points: i) HA4 exhibited antagonistic action against both TLR2 and TLR4 in vitro; ii) HA4 administration prevented hippocampal pyramidal neuron death for at least 7 days after neonatal H/I brain injury; iii) the main target of this inhibition was TLR2 but not TLR4 in vivo; iv) HA4 administration decreased the expression of proinflammatory cytokine IL-1b by inactivating the NF-kB/Rel family; and v) HA4 administration prevented inflammation. HA has a high molecular mass (>106 Da) under physiological conditions. However, in inflammatory tissues, the activation of hyaluronidase degrades HMW-HA into small
Figure 5 Inhibitory effect of HA4 on the expression of a proinflammatory cytokine, IL-1b, through inactivation of NF-kB after H/I injury. A: Quantification of IL-1b mRNA levels in ipsilateral hippocampi obtained from HA4- or saline-treated and TLR2-KO mice at indicated time periods after H/I injury. mRNA levels were measured by real-time quantitative PCR. B: Western blot analyses of phospho-p65/RelA (Ser536) and pan-p65/RelA in the ipsilateral hippocampi from HA4- or saline-administered and TLR2-KO mouse brains. Duplicated data are presented in protein extracts from the brains of mice injected with HA4 or saline and TLR2-KO. C: Ratios of protein amounts of phospho-p65/RelA (Ser536)/pan-p65/RelA in the ipsilateral hippocampi administered HA4 or saline and TLR2-KO. Data are expressed as means SEM (A and C). n Z 3 to 7 mice per group (A); n Z 4 mice per group (C). *P < 0.05.
administration diminished the expression of Iba1 to the steady state and Mac2-expressing cells were not observed (Figure 6B). Moreover, the expression of glial fibrillary acidic protein was strongly elevated only in the control hippocampus, suggesting that HA4 administration
The American Journal of Pathology
-
print & web 4C=FPO
745 746 747 748 749 750 751 752 753 754 755 756 757 758 759 760 761 762 763 764 765 766 767 768 769 770 771 772 773 774 775 776 777 778 779 780 781 782 783 784 785 786 787 788 789 790 791 792 793 794 795 796 797 798 799 800 801 802 803 804 805 806
HA4 Protects Neuron from H/I
Figure 6 The emergence of Mac2-expressing microglial cells and reactive astrocytes is suppressed by HA4 administration 24 hours after H/I injury. Representative demonstration of the ipsilateral hippocampi administered saline (A and C) and HA4 (B and D) stained with anti-Iba1 antibodies (green; A and B), antieglial fibrillary acidic protein (GFAP) antibodies (green; C and D), anti-Mac2 antibodies (red; AeD), and DAPI (blue; AeD). Scale bar Z 50 mm (AeD).
ajp.amjpathol.org
FLA 5.4.0 DTD AJPA2352_proof 18 June 2016 7:53 am EO: AJP15_0460
7
807 808 809 810 811 812 813 814 815 816 817 818 819 820 821 822 823 824 825 826 827 828 829 830 831 832 833 834 835 836 837 838 839 840 841 842 843 844 845 846 847 848 849 850 851 852 853 854 855 856 857 858 859 860 861 862 863 864 865 866 867 868
869 870 871 872 873 874 875 876 877 878 879 880 881 882 883 884 885 886 887 888 889 890 891 892 893 894 895 896 897 898 899 900 901 902 903 904 905 906 907 908 909 910 911 912 913 914 915 916 917 918 919 920 921 922 923 924 925 926 927 928 929 930
Sunabori et al fragments, whereas increased free radicals also depolymerize HMW-HA.31,32 Such fragmented or depolymerized LMW-HA molecules (1 to 2.5 104 Da) are known to be potent stimulators of inflammatory cytokines through TLR2 and TLR4. During acute lung injury, depolymerized LMWHA (1.35 to 2 104 Da) fragments will cause inflammation through both TLR2 and TLR4 in vivo.33 Conversely, forced expression of HMW-HA (>106 Da) exerts protective effects on lung injury and epithelial cell apoptosis.33 Our finding that the small HA oligomer (HA4) administration protected neurons from H/I injury (Figures 2 and 3) may explain the diverse biological effects of HA and its fragments. Our present data suggested that the neuroprotective effect of HA4 results from the antagonistic action against TLR to prevent the expression of proinflammatory cytokine IL-1b that leads to inflammation (Figures 1, 5, and 6). Our hypothesis concerning how HA4 can antagonize the TLR/ NF-kB pathway is as follows. Because HA is a straight chain of nonsulfated glycosaminoglycan that is composed of repeating polymeric disaccharides, D-glucuronic acid and N-acetyl glucosamine, it is plausible that HA4 can be accessible to TLR2 and TLR4 as well as LMW-HA. Ligandinduced receptor dimerization of TLR is a crucial step to launch the cascade that finally results in the activation of several transcription factors, including NF-kB.7 We speculate that HA4 binds to TLR2 and TLR4 and fills the pocket to block the binding of stimulants, but that it is too small to couple the receptors to activate the cascade. Therefore, HA4 can suppress the expression of proinflammatory cytokines by inactivating the TLR/NF-kB pathway. Consistent with our hypothesis, several groups have suggested that HA oligomers could attenuate the hyaluronan signaling by displacing multivalent HA polymers and inhibiting the malignant progression in various kinds of cancer.34 We have previously reported that HA4 up-regulates the expression of Hsp72 (an inducible form of Hsp70) and heat shock factor-1, and that it can prevent cell death under serum deprivation of neuronally differentiated PC12 cells.35 In the current study, we did not observe the expression of Hsp72. However, it is known that overexpression of Hsp70 attenuates brain damage after permanent and transient middle cerebral artery occlusion by reducing inflammation in adult mice.36,37 In addition, overexpression of Hsp72 decreases the release of IL-1b when macrophages were stimulated with high mobility group box 1.38 Although the regulatory mechanism is yet unclear, we will not exclude the possibility that the recovery observed by HA4 administration is because of an increase in Hsp70 expression. The number of TUNEL-positive nuclei dramatically decreased by HA4 administration throughout the entire hippocampal pyramidal layer (Figure 2E), and the CA1 region showed comparable results (Figure 2F). However, despite the slight reduction (61 24 to 17 6 by HA4 administration) (Figure 2F), no significant differences were observed in the CA3 region. Because the number of TUNEL-positive nuclei were limited and distinction
8
between samples was extensive, one interpretation could be the insufficiency of samples; however, this could be also rationalized by the difference of the cell death mode between CA1 and CA3 pyramidal cells.5 TLR2 and TLR4 are both known to be involved in neuronal cell death after cerebral ischemia-reperfusion in adults.25 In addition, HA4 administration antagonized specific agonists against both TLR2 and TLR4 in primary hippocampal neurons (Figure 1). However, the neonatal H/I injury model revealed that the major target of the neuroprotective role of HA4 was TLR2 but not TLR4 (Figure 4). This discrepancy could be because of the expression patterns of TLR2 and TLR4 in vitro and in vivo. Although we confirmed that TLR2 and TLR4 were both expressed in primary hippocampal neurons and the neonatal hippocampus by reverse transcriptase-PCR (Figure 1), we could not quantify the precise expression amounts of either TLR2 or TLR4, nor could we determine which cells were responsible for the promotion of inflammation. However, because TLR4-KO mice showed no significant recovery against the control mice after H/I injury, HA4 administration and TLR2KO could protect hippocampal pyramidal cell death to the equivalent level (Figure 4), it is possible to predict that the expression of TLR4 has little effect on hippocampal neuronal cell death after neonatal H/I injury. A recent report has shown that the peroxiredoxin family proteins released extracellularly after brain ischemiareperfusion injury in adult mice stimulate TLR2 and TLR4 as a DAMP.25 Interestingly, the blocking antibodies attenuated ischemic brain damage and suppressed inflammatory cytokine expression when systemically administered even 12 hours after ischemia.25 These data suggest that targeting TLRs could expand the therapeutic window after stroke. A large body of evidence suggests that TLR2 and/or TLR4 play a pivotal role in ischemia-reperfusion injuries in various organs (ie, hepatic, renal, cerebral, intestinal, and myocardial).30 Thus, HA4 could be an attractive candidate for clinical applications in ischemic insult. Moreover, although the recurrence rate after perinatal stroke is lower than in older children and adults,2 prophylactic efficacy of HA4 could be expected against stroke in newborn infants.
References 1. Ferriero DM: Neonatal brain injury. N Engl J Med 2004, 351: 1985e1995 2. Nelson KB, Lynch JK: Stroke in newborn infants. Lancet Neurol 2004, 3:150e158 3. Sheldon RA, Hall JJ, Noble LJ, Ferriero DM: Delayed cell death in neonatal mouse hippocampus from hypoxia-ischemia is neither apoptotic nor necrotic. Neurosci Lett 2001, 304:165e168 4. Uchiyama Y, Koike M, Shibata M: Autophagic neuron death in neonatal brain ischemia/hypoxia. Autophagy 2008, 4:404e408 5. Ginet V, Puyal J, Clarke PG, Truttmann AC: Enhancement of autophagic flux after neonatal cerebral hypoxia-ischemia and its regionspecific relationship to apoptotic mechanisms. Am J Pathol 2009, 175:1962e1974
ajp.amjpathol.org
-
The American Journal of Pathology
FLA 5.4.0 DTD AJPA2352_proof 18 June 2016 7:53 am EO: AJP15_0460
931 932 933 934 935 936 937 938 939 940 941 942 943 944 945 946 947 948 949 950 951 952 953 954 955 956 957 958 959 960 961 962 963 964 965 966 967 968 969 970 971 972 973 974 975 976 977 978 979 980 981 982 983 984 985 986 987 988 989 990 991 992
993 994 995 996 997 998 999 1000 1001 1002 1003 1004 1005 1006 1007 1008 1009 1010 1011 1012 1013 1014 1015 1016 1017 1018 1019 1020 1021 1022 1023 1024 1025 1026 1027 1028 1029 1030 1031 1032 1033 1034 1035 1036 1037 1038 1039 1040 1041 1042 1043 1044 1045 1046 1047
HA4 Protects Neuron from H/I 6. Hagberg H, Gressens P, Mallard C: Inflammation during fetal and neonatal life: implications for neurologic and neuropsychiatric disease in children and adults. Ann Neurol 2012, 71:444e457 7. Kumar H, Kawai T, Akira S: Pathogen recognition in the innate immune response. Biochem J 2009, 420:1e16 8. Bianchi ME: DAMPs, PAMPs and alarmins: all we need to know about danger. J Leukoc Biol 2007, 81:1e5 9. Piccinini AM, Midwood KS: DAMPening inflammation by modulating TLR signalling. Mediators Inflamm 2010, 2010. pii:672395 10. Scheibner KA, Lutz MA, Boodoo S, Fenton MJ, Powell JD, Horton MR: Hyaluronan fragments act as an endogenous danger signal by engaging TLR2. J Immunol 2006, 177:1272e1281 11. Termeer C, Benedix F, Sleeman J, Fieber C, Voith U, Ahrens T, Miyake K, Freudenberg M, Galanos C, Simon JC: Oligosaccharides of Hyaluronan activate dendritic cells via toll-like receptor 4. J Exp Med 2002, 195:99e111 12. Day AJ, de la Motte CA: Hyaluronan cross-linking: a protective mechanism in inflammation? Trends Immunol 2005, 26:637e643 13. Wakao N, Imagama S, Zhang H, Tauchi R, Muramoto A, Natori T, Takeshita S, Ishiguro N, Matsuyama Y, Kadomatsu K: Hyaluronan oligosaccharides promote functional recovery after spinal cord injury in rats. Neurosci Lett 2011, 488:299e304 14. Winkler CW, Foster SC, Itakura A, Matsumoto SG, Asari A, McCarty OJ, Sherman LS: Hyaluronan oligosaccharides perturb lymphocyte slow rolling on brain vascular endothelial cells: implications for inflammatory demyelinating disease. Matrix Biol 2013, 32: 160e168 15. Committee for the Update of the Guide for the Care and Use of Laboratory AnimalsNational Research Council: Guide for the Care and Use of Laboratory Animals. Eighth Edition. Washington, DC, National Academies Press, 2011 16. Rice JE, Vannucci RC, Brierley JB: The influence of immaturity on hypoxic-ischemic brain damage in the rat. Ann Neurol 1981, 9: 131e141 17. Koike M, Shibata M, Tadakoshi M, Gotoh K, Komatsu M, Waguri S, Kawahara N, Kuida K, Nagata S, Kominami E, Tanaka K, Uchiyama Y: Inhibition of autophagy prevents hippocampal pyramidal neuron death after hypoxic-ischemic injury. Am J Pathol 2008, 172: 454e469 18. Sheldon RA, Sedik C, Ferriero DM: Strain-related brain injury in neonatal mice subjected to hypoxia-ischemia. Brain Res 1998, 810: 114e122 19. Ness JM, Harvey CA, Strasser A, Bouillet P, Klocke BJ, Roth KA: Selective involvement of BH3-only Bcl-2 family members Bim and Bad in neonatal hypoxia-ischemia. Brain Res 2006, 1099: 150e159 20. Kaech S, Banker G: Culturing hippocampal neurons. Nat Protoc 2006, 1:2406e2415 21. Nitatori T, Sato N, Waguri S, Karasawa Y, Araki H, Shibanai K, Kominami E, Uchiyama Y: Delayed neuronal death in the CA1 pyramidal cell layer of the gerbil hippocampus following transient ischemia is apoptosis. J Neurosci 1995, 15:1001e1011 22. Tang SC, Arumugam TV, Xu X, Cheng A, Mughal MR, Jo DG, Lathia JD, Siler DA, Chigurupati S, Ouyang X, Magnus T, Camandola S, Mattson MP: Pivotal role for neuronal Toll-like
The American Journal of Pathology
-
23.
24. 25.
26.
27.
28.
29. 30.
31. 32.
33.
34. 35.
36.
37.
38.
receptors in ischemic brain injury and functional deficits. Proc Natl Acad Sci U S A 2007, 104:13798e13803 Stridh L, Smith PL, Naylor AS, Wang X, Mallard C: Regulation of toll-like receptor 1 and -2 in neonatal mice brains after hypoxiaischemia. J Neuroinflammation 2011, 8:45 Mallard C, Wang X, Hagberg H: The role of Toll-like receptors in perinatal brain injury. Clin Perinatol 2009, 36:763e772, v-vi Shichita T, Hasegawa E, Kimura A, Morita R, Sakaguchi R, Takada I, Sekiya T, Ooboshi H, Kitazono T, Yanagawa T, Ishii T, Takahashi H, Mori S, Nishibori M, Kuroda K, Akira S, Miyake K, Yoshimura A: Peroxiredoxin family proteins are key initiators of post-ischemic inflammation in the brain. Nat Med 2012, 18:911e917 Hagberg H, Gilland E, Bona E, Hanson LA, Hahin-Zoric M, Blennow M, Holst M, McRae A, Söder O: Enhanced expression of interleukin (IL)-1 and IL-6 messenger RNA and bioactive protein after hypoxia-ischemia in neonatal rats. Pediatr Res 1996, 40:603e609 Hedtjärn M, Leverin AL, Eriksson K, Blomgren K, Mallard C, Hagberg H: Interleukin-18 involvement in hypoxic-ischemic brain injury. J Neurosci 2002, 22:5910e5919 Liu XH, Kwon D, Schielke GP, Yang GY, Silverstein FS, Barks JD: Mice deficient in interleukin-1 converting enzyme are resistant to neonatal hypoxic-ischemic brain damage. J Cereb Blood Flow Metab 1999, 19:1099e1108 Baeuerle PA, Henkel T: Function and activation of NF-kappa B in the immune system. Annu Rev Immunol 1994, 12:141e179 Arumugam TV, Okun E, Tang SC, Thundyil J, Taylor SM, Woodruff TM: Toll-like receptors in ischemia-reperfusion injury. Shock 2009, 32:4e16 Fraser JR, Laurent TC, Laurent UB: Hyaluronan: its nature, distribution, functions and turnover. J Intern Med 1997, 242:27e33 Deguine V, Menasche M, Ferrari P, Fraisse L, Pouliquen Y, Robert L: Free radical depolymerization of hyaluronan by Maillard reaction products: role in liquefaction of aging vitreous. Int J Biol Macromol 1998, 22:17e22 Jiang D, Liang J, Fan J, Yu S, Chen S, Luo Y, Prestwich GD, Mascarenhas MM, Garg HG, Quinn DA, Homer RJ, Goldstein DR, Bucala R, Lee PJ, Medzhitov R, Noble PW: Regulation of lung injury and repair by Toll-like receptors and hyaluronan. Nat Med 2005, 11: 1173e1179 Toole BP: Hyaluronan: from extracellular glue to pericellular cue. Nat Rev Cancer 2004, 4:528e539 Xu H, Ito T, Tawada A, Maeda H, Yamanokuchi H, Isahara K, Yoshida K, Uchiyama Y, Asari A: Effect of hyaluronan oligosaccharides on the expression of heat shock protein 72. J Biol Chem 2002, 277:17308e17314 Rajdev S, Hara K, Kokubo Y, Mestril R, Dillmann W, Weinstein PR, Sharp FR: Mice overexpressing rat heat shock protein 70 are protected against cerebral infarction. Ann Neurol 2000, 47:782e791 Zheng Z, Kim JY, Ma H, Lee JE, Yenari MA: Anti-inflammatory effects of the 70 kDa heat shock protein in experimental stroke. J Cereb Blood Flow Metab 2008, 28:53e63 Tang D, Kang R, Xiao W, Wang H, Calderwood SK, Xiao X: The antiinflammatory effects of heat shock protein 72 involve inhibition of high-mobility-group box 1 release and proinflammatory function in macrophages. J Immunol 2007, 179:1236e1244
ajp.amjpathol.org
FLA 5.4.0 DTD AJPA2352_proof 18 June 2016 7:53 am EO: AJP15_0460
9
1048 1049 1050 1051 1052 1053 1054 1055 1056 1057 1058 1059 1060 1061 1062 1063 1064 1065 1066 1067 1068 1069 1070 1071 1072 1073 1074 1075 1076 1077 1078 1079 1080 1081 1082 1083 1084 1085 1086 1087 1088 1089 1090 1091 1092 1093 1094 1095 1096 1097 1098 1099 1100 1101 1102
The American Journal of Pathology, Vol. 186, No. 8, August 2016
ajp.amjpathol.org
Suppression of Ischemia-Induced Hippocampal Pyramidal Neuron Death by Hyaluronan Tetrasaccharide through Inhibition of Toll-Like Receptor 2 Signaling Pathway Q35
Q2
Takehiko Sunabori,*y Masato Koike,* Akira Asari,z Yoji Oonuki,x and Yasuo Uchiyamay From the Departments of Cell Biology and Neuroscience* and Cellular and Molecular Neuropathology,y Juntendo University Graduate School of Medicine, Bunkyo-Ku, Tokyo, Japan; the Department of Biomedical Engineering (ND20),z Lerner Research Institute, Cleveland Clinic Foundation, Cleveland, Ohio; and Glycoscience Laboratories Inc.,x Tokyo, Japan Accepted for publication March 28, 2016.
Q5
Q6
Address correspondence to Yasuo Uchiyama, M.D., Ph.D., Department of Cellular and Molecular Neuropathology, Juntendo University Graduate School of Medicine, 2-1-1 Hongo, Bunkyo-Ku, Tokyo 1138421, Japan. E-mail: y-uchi@ juntendo.ac.jp.
Toll-like receptors (TLRs) are one of the main contributors that induce inflammation under tissue injury and infection. Because excessive inflammation can aggravate disease states, it is important to control inflammation at a moderate level. Herein, we show that hyaluronan (HA) oligomer, HA tetrasaccharide (HA4), could suppress the expression of proinflammatory cytokine IL-1b when stimulated with both TLR2- and TLR4-specific agonists in primary hippocampal neurons. To understand the effect of HA4 against ischemic insult, we performed hypoxic-ischemic (H/I) brain injury against neonatal mice. HA4 treatment significantly prevented hippocampal pyramidal cell death even 7 days after H/I injury, compared with the control mice. Although TLR2 and TLR4 are known as receptors for HA and also act as a receptor for inducing inflammation, only TLR2-deficient mice showed tolerance against H/I injury. Moreover, HA4 administration suppressed gliosis by inhibiting the activation of NF-kB, the downstream target of TLR2, which led to the suppression of IL-1b expression. Taken together, our data suggest that the neuroprotective effect of HA4 relies on antagonizing the TLR2/NF-kB pathway to reduce inflammation through suppressing the expression of proinflammatory cytokines after neonatal H/I brain injury. (Am J Pathol 2016, 186: 1e9; http://dx.doi.org/10.1016/j.ajpath.2016.03.016)
Hypoxic-ischemic (H/I) brain injury in the perinatal period is known to link to subsequent neurological complications, such as seizure, cerebral palsy, mental retardation, and epilepsy.1 As recognized in neonates, symptomatic perinatal stroke occurs in one in 4000 live births.2 Morphological, biochemical, and genetic approaches have revealed that both apoptotic and nonapoptotic neuronal death takes place in the pyramidal layer of the hippocampus after H/I injury.3e5 Oxidative stress and glutamate excitotoxicity are suspected to be major executors that lead to cell death in the initial stage.1 However, reducing inflammation could prevent the progression of damage in the following step.6 An understanding of the molecular pathways underlying neuronal cell death induced by neonatal H/I brain injury could pave the way for new therapeutic approaches.
Toll-like receptors (TLRs) are the key receptors that initiate innate immune responses by recognizing specific molecular structures of pathogens known as pathogenassociated molecular patterns.7 However, TLRs are also stimulated by endogenous intracellular molecules released by activated or necrotic cells and extracellular matrix molecules that are up-regulated on injury or degraded after tissue damage, namely damage-associated molecular Supported by Japan Society for the Promotion of Science Kakenhi grants Q3 23390041 and 23659102 (Y.U.) and grants 23790239 and 26860139 (T.S.); the Scientific Research on Innovative Areas from the Ministry of Education, Science, Sports and Culture grants 23111004 and 23110517 (Y.U.); the MEXT-supported Program for the Strategic Research Foundation at Private Universities grant S1101009 (Y.U. and T.S.); and the Juntendo University Project Research grant-in-aid 2317 (T.S.). Q4 Disclosures: Y.O. is employed by Glycoscience Laboratories Inc.
Copyright ª 2016 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.ajpath.2016.03.016
FLA 5.4.0 DTD AJPA2352_proof 18 June 2016 7:53 am EO: AJP15_0460
63 64 65 66 67 68 69 70 71 72 73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 91 92 93 94 95 96 97 98 99 100 101 102 103 104 105 106 107 108 109 110 111 112 113 114 115 116 117 118 119 120 121 122 123 124
125 126 127 128 129 130 131 132 133 134 135 136 137 138 139 140 141 142 143 144 145 146 147 148 149 150 151 152 153 154 155 156 157 158 159 160 161 162 163 164 165 166 167 168 169 170 171 172 173 174 175 176 177 178 179 180 181 182 183 184 185 186
Sunabori et al patterns (DAMPs), even in the absence of infection.8,9 Accumulation of DAMPs initiates positive feedback loops of inflammation that increases tissue damage after impairment. Therefore, regulating the TLR signaling pathway is important for managing pathological conditions. Although hyaluronan (HA) is known as one of the DAMPs that stimulates TLR2 and TLR4,10,11 interestingly, the activity of HA depends on size. In general, high-molecularweight-HA (HMW-HA; 6 105 to 1 106 Da) is antiangiogenic, anti-inflammatory, and immunosuppressive, whereas low-molecular-weight-HA (LMW-HA; <5 104 Da) is inflammatory, immunostimulatory, and angiogenic.12 However, the definition of LMW-HA is ambiguous and little is known about small HA oligomers. Herein, we revealed the effect of a small HA oligomer, HA tetrasaccharide (HA4; 820.24 Da) against neonatal H/I brain injury. Several recent articles reported that HA4 treatment has improvement in a symptom after spinal cord injury13 or experimental autoimmune encephalomyelitis.14 However, little is known about the underlying molecular mechanisms. To further elucidate the effects of HA4 after neonatal H/I brain injury, we focused on the relationship between TLRs and the induction of inflammation.
Materials and Methods Animals
Q7 Q8 Q9
The procedures involving animal care, surgery, and sample preparation were approved by the Animal Experimental Committee of Juntendo University (Tokyo, Japan) and performed in accordance with the Guide for the Care and Use of Laboratory Animals.15 Neonatal C57BL/6J mice were obtained from Japan SLC (Shizuoka, Japan), and the C57BL/6 background TLR2 and TLR4 knockout (KO) mice were purchased from Oriental BioService (Kyoto, Japan). Mice were subsequently housed under specific pathogen-free conditions with a 12-hour light-dark cycle at Juntendo University.
Preparation of Hyaluronan Tetrasaccharide Preparation of HA4 was as follows. Hyaluronan (Shiseido, Tokyo, Japan) was digested in 0.3 mol/L methanesulfonic Q10 acid at 80 C for several hours (18 to 20 hours). The digest was diluted in water and then adjusted to a pH between 5 and 7 with sodium hydroxide before being loaded onto a column containing Dowex 1 4 anion exchange resin (Wako Chemical, Osaka, Japan; 200 to 400 mesh). The Q11 1 4 column was washed with an aqueous 40 mmol/L sodium chloride solution, and HA4 was eluted with a linear gradient consisting of 40 to 100 mmol/L sodium chloride. The Dowex 1 4 purified HA4 was adjusted to between pH 4 and pH 5 with HCl, diluted with water, and loaded onto a column containing BioRad High Q (anion exchange) resin (Bio-Rad Laboratories, Hercules, CA). The High Q column Q12 was washed with water, and HA4 was eluted with 0.5 mol/L sodium chloride. The pH of the eluted HA4 solution was adjusted to pH approximately 5 with HCl, concentrated by rotary evaporation, and then desalted by passing it through a BioGel P2 size exclusion column (Bio-Rad Laboratories) using water as the eluant. The desalted HA4 was purified with a Dowex 50Wx4 column (Wako Chemical) in the acid form. This purified HA4 was passed through ultrafilter membranes with a molecular weight cutoff of 10,000 Da (Merck Millipore, Darmstadt, Germany), concentrated, and then dried to a Q13 powder in a rotary evaporator. The purity of HA4 was determined by high-performance liquid chromatography.
Hyaluronan Administration HA4 was administered i.p. to P6 mouse pups 12 hours before, and just before, H/I injury at P7 with a concentration of 10 mg/kg body weight. Saline administration was performed against littermates or age-matched mice as a control.
Sample Preparation for Histochemical and Morphological Analyses
Hypoxic-Ischemic Injury Neonatal H/I brain injury was induced in mice on post-natal day 7 (P7), essentially according to the Rice-Vannucci model16 with minor modifications.17 After the mice were deeply anesthetized with isoflurane (2%), the left common carotid artery was dissected and ligated with silk sutures (6/0), followed by a recovery for 1 hour at 37 C. They were then placed in chambers maintained at 37 C with a flow purged with nitrogen gas to control oxygen concentration to 8% for 25 minutes. After hypoxic exposure, the pups were returned to their dams and were allowed to recover for 1, 3, 6, 12, and 24 hours, and 7 days. At each stage, the brains were processed for molecular, biochemical, and morphological analyses. This procedure resulted in brain injury in the ipsilateral hemisphere, consisting of cerebral infarction mainly in the hippocampus.17e19 Pups with severe damage in the cortex
2
were eliminated from the experiment. Control littermates were neither operated on nor subjected to hypoxia.
After H/I injury, mice were deeply anesthetized and fixed by cardiac perfusion with 4% paraformaldehyde buffered with 0.1 mol/L phosphate buffer (pH 7.2) containing 4% sucrose. Brain tissues were quickly removed from the mice and further immersed in the same fixative overnight at 4 C. Samples processed for paraffin embedding were cut into sections (4-mm thick) with a semimotorized rotary microtome (RM2245; Leica, Wetzlar, Germany) and placed on Q14 silane-coated glass slides. For routine histological studies, paraffin sections were stained with hematoxylin and eosin.
Immunoblot Analysis Each tissue was independently homogenized in RIPA buffer [50 mmol/L Tris-HCl (pH 7.6) with 150 mmol/L
ajp.amjpathol.org
-
The American Journal of Pathology
FLA 5.4.0 DTD AJPA2352_proof 18 June 2016 7:53 am EO: AJP15_0460
187 188 189 190 191 192 193 194 195 196 197 198 199 200 201 202 203 204 205 206 207 208 209 210 211 212 213 214 215 216 217 218 219 220 221 222 223 224 225 226 227 228 229 230 231 232 233 234 235 236 237 238 239 240 241 242 243 244 245 246 247 248
249 250 251 252 253 254 255 256 257 258 259 260 261 262 263 264 265 266 267 268 269 270 271 272 273 274 275 276 277 278 279 280 281 282 283 284 285 286 287 288 289 290 291 292 293 294 295 296 297 298 299 300 301 302 303 304 305 306 307 308 309 310
HA4 Protects Neuron from H/I Q1
Q15
Q16
Q17
NaCl, 1% NP-40, 0.5% sodium deoxycholate, and 0.1% SDS] containing a protease inhibitor-cocktail (Nacalai, Kyoto, Japan) and a phosphatase inhibitor-cocktail (Nacalai). After centrifugation at 20,000 g for 10 minutes at 4 C, the protein concentrations in the supernatants were determined using the BCA protein assay system (Pierce, Rockford, IL), and were used for immunoblotting. The lysates were eluted by boiling in loading buffer, separated by SDS-PAGE, and transferred to an Immobilon P-membrane (Millipore). The blots were probed with antieNF-kB p65 antibodies (1:1000 dilution; Cell Signaling Technology, Danvers, MA) and antiephospho-NF-kB p65 (Ser536) antibodies (1:500; Cell Signaling Technology).
Primary Cultures of Hippocampal Neurons Primary hippocampal neurons were prepared for culturing, as previously described.20 Briefly, the hippocampi were dissected and dissociated from 18-day-old fetal mice. Dissociated hippocampal neurons were plated on poly-Lornithine (Sigma) coated 6-well plates with 2% B27 (Invi- Q27 trogen, San Diego, CA) and 2 mmol/L L-glutamine (Nacalai) Q28 in Neurobasal medium (Invitrogen). Two days later, 10 mmol/L cytosine arabinoside (Nacalai) was added to inhibit nonneuronal growth. After 14 days, hippocampal neurons were stimulated with 1 mg/mL Pam3CSK4 (Invivogen) and 1 mg/mL LPS-SM Ultrapure (Invivogen) in the presence (50 ng/mL, 5 mg/mL) or absence of HA4 for 6 hours on day 14.
Immunohistochemical Analysis Q18
Q19 Q20
Q21 Q22
Q23
Deparaffinized sections were incubated overnight at 4 C with anti-NeuN antibodies (1:200; Merck Millipore), anti-Iba1 antibodies (1:200; Wako), anti-Mac2 antibodies (1:200; Cedarlane, Burlington, NC), and antieglial fibrillary acidic protein antibodies (1:200; Sigma, St. Louis, MO). Sections were further reacted with peroxidase-conjugated secondary antibodies at room temperature for 2 hours and washed three more times. To visualize the sections, they were reacted with peroxidase, using 0.0125% 3,30 -diaminobenzidine tetrahydrochloride and 0.002% H2O2 in 0.05 mol/L Tris-HCl buffer (pH 7.6) for 5 minutes. For immunofluorescence staining antimouse, anti-rabbit IgG Alexa Fluor 488 antibodies (Thermo Fisher Scientific, Rockford, IL), and anti-rat Cy3 antibodies (Jackson ImmunoResearch Laboratories, West Grove, PA) were used as secondary antibodies. Immunostained sections were observed using a BX50 (Olympus, Tokyo, Japan) and confocal laser scanning microscope FV1000 (Olympus).
qPCR and RT-PCR
Q24 Q25
Q26
For real-time quantitative PCR (qPCR) and RT-PCR analyses, total RNA was prepared using RNeasy (Qiagen, Hilden, Germany) and cDNA was synthesized using a PrimeScript master mix (Takara, Shiga, Japan), according to the manufacturer’s recommendations. The expression level of IL-1b for qPCR was analyzed using a Thunderbird SYBR qPCR mix (Toyobo, Osaka, Japan) on thermal cycler dice (Takara). The primer pairs were as follows: IL-1b, 50 -ACCTGTGGCCTTGGGCCTCAAA-30 (forward) and 50 -TTGGGATCCACACTCTCCAGCTGC-30 (reverse); b-actin, 50 -GGTGGGCCGCCCTAGGCACCA-30 (forward) and 50 -TTGGCCTTAGGGTTCAGGGGG-30 (reverse). The expressions of TLR2, TLR4, and b-actin were analyzed using KOD-plus (Toyobo). The primer pairs were as follows: TLR2, 50 -TGGTGTCTGGAGTCTGCTGTG-30 (forward) and 50 -CGCTCCGTACGAAGTTCTCAG-30 (reverse); TLR4, 50 -CAGTGGTCAGTGTGATTGTGG-30 (forward) and 50 -TTCCTGGATGATGTTGGCAGC-30 (reverse); and b-actin, 50 -GGTGGGCCGCCCTAGGCACCA-30 (forward) and 50 -TTGGCCTTAGGGTTCAGGGGG-30 (reverse).
The American Journal of Pathology
-
TUNEL Staining Terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick end labeling (TUNEL) staining was applied to deparaffinized sections as previously described.21 Briefly, sections were incubated with 100 U/mL TdT (Promega, Madison, WI) and 10 nmol/L biotinylated 16-20 -dUTP Q29 (Roche Diagnostics, Basel, Switzerland) in TdT buffer (100 Q30 mmol/L sodium cacodylate, pH 7.0, 1 mmol/L cobalt chloride, 50 mg/mL gelatin) in a humid atmosphere at 37 C for 1 hour, followed by further incubation with peroxidaseconjugated streptavidin (Vector Laboratories, Burlingame, CA) for 1 hour at room temperature. Staining for peroxidase Q31 was performed as stated above.
Cell Counting The number of TUNEL-positive cells was counted in the whole pyramidal regions of the hippocampus, located in the same portion (bregma: -1.82 mm of adult C57BL/J mice), according to the previous report.17
Neuropathological Evaluation H/I injury and neuroprotection were evaluated by the number of TUNEL-positive (dead) cells in the pyramidal layers of the hippocampus 24 hours after H/I injury using brain sections corresponding to the same portion of the hippocampus as mentioned above. The damaged area in the hippocampal pyramidal layers was also examined 7 days after H/I injury, in which time clear-cut shrinkage occurred in the ipsilateral hippocampus of the control mice, resulting in a loss of ipsilateral hippocampal neurons. To quantitate these damaged areas after neonatal H/I, the areas of lesioned and unlesioned hippocampi were measured and compared within the same section corresponding to the same portions mentioned above, using ImageJ software version 1.38 (NIH, Bethesda, MD). The Q32 area loss of the lesioned ipsilateral hippocampus was calculated as a percentage of the intact contralateral hippocampus, and was determined for each animal.
ajp.amjpathol.org
FLA 5.4.0 DTD AJPA2352_proof 18 June 2016 7:53 am EO: AJP15_0460
3
311 312 313 314 315 316 317 318 319 320 321 322 323 324 325 326 327 328 329 330 331 332 333 334 335 336 337 338 339 340 341 342 343 344 345 346 347 348 349 350 351 352 353 354 355 356 357 358 359 360 361 362 363 364 365 366 367 368 369 370 371 372
Sunabori et al 373 Statistical Analysis 374 375 All quantified data were expressed as the means SEM. 376 Differences between two mean values were evaluated using 377 an unpaired Student’s t-test and two-way analysis of vari378 ance post-hoc tests that were considered statistically sig379 nificant when the P value was <0.05. 380 381 382 Results 383 384 HA4 Antagonizes Both TLR2 and TLR4 Signaling 385 Pathway in Primary Hippocampal Neurons 386 387 The hippocampus is the brain region that is most vulnerable 388 to cerebral ischemic insult,18,19 and Toll-like receptors 389 (TLRs) are known to be involved in neuronal cell death after 390 ischemia.22e25 To determine the effect of a small HA 391 oligomer, HA tetrasaccharide (HA4; 820.24 Da), we 392 focused on the TLR signaling pathway for three reasons: i) 393 TLR2 and TLR4 are known as the receptors for HA10,11; ii) 394 stimulation of TLRs would activate NF-kB and induce 395 proinflammatory cytokines7; and iii) primary cortical neu396 397 rons generated from TLR2-KO and TLR4-KO mice are 398 resistant to glucose deprivation.22 Therefore, we first 399 explored whether HA4 could participate in the TLR/NF-kB 400 signaling pathway in primary hippocampal neurons. To 401 confirm the expressions of TLR2 and TLR4 in primary 402 hippocampal neurons, we performed reverse transcriptase403 PCR. Primary hippocampal neurons cultured for 14 days 404 in vitro expressed both TLR2 and TLR4 as well as the 405 microglial cell line BV2 (Figure 1A). Thus, we stimulated ½F1 406 TLR2 and TLR4 with specific agonists, Pam3CSK4 and 407 LPS, respectively, and observed the expression of the 408 409 proinflammatory cytokine IL-1b by qPCR in the presence 410 and absence of HA4. The increased expression of IL-1b by 411 Pam3CSK4 stimulation (2.7-fold) and LPS stimulation (2.8412 fold) was suppressed in a dose-dependent manner by HA4 413 administration (50 ng/mL, 5 mg/mL of HA4, respectively), 414 whereas no change was observed when HA4 was adminis415 tered alone (Figure 1B). These data suggest that HA4 can 416 play an antagonistic role against TLR2 and TLR4 in the 417 hippocampal neuron in vitro. 418 419 420 HA4 Administration Protects Hippocampal Pyramidal 421 Neurons from Neonatal H/I Injury 422 423 To elucidate the effect that HA4 administration exerts 424 against cerebral ischemic insult in vivo, we next examined 425 the damage in the pyramidal regions of the hippocampus in 426 neonatal mouse brains after H/I injury. HA4 (10 mg/kg 427 body weight) was administered i.p., 12 hours, and imme428 diately before H/I injury, and physiological saline was 429 injected as a control. The degree of damage in the hippo430 431 campal pyramidal cells, quantified as the number of 432 TUNEL-positive nuclei, was extremely reduced in mice 433 treated with HA4, compared with those treated with saline 434
4
Figure 1
HA4 antagonizes both TLR2 and TLR4 signaling pathway in primary hippocampal neurons. A: Expression of TLR2 and TLR4 mRNA was confirmed by reverse transcriptase-PCR in lysates from BV2 cells (lane 1; a microglial cell line), primary hippocampal neurons (lane 2), TLR2-KO mouse brain (lane 3), and TLR4-KO mouse brain (lane 4). B: Quantification of expression levels of IL-1b mRNA in primary hippocampal neurons cultured in the presence of Pam3CSK4 and lipopolysaccharide (LPS) with 50 ng/mL and 5 mg/mL of HA4 or without HA4, as measured by real-time quantitative PCR. Data are expressed as means SEM (B). n Z 4 independent experiments (B). *P < 0.05.
Q34
24 hours after H/I injury (Figure 2, AeD). The number of ½F2 TUNEL-positive nuclei within the pyramidal layer of mouse brains (Figure 2, B and D) that were administered with HA4 was significantly decreased (179 19) to fewer than half the number of those treated with saline (377 51) (Figure 2E). We farther evaluated the number of TUNELpositive nuclei, in the CA1 and CA3 region of the hippocampal pyramidal cell layer. The number of TUNELpositive nuclei within CA1 region (Figure 2F) significantly decreased by HA4 administration (44 10) compared with saline (214 30). However, although slight reduction of the number of TUNEL-positive nuclei was also observed, no significance was detected between HA4 (17 6) and saline (61 24) administration in the CA3 region (Figure 2F). These data suggest that HA4 administration can protect hippocampal pyramidal cells after neonatal H/I brain injury in a region-specific manner.
Pathological Alterations Are Not Detected in the Hippocampal Pyramidal Neurons of a HA4-Administered Neonatal Hippocampus 7 Days after H/I Injury Delayed neuronal death reportedly occurs in the pyramidal layers of the hippocampus within 7 days after transient brain ischemia, and damaged areas are replaced by glial cells.19
ajp.amjpathol.org
-
The American Journal of Pathology
FLA 5.4.0 DTD AJPA2352_proof 18 June 2016 7:53 am EO: AJP15_0460
435 436 437 438 439 440 441 442 443 444 445 446 447 448 449 450 451 452 453 454 455 456 457 458 459 460 461 462 463 464 465 466 467 468 469 470 471 472 473 474 475 476 477 478 479 480 481 482 483 484 485 486 487 488 489 490 491 492 493 494 495 496
print & web 4C=FPO
Figure 2
HA4 administration protects hippocampal pyramidal cells from neonatal H/I injury. Representative demonstration of the ipsilateral hippocampi of mice administered saline (A and B) and HA4 (C and D), respectively, stained with hematoxylin and eosin (H&E; A and C) and for terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL; B and D) 24 hours after H/I injury. E: Quantitative analysis of the number of TUNELpositive cells in the pyramidal layer of an identical portion (bregma: -1.82 mm of adult C57BL/J mice) of the hippocampus, administered saline and HA4, 24 hours after H/I injury. F: Quantitative analysis of the number of TUNEL-positive cells in the CA1 and CA3 region of an identical portion (bregma: -1.82 mm of adult C57BL/J mice) of the hippocampus, administered saline and HA4, 24 hours after H/I injury. Data are expressed as means SEM (E and F). n Z 13 and 14 mice per group (E); n Z 8 and 6 mice per group (F). **P < 0.01, ***P < 0.001. Scale bar Z 200 mm (AeD).
print & web 4C=FPO
497 498 499 500 501 502 503 504 505 506 507 508 509 510 511 512 513 514 515 516 517 518 519 520 521 522 523 524 525 526 527 528 529 530 531 532 533 534 535 536 537 538 539 540 541 542 543 544 545 546 547 548 549 550 551 552 553 554 555 556 557 558
HA4 Protects Neuron from H/I 559 control littermate mice 7 days after H/I injury. Loss of 560 NeuN-positive neurons with their neurites that was elimi561 nated by microglial cells contributed to the areal reduction 562 of pyramidal layers of the control mouse hippocampus 563 (Figure 3, C and D). The total area of the ipsilateral hip- ½F3 564 pocampus was significantly diminished, compared with the 565 contralateral hippocampus (Figure 3, A and B). Meanwhile, 566 in the HA4-administered mice, the remaining pyramidal 567 neurons were largely TUNEL-negative (data not shown), 568 569 and staining for hematoxylin and eosin, and NeuN showed 570 those neurons to be intact (Figure 3, AeF). The ipsilateral 571 hippocampal area in the HA4-administered mice was only 572 slightly diminished to 96% 9% compared with the 573 contralateral hippocampi (Figure 3, B and G). However, in 574 the physiological saline-injected littermate controls, the 575 ipsilateral hippocampal area was reduced to nearly half the 576 contralateral hippocampal area 7 days after H/I injury 577 (53% 7%) (Figure 3, A and G). These data indicate that 578 the prevention of hippocampal pyramidal cell death by HA4 579 administration is sustained at least until 7 days after H/I 580 581 injury.
582 583 584 The Neuroprotective Effect of HA4 Largely Relies on the 585 Inhibition of TLR2, but Not TLR4 in Neonatal H/I Injury 586 587 HA4 antagonized both TLR2 and TLR4 in primary hippo588 campal neurons in vitro (Figure 1) and protected hippo589 campal pyramidal cells after H/I injury in vivo (Figures 2 590 and 3). However, the major target of the neuroprotective 591 role of HA4 after neonatal H/I injury remained unclear. To 592 confirm the relationship between TLR2, TLR4, and HA4 593 To exclude the possibility that the administration of HA4 in vivo, we used TLR2-KO and TLR4-KO mice and applied 594 simply delayed the onset of pyramidal cell death, we further them to H/I injury. When saline was administered, the 595 analyzed the degree of damage in the neurons of the degree of damage in the hippocampal pyramidal cells was 596 extremely reduced in TLR2-KO mice (Figure 4C), compared ½F4 597 hippocampal pyramidal layers in HA4-administered and 598 599 600 601 602 603 604 605 606 607 608 609 610 611 612 613 614 615 Figure 3 Prevention of neuronal loss in the hippocampus by HA4 administration is further sustained up to 7 days after H/I injury. Representative 616 demonstration of the hippocampi of mice administered saline (A, C, and E) or HA4 (B, D, and F) stained with hematoxylin and eosin (H&E; A and B) and anti617 NeuN antibodies (CeF) 7 days after H/I injury. G: Evaluation of the area of ipsilateral (Ipsi) against contralateral (Contra) hippocampi of HA4-treated and 618 littermate control mouse brains of an identical portion (bregma: -1.82 mm of adult C57BL/J mice) of the hippocampus 7 days after H/I injury. Data are 619 expressed as means SEM (G). n Z 9 and 7 mice per group (G). **P < 0.01. Scale bars: 400 mm (AeD); 200 mm (E and F). 620
The American Journal of Pathology
-
ajp.amjpathol.org
FLA 5.4.0 DTD AJPA2352_proof 18 June 2016 7:53 am EO: AJP15_0460
5
together, these data indicate that the major target of HA4 after neonatal H/I brain injury was TLR2 but not TLR4.
HA4 Administration Inhibits the Expression of Proinflammatory Cytokine IL-1b through Inactivating NF-kB after H/I Injury
print & web 4C=FPO
621 622 623 624 625 626 627 628 629 630 631 632 633 634 635 636 637 638 639 640 641 642 643 644 645 646 647 648 649 650 651 652 653 654 655 656 657 658 659 660 661 662 663 664 665 666 667 668 669 670 671 672 673 674 675 676 677 678 679 680 681 682
Sunabori et al
Figure 4 TLR2 is the major target for the neuroprotective effect of HA4 after H/I injury in vivo. Representative demonstration of the ipsilateral hippocampus of wild-type (A and B), TLR2-KO (CeF), and TLR4-KO (GeJ) mice administered saline (AeD, G, and H) or HA4 (E, F, I, and J), and obtained 24 hours after H/I injury. A, C, E, G, and I: Hematoyxlin and eosin (H&E) staining. B, D, F, H, and J: Terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) staining. K: Quantification of the number of TUNELpositive cells in the pyramidal layer of an identical portion (bregma: -1.82 mm of adult C57BL/J mice) of the hippocampus of wild-type, TLR2-KO, and TLR4-KO mice administered saline or HA4 and obtained 24 hours after H/I injury. Data are expressed as means SEM (K). n Z 6 or 7 mice per group (K). *P < 0.05, **P < 0.01. Scale bar Z 200 mm (AeJ).
with both TLR4-KO (Figure 4G) and wild-type mice (Figure 4A) 24 hours after H/I injury, which was consistent with previous reports.23,24 Moreover, the improvement after administration of HA4 was observed only in TLR4-KO mice, but not in TLR2-KO mice (Figure 4, E and I). The TUNEL-positive cells within the ipsilateral hippocampal pyramidal layer (Figure 4, B, D, F, H, and J) were significantly decreased in TLR2-KO mice (130 16), compared with wild-type mice (280 19), and no additional improvement was observed after the administration of HA4 (113 24) (Figure 4K). In contrast, the TUNEL-positive cells in TLR4-KO mice (268 26) showed comparable level to the control mice, whereas the TUNEL positivity was significantly suppressed and the insult was improved by HA4 administration (194 15) (Figure 4K). Taken
6
H/I injury induces an inflammatory reaction in the neonatal brain, and the early proinflammatory phase is thought to deteriorate pathological states.6 Inhibition of proinflammatory cytokines, such as IL-1b26 and IL-18,27 and the activator of IL-1 and IL-18, caspase-1,28 reduces H/I brain injury to a moderate degree. Therefore, we evaluated the expression level of IL-1b in the ipsilateral hippocampus with or without HA4 administration and compared with TLR2-KO mice by qPCR. The expression level of IL-1b increased within an hour and peaked 6 hours after H/I injury in the control mice (Figure 5A). However, HA4 adminis- ½F5 tration, as well as TLR2-KO mice, significantly inhibited IL-1b expression 3 and 6 hours after H/I injury (Figure 5A). To further confirm the molecular mechanism regulating the expression of IL-1b, we next verified the activity of the NF-kB/Rel family. The Ser536 residue of p65/RelA, a member of the NF-kB/Rel family, is phosphorylated when NF-kB is activated and translocates to the nucleus to regulate gene expression including IL-1b.29 Western blot analyses revealed that the activation of NF-kB in the ipsilateral hippocampi, represented as an expression ratio of phospho-p65/RelA (Ser536) per pan-p65/RelA, increased 5.2-fold within an hour in control mice when compared with sham-operated mice, whereas the activation was significantly suppressed to 1.4-fold by HA4 administration and 0.98-fold in TLR2-KO mice (Figure 5, B and C). These data indicate that HA4 administration can inhibit the expression of proinflammatory cytokine IL-1b in vivo, in a closely related manner to TLR2-KO mice, by inactivating NF-kB in the early stages after H/I Injury.
HA4 Administration Inhibits Gliosis after H/I Injury Although the expression of IL-1b mRNA was suppressed by HA4 administration (Figure 5), the activation (cleavage for the secretion form) of IL-1b, which causes inflammation, was undetectable by both Western blot analyses and enzyme-linked immunosorbent assay analyses after H/I brain injury. Therefore, we focused on gliosis as the hallmark of inflammation. Gliosis consists of the activation of astrocytes and microglial cells, stimulated by proinflammatory cytokines, including IL-1b. The activation of microglial cells and astrocytes was investigated by evaluating the expression of Mac2 (for reactive microglial cells), Iba1 (for pan-microglial cells), and glial fibrillary acidic protein (for reactive astrocytes) 24 hours after H/I injury (Figure 6). Interestingly, the Iba1-positive microglial cells ½F6 slightly increased the number and many coexpressed Mac2 in the control hippocampus (Figure 6A), whereas HA4
ajp.amjpathol.org
-
The American Journal of Pathology
FLA 5.4.0 DTD AJPA2352_proof 18 June 2016 7:53 am EO: AJP15_0460
683 684 685 686 687 688 689 690 691 692 693 694 695 696 697 698 699 700 701 702 703 704 705 706 707 708 709 710 711 712 713 714 715 716 717 718 719 720 721 722 723 724 725 726 727 728 729 730 731 732 733 734 735 736 737 738 739 740 741 742 743 744
suppressed the emergence of reactive astrocytes (Figure 6, C and D). Taken together, these data indicate that HA4 Q33 administration protects neuronal cell death through suppressing inflammation via inhibiting the expression of proinflammatory cytokine IL-1b.
Discussion Inflammation is induced by both tissue injury and infection for facilitating wound healing and stimulating innate immunity. However, excessive inflammation can also aggravate symptoms. Although lines of evidence from KO animals have revealed that TLRs are one of the main contributors not only to pathogen-induced inflammation, but also to injury-induced inflammation in various organs,30 the strategies to attenuate inflammation after tissue damage have been limited. Herein, we demonstrated the following five points: i) HA4 exhibited antagonistic action against both TLR2 and TLR4 in vitro; ii) HA4 administration prevented hippocampal pyramidal neuron death for at least 7 days after neonatal H/I brain injury; iii) the main target of this inhibition was TLR2 but not TLR4 in vivo; iv) HA4 administration decreased the expression of proinflammatory cytokine IL-1b by inactivating the NF-kB/Rel family; and v) HA4 administration prevented inflammation. HA has a high molecular mass (>106 Da) under physiological conditions. However, in inflammatory tissues, the activation of hyaluronidase degrades HMW-HA into small
Figure 5 Inhibitory effect of HA4 on the expression of a proinflammatory cytokine, IL-1b, through inactivation of NF-kB after H/I injury. A: Quantification of IL-1b mRNA levels in ipsilateral hippocampi obtained from HA4- or saline-treated and TLR2-KO mice at indicated time periods after H/I injury. mRNA levels were measured by real-time quantitative PCR. B: Western blot analyses of phospho-p65/RelA (Ser536) and pan-p65/RelA in the ipsilateral hippocampi from HA4- or saline-administered and TLR2-KO mouse brains. Duplicated data are presented in protein extracts from the brains of mice injected with HA4 or saline and TLR2-KO. C: Ratios of protein amounts of phospho-p65/RelA (Ser536)/pan-p65/RelA in the ipsilateral hippocampi administered HA4 or saline and TLR2-KO. Data are expressed as means SEM (A and C). n Z 3 to 7 mice per group (A); n Z 4 mice per group (C). *P < 0.05.
administration diminished the expression of Iba1 to the steady state and Mac2-expressing cells were not observed (Figure 6B). Moreover, the expression of glial fibrillary acidic protein was strongly elevated only in the control hippocampus, suggesting that HA4 administration
The American Journal of Pathology
-
print & web 4C=FPO
745 746 747 748 749 750 751 752 753 754 755 756 757 758 759 760 761 762 763 764 765 766 767 768 769 770 771 772 773 774 775 776 777 778 779 780 781 782 783 784 785 786 787 788 789 790 791 792 793 794 795 796 797 798 799 800 801 802 803 804 805 806
HA4 Protects Neuron from H/I
Figure 6 The emergence of Mac2-expressing microglial cells and reactive astrocytes is suppressed by HA4 administration 24 hours after H/I injury. Representative demonstration of the ipsilateral hippocampi administered saline (A and C) and HA4 (B and D) stained with anti-Iba1 antibodies (green; A and B), antieglial fibrillary acidic protein (GFAP) antibodies (green; C and D), anti-Mac2 antibodies (red; AeD), and DAPI (blue; AeD). Scale bar Z 50 mm (AeD).
ajp.amjpathol.org
FLA 5.4.0 DTD AJPA2352_proof 18 June 2016 7:53 am EO: AJP15_0460
7
807 808 809 810 811 812 813 814 815 816 817 818 819 820 821 822 823 824 825 826 827 828 829 830 831 832 833 834 835 836 837 838 839 840 841 842 843 844 845 846 847 848 849 850 851 852 853 854 855 856 857 858 859 860 861 862 863 864 865 866 867 868
869 870 871 872 873 874 875 876 877 878 879 880 881 882 883 884 885 886 887 888 889 890 891 892 893 894 895 896 897 898 899 900 901 902 903 904 905 906 907 908 909 910 911 912 913 914 915 916 917 918 919 920 921 922 923 924 925 926 927 928 929 930
Sunabori et al fragments, whereas increased free radicals also depolymerize HMW-HA.31,32 Such fragmented or depolymerized LMW-HA molecules (1 to 2.5 104 Da) are known to be potent stimulators of inflammatory cytokines through TLR2 and TLR4. During acute lung injury, depolymerized LMWHA (1.35 to 2 104 Da) fragments will cause inflammation through both TLR2 and TLR4 in vivo.33 Conversely, forced expression of HMW-HA (>106 Da) exerts protective effects on lung injury and epithelial cell apoptosis.33 Our finding that the small HA oligomer (HA4) administration protected neurons from H/I injury (Figures 2 and 3) may explain the diverse biological effects of HA and its fragments. Our present data suggested that the neuroprotective effect of HA4 results from the antagonistic action against TLR to prevent the expression of proinflammatory cytokine IL-1b that leads to inflammation (Figures 1, 5, and 6). Our hypothesis concerning how HA4 can antagonize the TLR/ NF-kB pathway is as follows. Because HA is a straight chain of nonsulfated glycosaminoglycan that is composed of repeating polymeric disaccharides, D-glucuronic acid and N-acetyl glucosamine, it is plausible that HA4 can be accessible to TLR2 and TLR4 as well as LMW-HA. Ligandinduced receptor dimerization of TLR is a crucial step to launch the cascade that finally results in the activation of several transcription factors, including NF-kB.7 We speculate that HA4 binds to TLR2 and TLR4 and fills the pocket to block the binding of stimulants, but that it is too small to couple the receptors to activate the cascade. Therefore, HA4 can suppress the expression of proinflammatory cytokines by inactivating the TLR/NF-kB pathway. Consistent with our hypothesis, several groups have suggested that HA oligomers could attenuate the hyaluronan signaling by displacing multivalent HA polymers and inhibiting the malignant progression in various kinds of cancer.34 We have previously reported that HA4 up-regulates the expression of Hsp72 (an inducible form of Hsp70) and heat shock factor-1, and that it can prevent cell death under serum deprivation of neuronally differentiated PC12 cells.35 In the current study, we did not observe the expression of Hsp72. However, it is known that overexpression of Hsp70 attenuates brain damage after permanent and transient middle cerebral artery occlusion by reducing inflammation in adult mice.36,37 In addition, overexpression of Hsp72 decreases the release of IL-1b when macrophages were stimulated with high mobility group box 1.38 Although the regulatory mechanism is yet unclear, we will not exclude the possibility that the recovery observed by HA4 administration is because of an increase in Hsp70 expression. The number of TUNEL-positive nuclei dramatically decreased by HA4 administration throughout the entire hippocampal pyramidal layer (Figure 2E), and the CA1 region showed comparable results (Figure 2F). However, despite the slight reduction (61 24 to 17 6 by HA4 administration) (Figure 2F), no significant differences were observed in the CA3 region. Because the number of TUNEL-positive nuclei were limited and distinction
8
between samples was extensive, one interpretation could be the insufficiency of samples; however, this could be also rationalized by the difference of the cell death mode between CA1 and CA3 pyramidal cells.5 TLR2 and TLR4 are both known to be involved in neuronal cell death after cerebral ischemia-reperfusion in adults.25 In addition, HA4 administration antagonized specific agonists against both TLR2 and TLR4 in primary hippocampal neurons (Figure 1). However, the neonatal H/I injury model revealed that the major target of the neuroprotective role of HA4 was TLR2 but not TLR4 (Figure 4). This discrepancy could be because of the expression patterns of TLR2 and TLR4 in vitro and in vivo. Although we confirmed that TLR2 and TLR4 were both expressed in primary hippocampal neurons and the neonatal hippocampus by reverse transcriptase-PCR (Figure 1), we could not quantify the precise expression amounts of either TLR2 or TLR4, nor could we determine which cells were responsible for the promotion of inflammation. However, because TLR4-KO mice showed no significant recovery against the control mice after H/I injury, HA4 administration and TLR2KO could protect hippocampal pyramidal cell death to the equivalent level (Figure 4), it is possible to predict that the expression of TLR4 has little effect on hippocampal neuronal cell death after neonatal H/I injury. A recent report has shown that the peroxiredoxin family proteins released extracellularly after brain ischemiareperfusion injury in adult mice stimulate TLR2 and TLR4 as a DAMP.25 Interestingly, the blocking antibodies attenuated ischemic brain damage and suppressed inflammatory cytokine expression when systemically administered even 12 hours after ischemia.25 These data suggest that targeting TLRs could expand the therapeutic window after stroke. A large body of evidence suggests that TLR2 and/or TLR4 play a pivotal role in ischemia-reperfusion injuries in various organs (ie, hepatic, renal, cerebral, intestinal, and myocardial).30 Thus, HA4 could be an attractive candidate for clinical applications in ischemic insult. Moreover, although the recurrence rate after perinatal stroke is lower than in older children and adults,2 prophylactic efficacy of HA4 could be expected against stroke in newborn infants.
References 1. Ferriero DM: Neonatal brain injury. N Engl J Med 2004, 351: 1985e1995 2. Nelson KB, Lynch JK: Stroke in newborn infants. Lancet Neurol 2004, 3:150e158 3. Sheldon RA, Hall JJ, Noble LJ, Ferriero DM: Delayed cell death in neonatal mouse hippocampus from hypoxia-ischemia is neither apoptotic nor necrotic. Neurosci Lett 2001, 304:165e168 4. Uchiyama Y, Koike M, Shibata M: Autophagic neuron death in neonatal brain ischemia/hypoxia. Autophagy 2008, 4:404e408 5. Ginet V, Puyal J, Clarke PG, Truttmann AC: Enhancement of autophagic flux after neonatal cerebral hypoxia-ischemia and its regionspecific relationship to apoptotic mechanisms. Am J Pathol 2009, 175:1962e1974
ajp.amjpathol.org
-
The American Journal of Pathology
FLA 5.4.0 DTD AJPA2352_proof 18 June 2016 7:53 am EO: AJP15_0460
931 932 933 934 935 936 937 938 939 940 941 942 943 944 945 946 947 948 949 950 951 952 953 954 955 956 957 958 959 960 961 962 963 964 965 966 967 968 969 970 971 972 973 974 975 976 977 978 979 980 981 982 983 984 985 986 987 988 989 990 991 992
993 994 995 996 997 998 999 1000 1001 1002 1003 1004 1005 1006 1007 1008 1009 1010 1011 1012 1013 1014 1015 1016 1017 1018 1019 1020 1021 1022 1023 1024 1025 1026 1027 1028 1029 1030 1031 1032 1033 1034 1035 1036 1037 1038 1039 1040 1041 1042 1043 1044 1045 1046 1047
HA4 Protects Neuron from H/I 6. Hagberg H, Gressens P, Mallard C: Inflammation during fetal and neonatal life: implications for neurologic and neuropsychiatric disease in children and adults. Ann Neurol 2012, 71:444e457 7. Kumar H, Kawai T, Akira S: Pathogen recognition in the innate immune response. Biochem J 2009, 420:1e16 8. Bianchi ME: DAMPs, PAMPs and alarmins: all we need to know about danger. J Leukoc Biol 2007, 81:1e5 9. Piccinini AM, Midwood KS: DAMPening inflammation by modulating TLR signalling. Mediators Inflamm 2010, 2010. pii:672395 10. Scheibner KA, Lutz MA, Boodoo S, Fenton MJ, Powell JD, Horton MR: Hyaluronan fragments act as an endogenous danger signal by engaging TLR2. J Immunol 2006, 177:1272e1281 11. Termeer C, Benedix F, Sleeman J, Fieber C, Voith U, Ahrens T, Miyake K, Freudenberg M, Galanos C, Simon JC: Oligosaccharides of Hyaluronan activate dendritic cells via toll-like receptor 4. J Exp Med 2002, 195:99e111 12. Day AJ, de la Motte CA: Hyaluronan cross-linking: a protective mechanism in inflammation? Trends Immunol 2005, 26:637e643 13. Wakao N, Imagama S, Zhang H, Tauchi R, Muramoto A, Natori T, Takeshita S, Ishiguro N, Matsuyama Y, Kadomatsu K: Hyaluronan oligosaccharides promote functional recovery after spinal cord injury in rats. Neurosci Lett 2011, 488:299e304 14. Winkler CW, Foster SC, Itakura A, Matsumoto SG, Asari A, McCarty OJ, Sherman LS: Hyaluronan oligosaccharides perturb lymphocyte slow rolling on brain vascular endothelial cells: implications for inflammatory demyelinating disease. Matrix Biol 2013, 32: 160e168 15. Committee for the Update of the Guide for the Care and Use of Laboratory AnimalsNational Research Council: Guide for the Care and Use of Laboratory Animals. Eighth Edition. Washington, DC, National Academies Press, 2011 16. Rice JE, Vannucci RC, Brierley JB: The influence of immaturity on hypoxic-ischemic brain damage in the rat. Ann Neurol 1981, 9: 131e141 17. Koike M, Shibata M, Tadakoshi M, Gotoh K, Komatsu M, Waguri S, Kawahara N, Kuida K, Nagata S, Kominami E, Tanaka K, Uchiyama Y: Inhibition of autophagy prevents hippocampal pyramidal neuron death after hypoxic-ischemic injury. Am J Pathol 2008, 172: 454e469 18. Sheldon RA, Sedik C, Ferriero DM: Strain-related brain injury in neonatal mice subjected to hypoxia-ischemia. Brain Res 1998, 810: 114e122 19. Ness JM, Harvey CA, Strasser A, Bouillet P, Klocke BJ, Roth KA: Selective involvement of BH3-only Bcl-2 family members Bim and Bad in neonatal hypoxia-ischemia. Brain Res 2006, 1099: 150e159 20. Kaech S, Banker G: Culturing hippocampal neurons. Nat Protoc 2006, 1:2406e2415 21. Nitatori T, Sato N, Waguri S, Karasawa Y, Araki H, Shibanai K, Kominami E, Uchiyama Y: Delayed neuronal death in the CA1 pyramidal cell layer of the gerbil hippocampus following transient ischemia is apoptosis. J Neurosci 1995, 15:1001e1011 22. Tang SC, Arumugam TV, Xu X, Cheng A, Mughal MR, Jo DG, Lathia JD, Siler DA, Chigurupati S, Ouyang X, Magnus T, Camandola S, Mattson MP: Pivotal role for neuronal Toll-like
The American Journal of Pathology
-
23.
24. 25.
26.
27.
28.
29. 30.
31. 32.
33.
34. 35.
36.
37.
38.
receptors in ischemic brain injury and functional deficits. Proc Natl Acad Sci U S A 2007, 104:13798e13803 Stridh L, Smith PL, Naylor AS, Wang X, Mallard C: Regulation of toll-like receptor 1 and -2 in neonatal mice brains after hypoxiaischemia. J Neuroinflammation 2011, 8:45 Mallard C, Wang X, Hagberg H: The role of Toll-like receptors in perinatal brain injury. Clin Perinatol 2009, 36:763e772, v-vi Shichita T, Hasegawa E, Kimura A, Morita R, Sakaguchi R, Takada I, Sekiya T, Ooboshi H, Kitazono T, Yanagawa T, Ishii T, Takahashi H, Mori S, Nishibori M, Kuroda K, Akira S, Miyake K, Yoshimura A: Peroxiredoxin family proteins are key initiators of post-ischemic inflammation in the brain. Nat Med 2012, 18:911e917 Hagberg H, Gilland E, Bona E, Hanson LA, Hahin-Zoric M, Blennow M, Holst M, McRae A, Söder O: Enhanced expression of interleukin (IL)-1 and IL-6 messenger RNA and bioactive protein after hypoxia-ischemia in neonatal rats. Pediatr Res 1996, 40:603e609 Hedtjärn M, Leverin AL, Eriksson K, Blomgren K, Mallard C, Hagberg H: Interleukin-18 involvement in hypoxic-ischemic brain injury. J Neurosci 2002, 22:5910e5919 Liu XH, Kwon D, Schielke GP, Yang GY, Silverstein FS, Barks JD: Mice deficient in interleukin-1 converting enzyme are resistant to neonatal hypoxic-ischemic brain damage. J Cereb Blood Flow Metab 1999, 19:1099e1108 Baeuerle PA, Henkel T: Function and activation of NF-kappa B in the immune system. Annu Rev Immunol 1994, 12:141e179 Arumugam TV, Okun E, Tang SC, Thundyil J, Taylor SM, Woodruff TM: Toll-like receptors in ischemia-reperfusion injury. Shock 2009, 32:4e16 Fraser JR, Laurent TC, Laurent UB: Hyaluronan: its nature, distribution, functions and turnover. J Intern Med 1997, 242:27e33 Deguine V, Menasche M, Ferrari P, Fraisse L, Pouliquen Y, Robert L: Free radical depolymerization of hyaluronan by Maillard reaction products: role in liquefaction of aging vitreous. Int J Biol Macromol 1998, 22:17e22 Jiang D, Liang J, Fan J, Yu S, Chen S, Luo Y, Prestwich GD, Mascarenhas MM, Garg HG, Quinn DA, Homer RJ, Goldstein DR, Bucala R, Lee PJ, Medzhitov R, Noble PW: Regulation of lung injury and repair by Toll-like receptors and hyaluronan. Nat Med 2005, 11: 1173e1179 Toole BP: Hyaluronan: from extracellular glue to pericellular cue. Nat Rev Cancer 2004, 4:528e539 Xu H, Ito T, Tawada A, Maeda H, Yamanokuchi H, Isahara K, Yoshida K, Uchiyama Y, Asari A: Effect of hyaluronan oligosaccharides on the expression of heat shock protein 72. J Biol Chem 2002, 277:17308e17314 Rajdev S, Hara K, Kokubo Y, Mestril R, Dillmann W, Weinstein PR, Sharp FR: Mice overexpressing rat heat shock protein 70 are protected against cerebral infarction. Ann Neurol 2000, 47:782e791 Zheng Z, Kim JY, Ma H, Lee JE, Yenari MA: Anti-inflammatory effects of the 70 kDa heat shock protein in experimental stroke. J Cereb Blood Flow Metab 2008, 28:53e63 Tang D, Kang R, Xiao W, Wang H, Calderwood SK, Xiao X: The antiinflammatory effects of heat shock protein 72 involve inhibition of high-mobility-group box 1 release and proinflammatory function in macrophages. J Immunol 2007, 179:1236e1244
ajp.amjpathol.org
FLA 5.4.0 DTD AJPA2352_proof 18 June 2016 7:53 am EO: AJP15_0460
9
1048 1049 1050 1051 1052 1053 1054 1055 1056 1057 1058 1059 1060 1061 1062 1063 1064 1065 1066 1067 1068 1069 1070 1071 1072 1073 1074 1075 1076 1077 1078 1079 1080 1081 1082 1083 1084 1085 1086 1087 1088 1089 1090 1091 1092 1093 1094 1095 1096 1097 1098 1099 1100 1101 1102