Neuroscience Letters 591 (2015) 53–58
Contents lists available at ScienceDirect
Neuroscience Letters journal homepage: www.elsevier.com/locate/neulet
Research article
Neuroprotective effects of crocin against traumatic brain injury in mice: Involvement of notch signaling pathway Kai Wang a,1 , Lei Zhang a,1 , Wei Rao a,1 , Ning Su a , Hao Hui a , Li Wang a , Cheng Peng a , Yue Tu b , Sai Zhang b,∗∗ , Zhou Fei a,∗ a b
Department of Neurosurgery, Xijing Institute of Clinical Neuroscience, Xijing Hospital, Fourth Military Medical University, Xi’an, Shaanxi 710032, PR China Department of Neurosurgery, Affiliated Hospital of Logistics University of Chinese Armed Police Forces, Chenglin Road, Tianjian 300162, PR China
h i g h l i g h t s • Pretreatment of crocin (20 mg/kg) conferred neuroprotective effects on the mice against TBI. • Pretreatment of crocin (20 mg/kg) significantly increased the Notch signaling activation after TBI. • Inhibition of Notch signaling attenuated the ability of crocin to protect mice against TBI.
a r t i c l e
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Article history: Received 5 December 2014 Received in revised form 2 February 2015 Accepted 7 February 2015 Available online 11 February 2015 Keywords: Traumatic brain injury Crocin Notch
a b s t r a c t This study investigated the protective effects and mechanisms of crocin, an extract of saffron, on brain damage after traumatic brain injury (TBI) in mice. C57BL/6 mice were subjected to controlled cortical impact (CCI)-induced TBI. Pretreatment with crocin (20 mg/kg) had protective effects against TBI, demonstrated by improved neurological severity score (NSS) and brain edema, decreased microglial activation and release of several pro-inflammatory cytokines, and decreased cell apoptosis. TBI activated Notch signaling, as shown by upregulated levels of Notch intracellular domain (NICD) and Hes1 mRNA, and pretreatment with crocin further increased Notch activation. However, pretreatment with DAPT (100 mg/kg), a gamma-secretase inhibitor, significantly suppressed crocin-induced activation of Notch signaling and attenuated the ability of crocin to protect mice against TBI-induced inflammation and apoptosis. Therefore, these results suggest that crocin has neuroprotective effects against TBI in mice, and these effects are at least partially dependent on activation of Notch signaling. © 2015 Published by Elsevier Ireland Ltd.
1. Introduction Traumatic brain injury (TBI) represents physical injury to brain tissue by mechanical forces of shearing, tearing, or stretching, resulting in contusion, hemorrhage, and immediate clinical effects. TBI presents a heavy economic and social burden, and there are currently no effective neuroprotective agents available for TBI patients. Nowadays, it is widely accepted that brain damage following TBI can be divided into initial and secondary injury phases, and prevention of secondary injury is the major target in therapeutic management of TBI. Post-traumatic cerebral inflammation, which
∗ Corresponding author. Tel.: +86 29 84775323; fax: +86 29 84775567. ∗∗ Corresponding author. E-mail addresses: [email protected] (S. Zhang), [email protected] (Z. Fei). 1 These authors contributed equally to this work. http://dx.doi.org/10.1016/j.neulet.2015.02.016 0304-3940/© 2015 Published by Elsevier Ireland Ltd.
is triggered by the initial injury and is characterized by microglia activation, leukocyte recruitment, and a subsequent upregulation of cytokines, plays an important role in the progression of secondary injury [12]. This neuroinflammation has been reported to promote edema formation and neuronal death and to ultimately lead to functional defects [2]. Therefore, it is imperative to search for an effective treatment based on anti-inflammatory strategies. Crocin, a pharmacologically-active component of Crocus sativus L. (saffron), has been studied in many animal disease models. Crocin has a broad spectrum of pharmacological properties, including antihyperglycemic [24], anti-oxidant [24], and anti-tumor [10] effects, and it was recently found to exhibit anti-inflammatory activity in several rodent disease models. Specifically, crocin decreased the number of neutrophils in the carrageenan model of local inflammation in rats [4], alleviated inflammation by downregulating pro-inflammatory cytokines in a mouse colitis model when included in the diet [7], and improved spatial cognition
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after cerebral ischemia in rats [6]. However, whether crocin has effects on TBI and its related mechanisms has not been investigated. The Notch signaling pathway, a well-conserved signaling pathway in animals, is activated by continuous processes: Notch ligand-receptor binding, cleavage of the Notch intracellular domain (NICD), localization of NICD to the nucleus, and transcriptional regulation of genes (e.g., Hes1 and Hes5). Notch signaling is fundamental for neuronal development and specification [14]. Accumulating studies have shown the Notch pathway to be extensively involved in various types of brain injury [8,19]. However, the effect of crocin on Notch signaling is unknown. Therefore, this study investigated the effects of crocin and the Notch signaling pathway in a well-characterized TBI model, i.e., controlled cortical impact (CCI) in mice.
is awarded for failure of each of 10 tasks, such that the maximum score of 10 points represents severe neurological dysfunction, whereas 0 points indicates normal function.
2. Material and methods
Western blot analysis was used to detect expression of cleaved caspase3 and NICD. In brief, injured cerebral cortices were individually homogenized in cold lysis buffer, and protein concentrations were determined using a Bio-Rad Protein Assay kit (Bio-RadLos Angeles, CA, USA). Samples (40–50 g) were separated using 10% SDS-PAGE and blotted onto polyvinylidene fluoride membranes. Membranes were incubated with primary antibodies, and immunoreactivity was detected by HRP-conjugated secondary antibody and visualized using chemiluminescence (Amersham, Piscataway, NJ, USA). The following primary antibodies were used: anti-cleaved caspase3 (Cell signaling Technology, Danvers, MA, USA, 1:1000), anti-caspase3 (cell signaling technology, 1:1000), anti-NICD (Abcam, Cambridge, MA, USA, 1:500), and anti--Actin (Santa Cruz Biotechnology, Santa Cruz, USA, 1:1000). The secondary antibody was goat anti-rabbit IgG-B (Santa Cruz Biotechnology, 1:20,000). Western blot band intensity was analyzed using ImagePro Plus software.
2.1. Animals Male C57BL/6 mice (12–14 weeks of age, 28–32 g) were purchased from the Fourth Military Medical University Animal Services. Animals were housed under controlled conditions on a 12 h light/dark cycle at 21 ± 2 ◦ C and 60–70% humidity. All experimental protocols and animal handling procedures were performed in accordance with the National Institutes of Health (NIH) guidelines for the use of experimental animals and were approved by the Institutional Animal Care and Use Committee of the Fourth Military Medical University. 2.2. Drug treatments Crocin (Sigma–Aldrich Inc., St. Louis, MO, USA) was dissolved in normal saline, and the gamma-secretase inhibitor DAPT (Sigma–Aldrich Inc.) was dissolved in DMSO. Both crocin and DAPT were administered 30 min before TBI. The dose and method of crocin and DAPT infusion was chosen based on previous ischemia studies [13,18,23]. 2.3. Animal models TBI was produced via CCI (Hatteras Instruments, Cary, NC, USA) with some modification of previous methods [1]. Briefly, mice were anesthetized with 4% isoflurane in oxygen and placed in the stereotaxic frame on a thermostatically-controlled heating pad to maintain body temperature at 37 ± 0.5 ◦ C. A portable drill was used to create a 3.5 mm diameter craniotomy over the right parietal cortex between bregma and lambda, 1 mm lateral to the midline. The dura mater was kept intact. To induce injury, a pneumatic piston impactor device (3 mm diameter, rounded tip) was used to impact the brain at a depth of 1 mm (velocity 4.5 m/s). Sham-operated mice underwent identical surgical procedures, but did not receive a CCI. Testing at 24 h following TBI was completed as follows: (1) evaluation of neurological impairment (n = 5 per group); (2) evaluation of brain edema (n = 5 per group); (3) Western blot analysis (n = 5 per group); (4) real-time PCR (n = 5 per group); (5) terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) assay and immunofluorescence (n = 5 per group); (6) enzymelinked immunosorbent assay (n = 5 per group). 2.4. Evaluation of neurological impairment Neurological deficits were evaluated using the Neurological Severity Score (NSS) on a 10-point scale according to Chen’s method [21] by a researcher blinded to treatment. On this scale, one point
2.5. Evaluation of brain edema According to Hatashita’s wet-dry method [17], mice were killed by cervical dislocation, and their brains were immediately removed and placed onto a frozen plate, then weighed to determine wet weight. Next, brains were dried in a desiccating oven at 110 ◦ C for 24 h and weighed again to determine dry weight. Brain water content was calculated using the following formula: brain water content (%) = (wet weight − dry weight) × 100/wet weight. 2.6. Western blot analysis
2.7. Real-time PCR The total RNA of each ipsilateral hemisphere was extracted using RNAiso Plus (TaKaRa, Dalian, China), as described previously [22]. Quantitative PCR was completed with the Bio-Rad iQ5 Gradient Real-Time PCR system (Bio-Rad-Los Angeles, CA, USA). Quantified values of RNA were normalized with those of glyceraldehyde 3phosphate dehydrogenase (GAPDH). The following primers were used in the current study: Hes1: (Fwd: TGT CAA CAC GAC ACC GGA CA, Rev: GCC TCT TCT CCA TGA TAG GCT TTG); GAPDH: (Fwd: GGC ACA GTC AAG GCT GAG AAT G, Rev: ATG GTG GTG AAG ACG CCA GTA). 2.8. Enzyme-linked immunosorbent assay (ELISA) Mice were sacrificed 24 h after TBI, their brains were extracted, and the injured cerebral hemisphere was homogenized. The levels of tumor necrosis factor (TNF)-␣ and interleukin (IL)-1 were assessed using specific ELISA kits (Jiancheng Bioengineering Institute, Nanjing, China) according to manufacturer’s instructions. 2.9. Immunofluorescence Mice were anesthetized with isoflurane and intracardially perfused with 4% paraformaldehyde in phosphate buffered saline. Brains were extracted and sectioned coronally on a cryostat. Sections were permeabilized with 3% Triton X-100 for 10 min, blocked with 10% normal donkey serum in PBS for 60 min at room temperature, and incubated overnight with primary antibodies at 4 ◦ C. Activated microglia were detected using rabbit anti-mouse IBA1 antibody (Wako Pure Chemical Industries Ltd., Osaka, Japan; 1:800), followed by Alexa Fluor 488 donkey anti-rabbit secondary
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Fig. 1. Western blotting analysis was used to detect the expression of NICD, an activated form of Notch. (A) TBI upregulated NICD from 3 to 48 h. (**p < 0.05 vs. sham group, n = 5 per group). (B) Crocin and DAPT were injected 30 min before TBI. The enhanced activation of NICD stimulated by crocin was abrogated by DAPT at 24 h after TBI. (*p < 0.05 vs. TBI group; #p < 0.05 vs. T + C group, n = 5 per group). Real-time PCR analysis was used to detect Hes1 mRNA levels. (C) TBI upregulated Hes1 mRNA significantly from 6 h to 48 h. (**p < 0.05 vs. sham group, n = 5 per group). (D) Crocin and DAPT were injected 30 min before TBI. The upregulated Hes1 mRNA stimulated by crocin was abrogated by DAPT at 24 h after TBI. (*p < 0.05 vs. TBI group; #p < 0.05 vs. T + C group, n = 5 per group). T + C = TBI + crocin; T + Dm = TBI + DMSO; T + C + DA = TBI + crocin + DAPT.
Fig. 2. Crocin and DAPT were injected 30 min before TBI, and brain water content (A) and NSS (B) were measured at 24 h after TBI. *p < 0.05 vs. TBI group; #p < 0.05 vs. T + C group, n = 5 per group. T + DM = TBI + DMSO; T + C = TBI + crocin; T + C + DA = TBI + crocin + DAPT.
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antibodies (Molecular Probes, Eugene, OR, USA; 1:500). Sections were analyzed on a Nikon fluorescence microscope. The contusion edge in the cortex was used as an observation point. 2.10. TUNEL assay
activation of Notch, as demonstrated for both NICD and Hes1 mRNA levels (p < 0.05). 3.2. Crocin exerted neuroprotective effects on TBI-induced brain damage via Notch signaling activation
Apoptotic cell death was determined using TUNEL staining (Roche Applied Bioscience, Indianapolis, IN, USA) according to manufacturer’s instructions. After the TUNEL assay was complete, sections were counterstained using 4 -6-diamidino-2phenylindole (DAPI) (Vector Laboratories, Burlingame, CA, USA) for 5 min to obtain total cell counts. The labeled cells were visualized under the OLYMPUS IX70 inverted microscope. The contusion edge in the cortex was used as an observation point.
To determine the neuroprotective effects of crocin against TBI-induced brain damage, we examined the effects of crocin pretreatment on brain edema (Fig. 2A) and NSS (Fig. 2B) 24 h after TBI respectively. As expected, the TBI + crocin group had decreased brain water content compared to the TBI group (p < 0.05; Fig. 2A). Similarly, the TBI + crocin group showed improved NSS compared to the TBI group (p < 0.05; Fig. 2B). However, application of DAPT inhibited these neuroprotective effects of crocin, increasing water content and decreasing NSS (p < 0.05).
2.11. Statistical analysis
3.3. Crocin inhibited neuroinflammation after TBI via Notch signaling activation
Statistical analysis was performed using SPSS 16.0 software. All data are expressed as mean ± SD. Statistical evaluation of the data was performed by ANOVA, and between-group differences were detected with Tukey tests. A value of p < 0.05 was considered statistically significant. 3. Results 3.1. Crocin activated the Notch signaling pathway in mice after TBI To quantify activation of Notch signaling, western blot and realtime PCR were used to measure the level of NICD and Hes1 mRNA at different time points after TBI. As shown in Fig. 1A and C, NICD was up-regulated 3 h after TBI and peaked at 24 h, and Hes1 mRNA was up-regulated at 6 h after TBI and peaked at 48 h (p < 0.05). As shown in Fig. 1B and D, pretreatment with crocin 30 min before TBI caused a significant further up-regulation of NICD and Hes1 mRNA at 24 h compared to TBI alone (p < 0.05). However, addition of the gamma-secretase inhibitor DAPT abrogated crocin-induced
To assess the potential effects and mechanisms of crocin on neuroinflammation after TBI, we examined the microglial response using IBA-1, an activated microglia marker, and the levels of proinflammatory cytokines 24 h after TBI. As shown in Fig. 3A and B, crocin decreased the number of activated microglia at 24 h compared to TBI (p < 0.05), and DAPT reversed this effect (p < 0.05). Similarly, as shown in Fig. 3C and D, crocin reduced the release of IL-1 and TNF-␣ at 24 h compared to TBI (p < 0.05), and DAPT reversed these effects (p < 0.05). 3.4. Crocin exerted anti-apoptotic effects after TBI via Notch signaling activation To assess the anti-apoptotic effect of crocin and mechanism after TBI, we used TUNEL assay and Western blot analysis to detect the number of apoptotic cell and the expression of cleaved caspase3 24 h after TBI. As shown in Fig. 4A and B, crocin significantly reduced the number of TUNEL-positive apoptotic cells compared to TBI (p < 0.05). Similarly, as shown in Fig. 4C, crocin downregulated
Fig. 3. Crocin and DAPT were injected 30 min before TBI, immunohistochemical staining of injured cortices with the microglial-specific marker IBA-1 at 24 h after TBI (A and B). Brain tissue homogenates were obtained from the injured cerebral hemisphere at 24 h after TBI, and IL-1 (C) and TNF-␣ (D) were measured by ELISA. *p < 0.05 vs. TBI group; #p < 0.05 vs. T + C group, n = 5 per group. Scale bar = 20 m. T + DM = TBI + DMSO; T + C = TBI + crocin; T + C + DA = TBI + crocin + DAPT.
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Fig. 4. Effects of crocin and DAPT on TUNEL staining after TBI (A and B). Western blotting analysis was used to detect the cleaved form of caspase3 at 24 h after TBI (C). *p < 0.05 vs. TBI group; #p < 0.05 vs. T + C group, n = 5 per group. Scale bar = 20 m. T + DM = TBI + DMSO; T + C = TBI + crocin; T + C + DA = TBI + crocin + DAPT; C-caspase3 = cleaved caspase3; T-caspase3 = total caspase3.
the expression of cleaved caspase3 compared to TBI (p < 0.05). DAPT reversed these protective effects (p < 0.05).
4. Discussion TBI is a complex neurodegenerative process, involving many cellular and molecular events, including inflammation and apoptosis. Neuroinflammation is associated with activation of microglia and release of cytokines. Microglial cells play important roles in immune and inflammatory responses in the central nervous system. Activated microglial cells released various neurotoxic substances that might exacerbate neuronal death after TBI [5]. Suppression of excessive microglial activation is a therapeutic strategy to alleviate the progression of neurological diseases [15]. The inflammatory cascade activated by TBI is mediated by the release of pro- and anti-inflammatory cytokines. IL-1 and TNF-␣, two wellcharacterized pro-inflammatory cytokines, have been found to be elevated in the brain parenchyma within hours after brain injury in rodents [20]. Further, inhibiting TBI-induced expression of IL-1 exerted neuroprotective effects in mice [11], and inhibiting TBIinduced expression of TNF-␣ improved neurological outcome and reduced brain edema in rats [3]. A previous study revealed that crocin reduced LPS-stimulated production of TNF-␣ and IL-1 from cultured rat brain microglia and blocked LPS-induced cell death in organotypic hippocampal slice cultures [9], suggesting a neuroprotective effect by reducing the production of various neurotoxic molecules from activated microglia. In the present study, crocin pretreatment markedly reduced the number of activated microglia and simultaneously downregulated TNF-␣ and IL-1 after TBI.
Therefore, we conclude that crocin might significantly inhibit TBIinduced neuroinflammation. Apoptosis is another important component of secondary brain injury. Apoptosis has been divided into caspase-dependent and -independent processes. In caspase-dependent apoptosis, caspase3 acts as a final executer of the apoptotic process. A previous study showed that caspase3 inhibitor reduced brain tissue loss and improved neurological outcome after experimental TBI [16]. Our results showed that crocin significantly reduced the number of TUNEL-positive cells and the expression of cleaved caspase3, suggesting prevention of neuronal apoptosis after TBI in mice. Brain edema is believed to be one of the major factors leading to the high mortality associated with clinical TBI. We found that crocin significantly reduced brain water content after TBI in mice. Apoptotic signaling could induce cation accumulation, which would drive an influx of water into cells and induce cellular swelling, causing cytotoxic edema. Meanwhile, immune cells have been activated to remove apoptotic cells, and this cerebral inflammation might further damage the microvascular endothelium and lead to blood-brain barrier disruption and vasogenic edema. Therefore, the anti-edema effect of crocin observed in our study was likely related to its anti-apoptotic and anti-inflammatory effects. Activation of Notch signaling occurs in various brain injury models [8,19], but conflicting effects of Notch have been reported. For example, blockade of Notch improved functional outcome in a mouse model of stroke [19], but activation of Notch was also protective against transient cerebral ischemia by decreasing neuronal apoptosis in mice [13]. This discrepancy could be explained
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by the choice of animals and drug administrations. Although the role of Notch signaling after brain injury is controversial, our current study showed that crocin effectively activated Notch signaling and exerted neuroprotective effects in mice after TBI, and inhibition of Notch signaling significantly decreased the protective effects of crocin on neuronal apoptosis, microglial response, and release of pro-inflammatory cytokines. These results suggest that activation of Notch signaling mediated crocin-induced protective effects in TBI and provide an important therapeutic strategy for the treatment of TBI. In conclusion, this study demonstrated that crocin conferred neuroprotection to an in vivo experimental model of TBI by reducing brain edema and improving neurological recovery. More specifically, crocin protected mice against TBI-induced apoptotic cell death and inflammation. The Notch signaling pathway appears to play an important role in crocin-mediated neuroprotective effects. Conflict of interest There is no conflict of interest. Acknowledgements The work was supported by National Natural Science Foundation of China (Nos. 81430043), National Science and Technology Major Project of China (2013ZX 09J13109-02C), National Science and Technology Pillar Program of China (No. 2012BAI11B02), Science and Technology Project of Shanxi (No. 2013KTCQ03-01). References [1] C. Israelsson, H. Bengtsson, A. Kylberg, K. Kullander, A. Lewen, L. Hillered, T. Ebendal, Distinct cellular patterns of upregulated chemokine expression supporting a prominent inflammatory role in traumatic brain injury, J. Neurotrauma 25 (2008) 959–974. [2] E. Lloyd, K. Somera-Molina, L.J. Van Eldik, D.M. Watterson, M.S. Wainwright, Suppression of acute proinflammatory cytokine and chemokine upregulation by post-injury administration of a novel small molecule improves long-term neurologic outcome in a mouse model of traumatic brain injury, J. Neuroinflamma. 5 (2008) 28. [3] E. Shohami, R. Gallily, R. Mechoulam, R. Bass, T. Ben-Hur, Cytokine production in the brain following closed head injury: dexanabinol (HU-211) is a novel TNF-alpha inhibitor and an effective neuroprotectant, J. Neuroimmunol. 72 (1997) 169–177. [4] E. Tamaddonfard, A.A. Farshid, K. Eghdami, F. Samadi, A. Erfanparast, Comparison of the effects of crocin, safranal and diclofenac on local inflammation and inflammatory pain responses induced by carrageenan in rats, Pharmacol. Rep. 65 (2013) 1272–1280. [5] G.W. Kreutzberg, Microglia: a sensor for pathological events in the CNS, Trends Neurosci. 19 (1996) 312–318. [6] H. Hosseinzadeh, H.R. Sadeghnia, F.A. Ghaeni, V.S. Motamedshariaty, S.A. Mohajeri, Effects of saffron (Crocus sativus L.) and its active constituent, crocin, on recognition and spatial memory after chronic cerebral hypoperfusion in rats, Phytother. Res. 26 (2012) 381–386.
[7] K. Kawabata, N.H. Tung, Y. Shoyama, S. Sugie, T. Mori, T. Tanaka, Dietary crocin inhibits colitis and colitis-associated colorectal carcinogenesis in male ICR mice, Evid. Based Complement Alternat. Med. 2012 (2012) 820415. [8] K. Tatsumi, H. Okuda, M. Makinodan, T. Yamauchi, E. Makinodan, H. Matsuyoshi, T. Manabe, A. Wanaka, Transient activation of Notch signaling in the injured adult brain, J. Chem. Neuroanat. 39 (2010) 15–19. [9] K.N. Nam, Y.M. Park, H.J. Jung, J.Y. Lee, B.D. Min, S.U. Park, W.S. Jung, K.H. Cho, J.H. Park, I. Kang, J.W. Hong, E.H. Lee, Anti-inflammatory effects of crocin and crocetin in rat brain microglial cells, Eur. J. Pharmacol. 648 (2010) 110–116. [10] M. Rastgoo, H. Hosseinzadeh, H. Alavizadeh, A. Abbasi, Z. Ayati, M.R. Jaafari, Antitumor activity of PEGylated nanoliposomes containing crocin in mice bearing C26 colon carcinoma, Planta Med. 79 (2013) 447–451. [11] N.C. Jones, M.J. Prior, E. Burden-Teh, C.A. Marsden, P.G. Morris, S. Murphy, Antagonism of the interleukin-1 receptor following traumatic brain injury in the mouse reduces the number of nitric oxide synthase-2-positive cells and improves anatomical and functional outcomes, Eur. J. Neurosci. 22 (2005) 72–78. [12] O.I. Schmidt, C.E. Heyde, W. Ertel, P.F. Stahel, Closed head injury – an inflammatory disease, Brain Res. Brain Res. Rev. 48 (2005) 388–399. [13] Q. Yang, W. Yan, X. Li, L. Hou, H. Dong, Q. Wang, H. Dong, S. Wang, X. Zhang, L. Xiong, Activation of canonical notch signaling pathway is involved in the ischemic tolerance induced by sevoflurane preconditioning in mice, Anesthesiology 117 (2012) 996–1005. [14] R. Kopan, M.X. Ilagan, The canonical Notch signaling pathway: unfolding the activation mechanism, Cell 137 (2009) 216–233. [15] R.B. Rock, G. Gekker, S. Hu, W.S. Sheng, M. Cheeran, J.R. Lokensgard, P.K. Peterson, Role of microglia in central nervous system infections, Clin. Microbiol. Rev. 17 (2004) 942–964. [16] R.S. Clark, P.M. Kochanek, S.C. Watkins, M. Chen, C.E. Dixon, N.A. Seidberg, J. Melick, J.E. Loeffert, P.D. Nathaniel, K.L. Jin, S.H. Graham, Caspase-3 mediated neuronal death after traumatic brain injury in rats, J. Neurochem. 74 (2000) 740–753. [17] S. Hatashita, J.T. Hoff, S.M. Salamat, Ischemic brain edema and the osmotic gradient between blood and brain, J. Cereb. Blood Flow Metab. 8 (1988) 552–559. [18] T. Ochiai, H. Shimeno, K. Mishima, K. Iwasaki, M. Fujiwara, H. Tanaka, Y. Shoyama, A. Toda, R. Eyanagi, S. Soeda, Protective effects of carotenoids from saffron on neuronal injury in vitro and in vivo, Biochim. Biophys. Acta 1770 (2007) 578–584. [19] T.V. Arumugam, S.L. Chan, D.G. Jo, G. Yilmaz, S.C. Tang, A. Cheng, M. Gleichmann, E. Okun, V.D. Dixit, S. Chigurupati, M.R. Mughal, X. Ouyang, L. Miele, T. Magnus, S. Poosala, D.N. Granger, M.P. Mattson, Gamma secretase-mediated Notch signaling worsens brain damage and functional outcome in ischemic stroke, Nat. Med. 12 (2006) 621–623. [20] V. Taupin, S. Toulmond, A. Serrano, J. Benavides, F. Zavala, Increase in IL-6 IL-1 and TNF levels in rat brain following traumatic lesion. Influence of pre- and post-traumatic treatment with Ro5 4864, a peripheral-type (p site) benzodiazepine ligand, J. Neuroimmunol. 42 (1993) 177–185. [21] Y. Chen, S. Constantini, V. Trembovler, M. Weinstock, E. Shohami, An experimental model of closed head injury in mice: pathophysiology, histopathology, and cognitive deficits, J. Neurotrauma. 13 (1996) 557–568. [22] Y. Wang, W. Tu, Y. Lou, A. Xie, X. Lai, F. Guo, Z. Deng, Mesenchymal stem cells regulate the proliferation and differentiation of neural stem cells through Notch signaling, Cell Biol. Int. 33 (2009) 1173–1179. [23] Y.Q. Zheng, J.X. Liu, J.N. Wang, L. Xu, Effects of crocin on reperfusion-induced oxidative/nitrative injury to cerebral microvessels after global cerebral ischemia, Brain Res. 1138 (2007) 86–94. [24] Z. Rajaei, M.A. Hadjzadeh, H. Nemati, M. Hosseini, M. Ahmadi, S. Shafiee, Antihyperglycemic and antioxidant activity of crocin in streptozotocin-induced diabetic rats, J. Med. Food 16 (2013) 206–210.
Contents lists available at ScienceDirect
Neuroscience Letters journal homepage: www.elsevier.com/locate/neulet
Research article
Neuroprotective effects of crocin against traumatic brain injury in mice: Involvement of notch signaling pathway Kai Wang a,1 , Lei Zhang a,1 , Wei Rao a,1 , Ning Su a , Hao Hui a , Li Wang a , Cheng Peng a , Yue Tu b , Sai Zhang b,∗∗ , Zhou Fei a,∗ a b
Department of Neurosurgery, Xijing Institute of Clinical Neuroscience, Xijing Hospital, Fourth Military Medical University, Xi’an, Shaanxi 710032, PR China Department of Neurosurgery, Affiliated Hospital of Logistics University of Chinese Armed Police Forces, Chenglin Road, Tianjian 300162, PR China
h i g h l i g h t s • Pretreatment of crocin (20 mg/kg) conferred neuroprotective effects on the mice against TBI. • Pretreatment of crocin (20 mg/kg) significantly increased the Notch signaling activation after TBI. • Inhibition of Notch signaling attenuated the ability of crocin to protect mice against TBI.
a r t i c l e
i n f o
Article history: Received 5 December 2014 Received in revised form 2 February 2015 Accepted 7 February 2015 Available online 11 February 2015 Keywords: Traumatic brain injury Crocin Notch
a b s t r a c t This study investigated the protective effects and mechanisms of crocin, an extract of saffron, on brain damage after traumatic brain injury (TBI) in mice. C57BL/6 mice were subjected to controlled cortical impact (CCI)-induced TBI. Pretreatment with crocin (20 mg/kg) had protective effects against TBI, demonstrated by improved neurological severity score (NSS) and brain edema, decreased microglial activation and release of several pro-inflammatory cytokines, and decreased cell apoptosis. TBI activated Notch signaling, as shown by upregulated levels of Notch intracellular domain (NICD) and Hes1 mRNA, and pretreatment with crocin further increased Notch activation. However, pretreatment with DAPT (100 mg/kg), a gamma-secretase inhibitor, significantly suppressed crocin-induced activation of Notch signaling and attenuated the ability of crocin to protect mice against TBI-induced inflammation and apoptosis. Therefore, these results suggest that crocin has neuroprotective effects against TBI in mice, and these effects are at least partially dependent on activation of Notch signaling. © 2015 Published by Elsevier Ireland Ltd.
1. Introduction Traumatic brain injury (TBI) represents physical injury to brain tissue by mechanical forces of shearing, tearing, or stretching, resulting in contusion, hemorrhage, and immediate clinical effects. TBI presents a heavy economic and social burden, and there are currently no effective neuroprotective agents available for TBI patients. Nowadays, it is widely accepted that brain damage following TBI can be divided into initial and secondary injury phases, and prevention of secondary injury is the major target in therapeutic management of TBI. Post-traumatic cerebral inflammation, which
∗ Corresponding author. Tel.: +86 29 84775323; fax: +86 29 84775567. ∗∗ Corresponding author. E-mail addresses: [email protected] (S. Zhang), [email protected] (Z. Fei). 1 These authors contributed equally to this work. http://dx.doi.org/10.1016/j.neulet.2015.02.016 0304-3940/© 2015 Published by Elsevier Ireland Ltd.
is triggered by the initial injury and is characterized by microglia activation, leukocyte recruitment, and a subsequent upregulation of cytokines, plays an important role in the progression of secondary injury [12]. This neuroinflammation has been reported to promote edema formation and neuronal death and to ultimately lead to functional defects [2]. Therefore, it is imperative to search for an effective treatment based on anti-inflammatory strategies. Crocin, a pharmacologically-active component of Crocus sativus L. (saffron), has been studied in many animal disease models. Crocin has a broad spectrum of pharmacological properties, including antihyperglycemic [24], anti-oxidant [24], and anti-tumor [10] effects, and it was recently found to exhibit anti-inflammatory activity in several rodent disease models. Specifically, crocin decreased the number of neutrophils in the carrageenan model of local inflammation in rats [4], alleviated inflammation by downregulating pro-inflammatory cytokines in a mouse colitis model when included in the diet [7], and improved spatial cognition
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after cerebral ischemia in rats [6]. However, whether crocin has effects on TBI and its related mechanisms has not been investigated. The Notch signaling pathway, a well-conserved signaling pathway in animals, is activated by continuous processes: Notch ligand-receptor binding, cleavage of the Notch intracellular domain (NICD), localization of NICD to the nucleus, and transcriptional regulation of genes (e.g., Hes1 and Hes5). Notch signaling is fundamental for neuronal development and specification [14]. Accumulating studies have shown the Notch pathway to be extensively involved in various types of brain injury [8,19]. However, the effect of crocin on Notch signaling is unknown. Therefore, this study investigated the effects of crocin and the Notch signaling pathway in a well-characterized TBI model, i.e., controlled cortical impact (CCI) in mice.
is awarded for failure of each of 10 tasks, such that the maximum score of 10 points represents severe neurological dysfunction, whereas 0 points indicates normal function.
2. Material and methods
Western blot analysis was used to detect expression of cleaved caspase3 and NICD. In brief, injured cerebral cortices were individually homogenized in cold lysis buffer, and protein concentrations were determined using a Bio-Rad Protein Assay kit (Bio-RadLos Angeles, CA, USA). Samples (40–50 g) were separated using 10% SDS-PAGE and blotted onto polyvinylidene fluoride membranes. Membranes were incubated with primary antibodies, and immunoreactivity was detected by HRP-conjugated secondary antibody and visualized using chemiluminescence (Amersham, Piscataway, NJ, USA). The following primary antibodies were used: anti-cleaved caspase3 (Cell signaling Technology, Danvers, MA, USA, 1:1000), anti-caspase3 (cell signaling technology, 1:1000), anti-NICD (Abcam, Cambridge, MA, USA, 1:500), and anti--Actin (Santa Cruz Biotechnology, Santa Cruz, USA, 1:1000). The secondary antibody was goat anti-rabbit IgG-B (Santa Cruz Biotechnology, 1:20,000). Western blot band intensity was analyzed using ImagePro Plus software.
2.1. Animals Male C57BL/6 mice (12–14 weeks of age, 28–32 g) were purchased from the Fourth Military Medical University Animal Services. Animals were housed under controlled conditions on a 12 h light/dark cycle at 21 ± 2 ◦ C and 60–70% humidity. All experimental protocols and animal handling procedures were performed in accordance with the National Institutes of Health (NIH) guidelines for the use of experimental animals and were approved by the Institutional Animal Care and Use Committee of the Fourth Military Medical University. 2.2. Drug treatments Crocin (Sigma–Aldrich Inc., St. Louis, MO, USA) was dissolved in normal saline, and the gamma-secretase inhibitor DAPT (Sigma–Aldrich Inc.) was dissolved in DMSO. Both crocin and DAPT were administered 30 min before TBI. The dose and method of crocin and DAPT infusion was chosen based on previous ischemia studies [13,18,23]. 2.3. Animal models TBI was produced via CCI (Hatteras Instruments, Cary, NC, USA) with some modification of previous methods [1]. Briefly, mice were anesthetized with 4% isoflurane in oxygen and placed in the stereotaxic frame on a thermostatically-controlled heating pad to maintain body temperature at 37 ± 0.5 ◦ C. A portable drill was used to create a 3.5 mm diameter craniotomy over the right parietal cortex between bregma and lambda, 1 mm lateral to the midline. The dura mater was kept intact. To induce injury, a pneumatic piston impactor device (3 mm diameter, rounded tip) was used to impact the brain at a depth of 1 mm (velocity 4.5 m/s). Sham-operated mice underwent identical surgical procedures, but did not receive a CCI. Testing at 24 h following TBI was completed as follows: (1) evaluation of neurological impairment (n = 5 per group); (2) evaluation of brain edema (n = 5 per group); (3) Western blot analysis (n = 5 per group); (4) real-time PCR (n = 5 per group); (5) terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) assay and immunofluorescence (n = 5 per group); (6) enzymelinked immunosorbent assay (n = 5 per group). 2.4. Evaluation of neurological impairment Neurological deficits were evaluated using the Neurological Severity Score (NSS) on a 10-point scale according to Chen’s method [21] by a researcher blinded to treatment. On this scale, one point
2.5. Evaluation of brain edema According to Hatashita’s wet-dry method [17], mice were killed by cervical dislocation, and their brains were immediately removed and placed onto a frozen plate, then weighed to determine wet weight. Next, brains were dried in a desiccating oven at 110 ◦ C for 24 h and weighed again to determine dry weight. Brain water content was calculated using the following formula: brain water content (%) = (wet weight − dry weight) × 100/wet weight. 2.6. Western blot analysis
2.7. Real-time PCR The total RNA of each ipsilateral hemisphere was extracted using RNAiso Plus (TaKaRa, Dalian, China), as described previously [22]. Quantitative PCR was completed with the Bio-Rad iQ5 Gradient Real-Time PCR system (Bio-Rad-Los Angeles, CA, USA). Quantified values of RNA were normalized with those of glyceraldehyde 3phosphate dehydrogenase (GAPDH). The following primers were used in the current study: Hes1: (Fwd: TGT CAA CAC GAC ACC GGA CA, Rev: GCC TCT TCT CCA TGA TAG GCT TTG); GAPDH: (Fwd: GGC ACA GTC AAG GCT GAG AAT G, Rev: ATG GTG GTG AAG ACG CCA GTA). 2.8. Enzyme-linked immunosorbent assay (ELISA) Mice were sacrificed 24 h after TBI, their brains were extracted, and the injured cerebral hemisphere was homogenized. The levels of tumor necrosis factor (TNF)-␣ and interleukin (IL)-1 were assessed using specific ELISA kits (Jiancheng Bioengineering Institute, Nanjing, China) according to manufacturer’s instructions. 2.9. Immunofluorescence Mice were anesthetized with isoflurane and intracardially perfused with 4% paraformaldehyde in phosphate buffered saline. Brains were extracted and sectioned coronally on a cryostat. Sections were permeabilized with 3% Triton X-100 for 10 min, blocked with 10% normal donkey serum in PBS for 60 min at room temperature, and incubated overnight with primary antibodies at 4 ◦ C. Activated microglia were detected using rabbit anti-mouse IBA1 antibody (Wako Pure Chemical Industries Ltd., Osaka, Japan; 1:800), followed by Alexa Fluor 488 donkey anti-rabbit secondary
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Fig. 1. Western blotting analysis was used to detect the expression of NICD, an activated form of Notch. (A) TBI upregulated NICD from 3 to 48 h. (**p < 0.05 vs. sham group, n = 5 per group). (B) Crocin and DAPT were injected 30 min before TBI. The enhanced activation of NICD stimulated by crocin was abrogated by DAPT at 24 h after TBI. (*p < 0.05 vs. TBI group; #p < 0.05 vs. T + C group, n = 5 per group). Real-time PCR analysis was used to detect Hes1 mRNA levels. (C) TBI upregulated Hes1 mRNA significantly from 6 h to 48 h. (**p < 0.05 vs. sham group, n = 5 per group). (D) Crocin and DAPT were injected 30 min before TBI. The upregulated Hes1 mRNA stimulated by crocin was abrogated by DAPT at 24 h after TBI. (*p < 0.05 vs. TBI group; #p < 0.05 vs. T + C group, n = 5 per group). T + C = TBI + crocin; T + Dm = TBI + DMSO; T + C + DA = TBI + crocin + DAPT.
Fig. 2. Crocin and DAPT were injected 30 min before TBI, and brain water content (A) and NSS (B) were measured at 24 h after TBI. *p < 0.05 vs. TBI group; #p < 0.05 vs. T + C group, n = 5 per group. T + DM = TBI + DMSO; T + C = TBI + crocin; T + C + DA = TBI + crocin + DAPT.
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antibodies (Molecular Probes, Eugene, OR, USA; 1:500). Sections were analyzed on a Nikon fluorescence microscope. The contusion edge in the cortex was used as an observation point. 2.10. TUNEL assay
activation of Notch, as demonstrated for both NICD and Hes1 mRNA levels (p < 0.05). 3.2. Crocin exerted neuroprotective effects on TBI-induced brain damage via Notch signaling activation
Apoptotic cell death was determined using TUNEL staining (Roche Applied Bioscience, Indianapolis, IN, USA) according to manufacturer’s instructions. After the TUNEL assay was complete, sections were counterstained using 4 -6-diamidino-2phenylindole (DAPI) (Vector Laboratories, Burlingame, CA, USA) for 5 min to obtain total cell counts. The labeled cells were visualized under the OLYMPUS IX70 inverted microscope. The contusion edge in the cortex was used as an observation point.
To determine the neuroprotective effects of crocin against TBI-induced brain damage, we examined the effects of crocin pretreatment on brain edema (Fig. 2A) and NSS (Fig. 2B) 24 h after TBI respectively. As expected, the TBI + crocin group had decreased brain water content compared to the TBI group (p < 0.05; Fig. 2A). Similarly, the TBI + crocin group showed improved NSS compared to the TBI group (p < 0.05; Fig. 2B). However, application of DAPT inhibited these neuroprotective effects of crocin, increasing water content and decreasing NSS (p < 0.05).
2.11. Statistical analysis
3.3. Crocin inhibited neuroinflammation after TBI via Notch signaling activation
Statistical analysis was performed using SPSS 16.0 software. All data are expressed as mean ± SD. Statistical evaluation of the data was performed by ANOVA, and between-group differences were detected with Tukey tests. A value of p < 0.05 was considered statistically significant. 3. Results 3.1. Crocin activated the Notch signaling pathway in mice after TBI To quantify activation of Notch signaling, western blot and realtime PCR were used to measure the level of NICD and Hes1 mRNA at different time points after TBI. As shown in Fig. 1A and C, NICD was up-regulated 3 h after TBI and peaked at 24 h, and Hes1 mRNA was up-regulated at 6 h after TBI and peaked at 48 h (p < 0.05). As shown in Fig. 1B and D, pretreatment with crocin 30 min before TBI caused a significant further up-regulation of NICD and Hes1 mRNA at 24 h compared to TBI alone (p < 0.05). However, addition of the gamma-secretase inhibitor DAPT abrogated crocin-induced
To assess the potential effects and mechanisms of crocin on neuroinflammation after TBI, we examined the microglial response using IBA-1, an activated microglia marker, and the levels of proinflammatory cytokines 24 h after TBI. As shown in Fig. 3A and B, crocin decreased the number of activated microglia at 24 h compared to TBI (p < 0.05), and DAPT reversed this effect (p < 0.05). Similarly, as shown in Fig. 3C and D, crocin reduced the release of IL-1 and TNF-␣ at 24 h compared to TBI (p < 0.05), and DAPT reversed these effects (p < 0.05). 3.4. Crocin exerted anti-apoptotic effects after TBI via Notch signaling activation To assess the anti-apoptotic effect of crocin and mechanism after TBI, we used TUNEL assay and Western blot analysis to detect the number of apoptotic cell and the expression of cleaved caspase3 24 h after TBI. As shown in Fig. 4A and B, crocin significantly reduced the number of TUNEL-positive apoptotic cells compared to TBI (p < 0.05). Similarly, as shown in Fig. 4C, crocin downregulated
Fig. 3. Crocin and DAPT were injected 30 min before TBI, immunohistochemical staining of injured cortices with the microglial-specific marker IBA-1 at 24 h after TBI (A and B). Brain tissue homogenates were obtained from the injured cerebral hemisphere at 24 h after TBI, and IL-1 (C) and TNF-␣ (D) were measured by ELISA. *p < 0.05 vs. TBI group; #p < 0.05 vs. T + C group, n = 5 per group. Scale bar = 20 m. T + DM = TBI + DMSO; T + C = TBI + crocin; T + C + DA = TBI + crocin + DAPT.
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Fig. 4. Effects of crocin and DAPT on TUNEL staining after TBI (A and B). Western blotting analysis was used to detect the cleaved form of caspase3 at 24 h after TBI (C). *p < 0.05 vs. TBI group; #p < 0.05 vs. T + C group, n = 5 per group. Scale bar = 20 m. T + DM = TBI + DMSO; T + C = TBI + crocin; T + C + DA = TBI + crocin + DAPT; C-caspase3 = cleaved caspase3; T-caspase3 = total caspase3.
the expression of cleaved caspase3 compared to TBI (p < 0.05). DAPT reversed these protective effects (p < 0.05).
4. Discussion TBI is a complex neurodegenerative process, involving many cellular and molecular events, including inflammation and apoptosis. Neuroinflammation is associated with activation of microglia and release of cytokines. Microglial cells play important roles in immune and inflammatory responses in the central nervous system. Activated microglial cells released various neurotoxic substances that might exacerbate neuronal death after TBI [5]. Suppression of excessive microglial activation is a therapeutic strategy to alleviate the progression of neurological diseases [15]. The inflammatory cascade activated by TBI is mediated by the release of pro- and anti-inflammatory cytokines. IL-1 and TNF-␣, two wellcharacterized pro-inflammatory cytokines, have been found to be elevated in the brain parenchyma within hours after brain injury in rodents [20]. Further, inhibiting TBI-induced expression of IL-1 exerted neuroprotective effects in mice [11], and inhibiting TBIinduced expression of TNF-␣ improved neurological outcome and reduced brain edema in rats [3]. A previous study revealed that crocin reduced LPS-stimulated production of TNF-␣ and IL-1 from cultured rat brain microglia and blocked LPS-induced cell death in organotypic hippocampal slice cultures [9], suggesting a neuroprotective effect by reducing the production of various neurotoxic molecules from activated microglia. In the present study, crocin pretreatment markedly reduced the number of activated microglia and simultaneously downregulated TNF-␣ and IL-1 after TBI.
Therefore, we conclude that crocin might significantly inhibit TBIinduced neuroinflammation. Apoptosis is another important component of secondary brain injury. Apoptosis has been divided into caspase-dependent and -independent processes. In caspase-dependent apoptosis, caspase3 acts as a final executer of the apoptotic process. A previous study showed that caspase3 inhibitor reduced brain tissue loss and improved neurological outcome after experimental TBI [16]. Our results showed that crocin significantly reduced the number of TUNEL-positive cells and the expression of cleaved caspase3, suggesting prevention of neuronal apoptosis after TBI in mice. Brain edema is believed to be one of the major factors leading to the high mortality associated with clinical TBI. We found that crocin significantly reduced brain water content after TBI in mice. Apoptotic signaling could induce cation accumulation, which would drive an influx of water into cells and induce cellular swelling, causing cytotoxic edema. Meanwhile, immune cells have been activated to remove apoptotic cells, and this cerebral inflammation might further damage the microvascular endothelium and lead to blood-brain barrier disruption and vasogenic edema. Therefore, the anti-edema effect of crocin observed in our study was likely related to its anti-apoptotic and anti-inflammatory effects. Activation of Notch signaling occurs in various brain injury models [8,19], but conflicting effects of Notch have been reported. For example, blockade of Notch improved functional outcome in a mouse model of stroke [19], but activation of Notch was also protective against transient cerebral ischemia by decreasing neuronal apoptosis in mice [13]. This discrepancy could be explained
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by the choice of animals and drug administrations. Although the role of Notch signaling after brain injury is controversial, our current study showed that crocin effectively activated Notch signaling and exerted neuroprotective effects in mice after TBI, and inhibition of Notch signaling significantly decreased the protective effects of crocin on neuronal apoptosis, microglial response, and release of pro-inflammatory cytokines. These results suggest that activation of Notch signaling mediated crocin-induced protective effects in TBI and provide an important therapeutic strategy for the treatment of TBI. In conclusion, this study demonstrated that crocin conferred neuroprotection to an in vivo experimental model of TBI by reducing brain edema and improving neurological recovery. More specifically, crocin protected mice against TBI-induced apoptotic cell death and inflammation. The Notch signaling pathway appears to play an important role in crocin-mediated neuroprotective effects. Conflict of interest There is no conflict of interest. Acknowledgements The work was supported by National Natural Science Foundation of China (Nos. 81430043), National Science and Technology Major Project of China (2013ZX 09J13109-02C), National Science and Technology Pillar Program of China (No. 2012BAI11B02), Science and Technology Project of Shanxi (No. 2013KTCQ03-01). References [1] C. Israelsson, H. Bengtsson, A. Kylberg, K. Kullander, A. Lewen, L. Hillered, T. Ebendal, Distinct cellular patterns of upregulated chemokine expression supporting a prominent inflammatory role in traumatic brain injury, J. Neurotrauma 25 (2008) 959–974. [2] E. Lloyd, K. Somera-Molina, L.J. Van Eldik, D.M. Watterson, M.S. Wainwright, Suppression of acute proinflammatory cytokine and chemokine upregulation by post-injury administration of a novel small molecule improves long-term neurologic outcome in a mouse model of traumatic brain injury, J. Neuroinflamma. 5 (2008) 28. [3] E. Shohami, R. Gallily, R. Mechoulam, R. Bass, T. Ben-Hur, Cytokine production in the brain following closed head injury: dexanabinol (HU-211) is a novel TNF-alpha inhibitor and an effective neuroprotectant, J. Neuroimmunol. 72 (1997) 169–177. [4] E. Tamaddonfard, A.A. Farshid, K. Eghdami, F. Samadi, A. Erfanparast, Comparison of the effects of crocin, safranal and diclofenac on local inflammation and inflammatory pain responses induced by carrageenan in rats, Pharmacol. Rep. 65 (2013) 1272–1280. [5] G.W. Kreutzberg, Microglia: a sensor for pathological events in the CNS, Trends Neurosci. 19 (1996) 312–318. [6] H. Hosseinzadeh, H.R. Sadeghnia, F.A. Ghaeni, V.S. Motamedshariaty, S.A. Mohajeri, Effects of saffron (Crocus sativus L.) and its active constituent, crocin, on recognition and spatial memory after chronic cerebral hypoperfusion in rats, Phytother. Res. 26 (2012) 381–386.
[7] K. Kawabata, N.H. Tung, Y. Shoyama, S. Sugie, T. Mori, T. Tanaka, Dietary crocin inhibits colitis and colitis-associated colorectal carcinogenesis in male ICR mice, Evid. Based Complement Alternat. Med. 2012 (2012) 820415. [8] K. Tatsumi, H. Okuda, M. Makinodan, T. Yamauchi, E. Makinodan, H. Matsuyoshi, T. Manabe, A. Wanaka, Transient activation of Notch signaling in the injured adult brain, J. Chem. Neuroanat. 39 (2010) 15–19. [9] K.N. Nam, Y.M. Park, H.J. Jung, J.Y. Lee, B.D. Min, S.U. Park, W.S. Jung, K.H. Cho, J.H. Park, I. Kang, J.W. Hong, E.H. Lee, Anti-inflammatory effects of crocin and crocetin in rat brain microglial cells, Eur. J. Pharmacol. 648 (2010) 110–116. [10] M. Rastgoo, H. Hosseinzadeh, H. Alavizadeh, A. Abbasi, Z. Ayati, M.R. Jaafari, Antitumor activity of PEGylated nanoliposomes containing crocin in mice bearing C26 colon carcinoma, Planta Med. 79 (2013) 447–451. [11] N.C. Jones, M.J. Prior, E. Burden-Teh, C.A. Marsden, P.G. Morris, S. Murphy, Antagonism of the interleukin-1 receptor following traumatic brain injury in the mouse reduces the number of nitric oxide synthase-2-positive cells and improves anatomical and functional outcomes, Eur. J. Neurosci. 22 (2005) 72–78. [12] O.I. Schmidt, C.E. Heyde, W. Ertel, P.F. Stahel, Closed head injury – an inflammatory disease, Brain Res. Brain Res. Rev. 48 (2005) 388–399. [13] Q. Yang, W. Yan, X. Li, L. Hou, H. Dong, Q. Wang, H. Dong, S. Wang, X. Zhang, L. Xiong, Activation of canonical notch signaling pathway is involved in the ischemic tolerance induced by sevoflurane preconditioning in mice, Anesthesiology 117 (2012) 996–1005. [14] R. Kopan, M.X. Ilagan, The canonical Notch signaling pathway: unfolding the activation mechanism, Cell 137 (2009) 216–233. [15] R.B. Rock, G. Gekker, S. Hu, W.S. Sheng, M. Cheeran, J.R. Lokensgard, P.K. Peterson, Role of microglia in central nervous system infections, Clin. Microbiol. Rev. 17 (2004) 942–964. [16] R.S. Clark, P.M. Kochanek, S.C. Watkins, M. Chen, C.E. Dixon, N.A. Seidberg, J. Melick, J.E. Loeffert, P.D. Nathaniel, K.L. Jin, S.H. Graham, Caspase-3 mediated neuronal death after traumatic brain injury in rats, J. Neurochem. 74 (2000) 740–753. [17] S. Hatashita, J.T. Hoff, S.M. Salamat, Ischemic brain edema and the osmotic gradient between blood and brain, J. Cereb. Blood Flow Metab. 8 (1988) 552–559. [18] T. Ochiai, H. Shimeno, K. Mishima, K. Iwasaki, M. Fujiwara, H. Tanaka, Y. Shoyama, A. Toda, R. Eyanagi, S. Soeda, Protective effects of carotenoids from saffron on neuronal injury in vitro and in vivo, Biochim. Biophys. Acta 1770 (2007) 578–584. [19] T.V. Arumugam, S.L. Chan, D.G. Jo, G. Yilmaz, S.C. Tang, A. Cheng, M. Gleichmann, E. Okun, V.D. Dixit, S. Chigurupati, M.R. Mughal, X. Ouyang, L. Miele, T. Magnus, S. Poosala, D.N. Granger, M.P. Mattson, Gamma secretase-mediated Notch signaling worsens brain damage and functional outcome in ischemic stroke, Nat. Med. 12 (2006) 621–623. [20] V. Taupin, S. Toulmond, A. Serrano, J. Benavides, F. Zavala, Increase in IL-6 IL-1 and TNF levels in rat brain following traumatic lesion. Influence of pre- and post-traumatic treatment with Ro5 4864, a peripheral-type (p site) benzodiazepine ligand, J. Neuroimmunol. 42 (1993) 177–185. [21] Y. Chen, S. Constantini, V. Trembovler, M. Weinstock, E. Shohami, An experimental model of closed head injury in mice: pathophysiology, histopathology, and cognitive deficits, J. Neurotrauma. 13 (1996) 557–568. [22] Y. Wang, W. Tu, Y. Lou, A. Xie, X. Lai, F. Guo, Z. Deng, Mesenchymal stem cells regulate the proliferation and differentiation of neural stem cells through Notch signaling, Cell Biol. Int. 33 (2009) 1173–1179. [23] Y.Q. Zheng, J.X. Liu, J.N. Wang, L. Xu, Effects of crocin on reperfusion-induced oxidative/nitrative injury to cerebral microvessels after global cerebral ischemia, Brain Res. 1138 (2007) 86–94. [24] Z. Rajaei, M.A. Hadjzadeh, H. Nemati, M. Hosseini, M. Ahmadi, S. Shafiee, Antihyperglycemic and antioxidant activity of crocin in streptozotocin-induced diabetic rats, J. Med. Food 16 (2013) 206–210.
