International Journal of Neuroscience, 2014; 124(9): 657–665 Copyright © 2014 Informa Healthcare USA, Inc. ISSN: 0020-7454 print / 1543-5245 online DOI: 10.3109/00207454.2013.856009
RESEARCH ARTICLE
Ceftriaxone pretreatment protects rats against cerebral ischemic injury by attenuating microglial activation-induced IL-1β expression Yang Lujia,1 Li Xin,2 Wang Shiquan,2 Chen Yu,2 Zhang Shuzhuo,3 and Zhang Hong1 Int J Neurosci Downloaded from informahealthcare.com by Kainan University on 04/16/15 For personal use only.
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Department of Anesthesiology, Chinese PLA General Hospital, Beijing, China; 2 Department of Anesthesiology, Xijing Hospital, Fourth Military Medical University, Xi’an, China, and 3 Academy of Military Medical Sciences, Beijing, China; Background: Although the neuroprotective effect of ceftriaxone (CTX) has been reported, the underlying mechanisms are still uncertain. In this study, we investigated if rats recover better from CTX pretreatment against cerebral ischemia by inhibiting the inflammatory response. Methods: Rats were pretreated with CTX (200 mg/kg, 1/day, i.p.) for 5 d. At 24 h after the end of the last CTX pretreatment, focal cerebral ischemia was induced by middle cerebral artery occlusion (MCAO) for 120 min in male Sprague Dawley rats. The neurological deficit scores (NDS) and infarct volumes were evaluated. Microglia cells were observed by immunofluorescence staining and IL-1β was assayed by ELISA and Western Blot. Results: The results showed that CTX pretreatment improved the neurological deficit scores and decreased the infarct volumes 24 h after reperfusion. The activation of microglia cells was reduced and the expression of IL-1β was partially inhibited 24 h after reperfusion. Conclusion: These findings demonstrate that CTX pretreatment may provide a neuroprotective effect against transient cerebral ischemic injury, partially inhibit in microglial activation and reduce the expression of IL-1β. KEYWORDS: ceftriaxone, cerebral ischemia, interleukin-1β, microglia, pretreatment
Introduction Stroke is the leading cause of disabilities worldwide and imposes a major impact on the economic burden of our society [1]. To fulfill the increasing needs for better control of stroke, it is crucial to develop novel strategies and get more understanding of the neuroprotective mechanism in the brain. In clinic, multiple pharmacological preconditioning methods have been used to reduce the brain damage of stroke [2]. However, the underlying mechanisms are still unknown. Recent experimental studies have shown that βlactam antibiotic pretreatment, such as ceftriaxone (CTX), is likely neuroprotective effect in vitro and in vivo. It was found that under oxygen–glucose-deprived condition, the number of cell death was decrease by application of CTX [3]. When delivered in animals, CTX Received 15 December 2012; revised 13 October 2013; accepted 13 October 2013 Correspondence: Zhang Hong, Department of Anesthesiology, Chinese PLA General Hospital, Beijing 100853, China. Tel: 010-66938152. E-mail: [email protected]
was neuroprotective in mouse models of amyotropic lateral sclerosis and spinal muscular atrophy [4, 5]. These findings have proved that CTX may be a potential neuroprotectant. But the mechanism of CTX preconditioning remains unclear. Acute ischemic stroke involves a complex array of processes, the combined action of which determines the outcome of ischemic event. Among these processes, growing findings implicate a crucial role for inflammation, which occurs in the necrotic brain tissue, following the activation of astrocytes and microglia cells as well as infiltration of blood immune cells [6–8]. The overactivated astrocytes and microglia cells release plenty of inflammatory cytokines such as interleukin-1β (IL1β), tumor necrosis factor-α (TNF-α) and macrophage inflammatory protein-1, etc. [9–11]. Furthermore, the inflammatory cytokines promote the activation of astrocytes and microglia cells in return [12], and then facilitate the ischemic injury. It is known that IL-1β is secreted immediately after the ischemia, directly induces apoptosis of neuronal cells and enhances the expression of other inflammatory cytokines and neural excitotoxicity [13–15]. Therefore, IL-1β becomes a marker for 657
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predicting the inflammatory level after stroke [16]. Moreover, the recent study has shown that neuroprotection by CTX pretreatment may be mediated via inhibition of the inflammatory response in the animal models of traumatic brain injury [17]. We hypothesized that CTX pretreatment can exert neuroprotective effects in ischemic stroke by partially reducing the overactivation of microglia cells and inhibiting the expression of IL-1β. In present study, we tested this hypothesis in the middle cerebral artery occlusion (MCAO) model in rats.
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Materials and methods Animal care and experimental protocol The experimental protocol used in this study was approved by the Ethics Committee for Animal Experimentation of the Fourth Military Medical University and was conducted according to the Guidelines for Animal Experimentation of the Fourth Military Medical University (Xi’an, China). Male Sprague Dawley rats, weighing 230–250 g, were provided by the Experimental Animal Center of the Fourth Military Medical University. They were housed with 12-h light/dark cycle, with food and water ad libitum, temperature of 22 ± 2◦ C for at least one week before they received CTX pretreatment or surgery. To determine the CTX pretreatment effect, 72 rats were randomly assigned to the following experimental groups: Sham group (n = 12), control group (n = 30) and CTX pretreatment group (n = 30). There is no difference in weight between the above three groups. Rats in CTX pretreatment group underwent injection with CTX (200 mg/kg dissolved in 0.5 ml PBS, i.p.) (Shanghai Roche Pharmaceutical Ltd., China) once a day, for 5 consecutive days before the MCAO surgery [18]. Rats in Sham group were given injection of PBS (0.5 ml, i.p.) once a day, for 5 consecutive days and then received identical surgery without MCAO. The rats in control group were given injection of PBS (0.5 ml, i.p.) once a day, for 5 consecutive days before they received the MCAO surgery. The numbers of animals per group for various experiments are summarized in Table 1.
Transient middle cerebral artery occlusion with reperfusion We used the traditional transient MCAO model to induce focal cerebral ischemia [19, 20]. Under general anesthesia with intraperitoneal injection of pentobarbital 40 mg/kg, the right cervical carotid bifurcation was exposed through a midline neck incision. A 3–0 monofilament nylon suture (Ethicon, Inc., Osaka, Japan) was
gently inserted in the common carotid artery and advanced into the internal carotid artery up to middle cerebral artery. After 120 min, the filament was withdrawn from the internal carotid artery to allow reperfusion. Sham-operated rats underwent the same surgical procedures except that the filament was not inserted. Cerebral blood flow (CBF) was monitored during surgical intervention with a probe attached to the skull above the supply territory of the MCA (2 mm caudal to bregma and 4 mm lateral) by using Laser Doppler flowmetry (PeriFlux 5000, Perimed AB, Sweden). The monitoring time points were as followed: pre-MCAO, 1 min after occlusion, 120 min after occlusion and reperfusion. The cerebral blood flow reduced to less than 20% of the baseline after the filament inserted and rapidly restored to 70% of the preoperative during reperfusion. Animals were excluded if they did not meet the above requirement. The temperature of the rats was maintained at 37–38◦ C with a heating pad.
Neurological deficit scores In order to observe the change of neurological function, the animals were assessed by an investigator who was unaware to those experimental groups 24 h after reperfusion. The neurological deficit scores (NDS) were first reported by Garcia [21]. There are: (1) spontaneous activity, (2) symmetry in the movement of four limbs, (3) forepaw outstretching, (4) climbing, (5) body proprioception, and (6) response to vibrissae touch. The final scores given to each rat at the completion of the evaluation are the summation of all six individual test score. The higher the scores, the better the neurobehavioral function.
Infarct volumes measurement We measured infarct volumes 24 h after reperfusion using 2,3,5-triphenyltetrazolium chloride (TTC; Sigma–Aldrich, USA) staining. The rats were sacrificed using an overdose of pentobarbital by intraperitoneal injection (60 mg/kg), and brains were rapidly removed, cooled in iced saline for 10 min. Each brain was traced into 2-mm slices with the aid of brain matrix. All six sections were immersed in 2% TTC at 37◦ C for 10 min. Afterward, an image analysis system (Adobe Photoshop CS5) was used to measure the infarct volumes. To compensate for the effect of brain edema, corrected infarct volumes were calculated as previously described using the formula for the corrected infarct area. The area of infarction in one section was calculated by subtracting the area of the nonlesioned ipsilateral hemisphere from the total area of the contralateral hemisphere. The total volumes of infarction were calculated by integration of International Journal of Neuroscience
Neuroprotective effect of CTX Table 1.
Numbers of animals per group for different experiments.
Protocol
Experiment
TTC staininga and neurological deficit scores Activation of astrocytes, microglia Immunofluorescence staining and IL-1β Activation of microglia, astrocytes Immunofluorescence staining and IL-1β Expression of and IL-1β Western blot and ELISA
Effect of CTX retreatment
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Time point
Number
24 h
Sham (n = 2), MCAO and CTX (n = 6)
3h
Sham (n = 2), MCAO and CTX (n = 3)
24 h
Sham (n = 2), MCAO and CTX (n = 3)
3 h, 24 h, 24 h Sham (n = 2), MCAO and CTX (n = 6) Sham (n = 2), MCAO and CTX (n = 6) Sham (n = 2), MCAO and CTX (n = 6)
TTC staining: 2,3,5-triphenyltetrazolium chloride staining.
the lesion areas from all six sections. Infarct volume was expressed as percentages of total hemispheric volume.
Double immunofluorescence for astrocytes and IL-1β, microglia cells and IL-1β At 3 and 24 h after reperfusion, rats were anesthetized using an overdose of pentobarbital (60 mg/kg, i.p.) and transcardially perfused with 4% paraformaldehyde. At 3 h after paraformaldehyde perfusion, the brains were transferred to 30% sucrose in PB solution at 4◦ C until it sank. To ensure that homologous areas of injury were sampled between different groups, parallel sets of sections from –3.0 to –5.0 mm from bregma (covering the infarct area) were used. All 10-μm thick coronal tissue sections were incubated for 16 h at 4◦ C with the following primary antibodies: mouse anti-GFAP antibody (1:500 dilution, abcam, ab4648, UK) to label activated astrocytes; mouse anti-CD11b antibody (OX42) (1:400 dilution, abcam, ab78457, UK) to label activated microglia cells; and rabbit anti-IL1 beta antibody (1:200 dilution, abcam, ab9787, UK) to label IL-1β. After washed three times by PBS, sections were incubated for 2 h at room temperature in a solution containing an appropriate mixture of the corresponding secondary antibodies: goat anti-rabbit IgG FITC (1:200 dilution, cwbio, cw0114, China) and donkey anti-mouse IgG FITC (1:200 dilution, cwbio, cw0224, China). Six slices were selected in each group, and then counted the positive numbers of glial cells in two different fields of each slice. The sections were mounted on gelatin-coated slides. The images were captured by Olympus BX-60 fluorescence microscope (Olympus Corporation, Tokyo, Japan).
Western blot analysis of IL-1β After 3 and 24 h of reperfusion, rats were anesthetized by pentobarbital (60 mg/kg, i.p.) and the ischemic penumbras were microdissected according to the protocols in rodent models of unilateral proximal MCAO [22]. The C
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penumbra cortex was divided and homogenized into icecold lysis buffer for 20 min and then was centrifuged at 12 000 rpm at 4◦ C for 10 min. After separation, the electrophoretic blotting procedures were as previously described for 2 h. PVDF membrane was blocked for 1 h at room temperature in Tris-buffered saline (TBS) containing 0.1% Tween-20 and 5% dry milk, then incubated with the primary rabbit anti-IL-1β antibody (1:200 dilution, abcam, ab9787, UK) at 4◦ C overnight. After washed three times by TBST, the membranes were incubated with secondary horseradish peroxidaseconjugated goat anti-rabbit secondary antibody (1:5000 dilution, Pierce Biotechnology Inc., France) for 1 h at room temperature. The intensity of the band was visualized with the NIH image program (NIH Image Version 1.61, Bethesda, MD) and each sample calculated three times.
ELISA analysis of IL-1β At 24 h after reperfusion, rats were anesthetized by overdose pentobarbital as previously described. To determine whether there were differences between penumbra and the infarct core area, we rapidly removed brains and separated the ischemic penumbra cortex and the infarct core area of striatum. We used double antibody sandwich ABC-ELISA method by rat IL-1β kit (Westang biological science and technology company, Shanghai, China). Added 100 μl sample for 120 min at 37◦ C, then washed the plate five times. First antibody working solution (100 μl) was added and reacted for about 1 h at 37◦ C. Washed the plate and added HRP antibody solution 100 μl. Repeated the washing steps and added 100 μl substrate solution for 15 min at 37◦ C in the dark. We used enzyme-labeled instrument (Thermo, DENLEY DRAGON Wellscan MK 3) to measure the absorbance values at 450 nm.
Statistical analysis SPSS 13.0 for Windows (SPSS Inc., Chicago, IL) was used to conduct statistical analyses. All the data,
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except neurological deficit scores and the quantity of glial cells, were presented as mean ± SD and analyzed by Single-factor analysis of variance (ANOVA). To compare differences between groups, Tukey’s post hoc tests were used. The neurological deficit scores and the quantity of glial cells were expressed as median and tested by the Kruskal–Wallis test, if there were differences between the two groups, Mann–Whitney U-test with Bonferroni was used to correct the data. p Value of less than 0.05 was considered as statistically significant.
Results Figure 2. Exclusion of rats during the MCAO surgery. There
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Cerebral blood flow During the MCAO surgery, we monitored cerebral blood flow among three groups at four time points. After reperfusion, there was no difference between control and CTX pretreatment groups (p = 0.908) (Figure 1). Animals were excluded if they did not meet the reperfusion requirement (Figure 2). However, we used additional rats with similar weight to supplement the animal number to meet the scheduled design.
were five rats died of intracranial hemorrhage and three rats were eliminated due to bad reperfusion and one rat died of carotid artery rupture and bleeding.
in control and CTX pretreatment groups (Figure 4A). Compared with the control group, the percentage of infarct volumes in CTX pretreatment group was significantly smaller (p = 0.012). Moreover, the difference of infarct volume was most in the cortex, not in the striatum (Figure 4B).
Neurological deficit scores At 24 h after reperfusion, we observed the neurological behavior changes among three groups. After the MCAO surgery, the NDS decreased both in control and CTX groups. However, compared with the control group, the NDS of CTX pretreatment group were much higher (p = 0.001) (Figure 3).
Double immunofluorescence of glia cells and IL-1β We observed the activation of astrocytes and microglia cells at 3 h after reperfusion. It showed that in the early stage of acute ischemia, astrocytes were rapidly activated by the ischemic insult both in the control and CTX pretreatment groups. The number of astrocytes-positive
Infarct volumes At 24 h after reperfusion, the TTC staining results showed that the brain tissues were seriously injured
Figure 1. Cerebral blood flow during the MCAO surgery. Cere-
Figure 3. Neurological deficit scores at 24 h after ischemic-
bral blood flow (n = 12 in Sham group, n = 30 in MCAO and CTX group). No difference between MCAO and CTX group at 1 min (33.4 ± 11.5 vs. 36.5 ± 12.3), 120 min (35.2 ± 13.5 vs. 38.9 ± 10.1) and reperfusion (136.0 ± 18.4 vs. 146.4 ± 17.2).
reperfusion. Neurological deficit scores (n = 8 in Sham group, n = 21 in MCAO and CTX group). CTX pretreatment group (12.0 ± 0.4) was higher than control group (8.4 ± 0.3). ∗ p < 0.01 vs. MCAO group.
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Figure 4. Neuroprotective effect induced by CTX pretreatment at 24 h after ischemic-reperfusion. (A) TTC stain-
ing. (B) Percentage of infarct volumes. (n = 2 in Sham group, n = 6 in MCAO and CTX group). The percentage of infarct volumes in CTX pretreatment group (18.92 ± 1.977)% was smaller than control group (32.748 ± 1.802)%. ∗ p < 0.01 vs. MCAO group.
cells was less in CTX pretreatment group than that in control group (p = 0.028) (Figure 5B). In addition, IL1β and astrocytes showed little colocalization, as shown in Figure 4A. The quantity of microglia cells in the control group was not different from the CTX group (p = 0.762) (Figure 6B). At 24 h after reperfusion, the quantity of astrocytes showed no difference between control and CTX groups (p = 0.825) (Figure 7B). However, microglia cells were largely activated, as shown in Figure 8. The number of microglia-positive cells was significantly decreased in the CTX pretreatment group than that in the control group (p = 0.034) (Figure 8B). IL-1β positive cells were colocalized with microglia cells, as shown in double immunofluorescence images (Figure 8A).
The changes in expression of IL-1β We separately tested the changes in the expression of IL-1β at 3 and 24 h after reperfusion by western blot analysis. As shown in Figure 9, the expression of IL-1β was sharply increased both in control and CTX pretreatment groups at 3 h after reperfusion, but there was no difference between the two groups (p = 0.820). However, in the late period after ischemia, the expression of IL-1β in the CTX pretreatment group was lower than that in the control group at 24 h after reperfusion (p = 0.036). C
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In order to determine the difference of IL-1β in the cortex and striatum, we separately tested the expression of IL-1β in penumbra cortex and striatum by ELISA. The result (Figure 10) showed that the expression level of IL-1β in cortex in the CTX pretreatment group was less than the MCAO group but the difference was not statistically significant (p = 0.183). There was no difference between the two groups in the striatum either (p = 0.910).
Discussion Focal cerebral ischemia evokes a robust inflammatory response that begins within a few minutes after ischemia and can last couple of hours or even few days [23–26]. The inflammatory response is a composite process that involves various kinds of cell types, inflammatory mediators, and extracellular receptors. Among them, glia cells play important role in the process of inflammation. The activated astrocytes and microglia cells express various kinds of interleukins, such as IL-1β, TNF-α, to mediate further damage [27]. Innate immune response is a consequence of stroke but contributes to ischemic brain injury. Among all inflammatory signals, glia-interleukins pathway is one of the crucial targets to suppress the innate inflammation responses [28]. However, the role of inflammation in neuroprotection induced by ceftriaxone pretreatment is still unclear.
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Figure 5. Immunofluorescent double-labeling of astrocytes and IL-1β at 3 h after reperfusion. The arrowhead indicates doublelabeled cell. (A) Red color represents astrocytes and green color represents IL-1β in the penumbra at 3 h after reperfusion. (B) Quantitative analysis of the number of astrocytes-positive cells in the penumbra. (n = 2 in Sham group, n = 3 in MCAO and CTX group) ∗ p < 0.05 vs. MCAO group. Scale bars = 50 μm.
In the present study, we found that with CTX pretreatment for 5 consecutive days before the MCAO surgery, the brain damage of rats could be partially reversed. At 24 h after reperfusion, the neurological deficit scores shown that the CTX pretreatment group had less neurological deficit compared with the control group, which demonstrates the interventions by CTX could attenuate the damage of behavior after the MCAO surgery. The TTC-staining results demonstrated that the infarct volumes of CTX pretreatment group were smaller than control group and the infarct area was mainly in the striatum. This result indicates that CTX pretreatment could partially reverse the damage in the penumbra cortex. In the early period of ischemia, astrocytes can be immediately activated to mediate neurotoxicity and release large amount of inflammatory factors [29, 30]. Microglia cells are activated subsequently with the increasing expression of different kinds of interleukins, such as
Figure 6. Immunofluorescent double-labeling of microglia cells and IL-1β at 3 h after reperfusion. The arrowhead indicates double-labeled cell. (A) Red color represents microglia cells while green color represents IL-1β in the penumbra at 3 h after reperfusion. (B) Quantitative analysis of the number of microglia-positive cells in the penumbra. (n = 2 in Sham group, n = 3 in MCAO and CTX group). No difference between MCAO and CTX group. Scale bars = 50 μm.
IL-1β. Our immunofluorescent staining shown that astrocytes and microglia cells were involved in the expression of IL-1β and the activation of glia cells could be partially inhibited by CTX pretreatment. In addition, the expression level of IL-1β was significantly decreased after 24 h of reperfusion according to the western blot. These results are consistent with the possibility that the activation of glia cells and interleukins both play important role in inflammation response and the CTX pretreatment may attenuate the damage by preventing this inflammatory process. IL-1β is known elevated by ischemia and contributes to the apoptosis after stroke [31]. As a neurotoxic mediator, IL-1β is reported one of the effective targets to reduce the infarct size of brain [32]. Some studies have reported that IL-1β is produced from activation-microglia cells and astrocytes [33], whereas another data show that IL-1β is mostly produced from monocytes, which are International Journal of Neuroscience
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Figure 7. Immunofluorescent double-labeling of astrocytes and
Figure 8. Immunofluorescent double-labeling of microglia cells
IL-1β at 24 h after reperfusion. The arrowhead indicates doublelabeled cell. (A) Red color represents astrocytes and green color represents IL-1β in the penumbra at 24 h after reperfusion. (B) Quantitative analysis of the number of astrocytes-positive cells in the penumbra. (n = 2 in Sham group, n = 3 in MCAO and CTX group). No difference was observed in MCAO and CTX group. Scale bars = 50 μm.
and IL-1β at 24 h after reperfusion. The arrowhead indicates double-labeled cell. (A) Red color represents microglia cells while green color represents IL-1β in the penumbra at 24 h after reperfusion. (B) Quantitative analysis of the number of microgliapositive cells in the penumbra. (n = 2 in Sham group, n = 3 in MCAO and CTX group). ∗ p < 0.05 vs. MCAO group. Scale bars = 50 μm.
activated by endogenous Toll-like receptor (TLR) ligands [34]. Our findings indicated that both microglia cells and astrocytes could express IL-1β. Moreover, the expression of IL-1β could be suppressed by CTX. CTX pretreatment could partially attenuate the activation of microglia cells and downregulate the expression level of IL-1β at 24 h after the MCAO surgery. These results indicate that at the early phase of reperfusion, there may be other different mechanisms contribute to the expression of IL-1β rather than astrocytes-IL-1β pathway. But in the late phase of reperfusion, the activation of microglia may be the main source of the IL-1β. Thus, these findings confirm the role of microglia cells in IL-1β releasing, and suggest that controlling the expression of IL1β is one of the effective ways to decrease inflammatory factors in late phase after brain ischemia-reperfusion injury.
There is compelling evidence that CTX could provide a neuroprotective effect against central nervous system diseases, such as stroke, epilepsy and amyotrophic lateral sclerosis [35]. Some studies show that ceftriaxone could increase the expression of glutamate transporter (GLT-1), reduce the glutamate excitotoxicity in the ischemic area and inhibit glial glutamate transporters on astrocytes [18]. But the relation of glutamate transporter and inflammation is not fully understood. Activation astrocytes can express inflammatory factors and secrete proapoptotic mediators and downregulate GLT-1 [36]. In addition, it has been reported that microglia cells also contribute to the secretion of glutamate [37]. Therefore, we assume that the inhibition of astrocytes and microglia cells activation might result in a decrease in the glutamate release and the excitotoxicity damage, as well as downregulating the preapoptotic mediators.
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However, the mechanism of the microglia cells responding to CTX still need to be further explored. Clinically, CTX is used against bacterial infections, and administered in the perioperative period. Our findings indicate that CTX also play a neuroprotective role in the central nervous system. In the future, those with high risk of ischemic stroke situations, like cardiopulmonary bypass surgery, may choose CTX as rational antibiotic, which may induce ischemic tolerance in the perioperative period.
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Conclusion In summary, our results suggest that CTX pretreatment may provide a neuroprotective effect against cerebral ischemia injury and reduce the expression of IL-1β after the MCAO surgery. Our results also demonstrate that CTX pretreatment can partially inhibit the activation of astrocytes and microglia cells. Although further research is needed to elucidate how CTX affects the glia cells, our finding may represent a novel mechanism of CTX pretreatment-induced ischemia tolerance in rats.
Declaration of Interest
Figure 9. The expression changes of IL-1β at 3 and 24 h after
reperfusion. Western blot showing representative IL-1β protein expression in the penumbra cortex at 3 and 24 h after reperfusion. The density represents IL-1β expression relative to β-actin protein expression. (n = 2 in Sham group, n = 6 in MCAO and CTX group) ∗ p < 0.05 vs. MCAO group.
Figure 10. The expression level of IL-1β in different parts of the brain at 24 h after reperfusion. The result of ELISA represents the expression levels of IL-1β in the brain. But the difference was not significant in statistics. (n = 2 in Sham group, n = 6 in MCAO and CTX group).
The authors declare that they have no competing interests. The authors alone are responsible for the content and writing of this paper.
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RESEARCH ARTICLE
Ceftriaxone pretreatment protects rats against cerebral ischemic injury by attenuating microglial activation-induced IL-1β expression Yang Lujia,1 Li Xin,2 Wang Shiquan,2 Chen Yu,2 Zhang Shuzhuo,3 and Zhang Hong1 Int J Neurosci Downloaded from informahealthcare.com by Kainan University on 04/16/15 For personal use only.
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Department of Anesthesiology, Chinese PLA General Hospital, Beijing, China; 2 Department of Anesthesiology, Xijing Hospital, Fourth Military Medical University, Xi’an, China, and 3 Academy of Military Medical Sciences, Beijing, China; Background: Although the neuroprotective effect of ceftriaxone (CTX) has been reported, the underlying mechanisms are still uncertain. In this study, we investigated if rats recover better from CTX pretreatment against cerebral ischemia by inhibiting the inflammatory response. Methods: Rats were pretreated with CTX (200 mg/kg, 1/day, i.p.) for 5 d. At 24 h after the end of the last CTX pretreatment, focal cerebral ischemia was induced by middle cerebral artery occlusion (MCAO) for 120 min in male Sprague Dawley rats. The neurological deficit scores (NDS) and infarct volumes were evaluated. Microglia cells were observed by immunofluorescence staining and IL-1β was assayed by ELISA and Western Blot. Results: The results showed that CTX pretreatment improved the neurological deficit scores and decreased the infarct volumes 24 h after reperfusion. The activation of microglia cells was reduced and the expression of IL-1β was partially inhibited 24 h after reperfusion. Conclusion: These findings demonstrate that CTX pretreatment may provide a neuroprotective effect against transient cerebral ischemic injury, partially inhibit in microglial activation and reduce the expression of IL-1β. KEYWORDS: ceftriaxone, cerebral ischemia, interleukin-1β, microglia, pretreatment
Introduction Stroke is the leading cause of disabilities worldwide and imposes a major impact on the economic burden of our society [1]. To fulfill the increasing needs for better control of stroke, it is crucial to develop novel strategies and get more understanding of the neuroprotective mechanism in the brain. In clinic, multiple pharmacological preconditioning methods have been used to reduce the brain damage of stroke [2]. However, the underlying mechanisms are still unknown. Recent experimental studies have shown that βlactam antibiotic pretreatment, such as ceftriaxone (CTX), is likely neuroprotective effect in vitro and in vivo. It was found that under oxygen–glucose-deprived condition, the number of cell death was decrease by application of CTX [3]. When delivered in animals, CTX Received 15 December 2012; revised 13 October 2013; accepted 13 October 2013 Correspondence: Zhang Hong, Department of Anesthesiology, Chinese PLA General Hospital, Beijing 100853, China. Tel: 010-66938152. E-mail: [email protected]
was neuroprotective in mouse models of amyotropic lateral sclerosis and spinal muscular atrophy [4, 5]. These findings have proved that CTX may be a potential neuroprotectant. But the mechanism of CTX preconditioning remains unclear. Acute ischemic stroke involves a complex array of processes, the combined action of which determines the outcome of ischemic event. Among these processes, growing findings implicate a crucial role for inflammation, which occurs in the necrotic brain tissue, following the activation of astrocytes and microglia cells as well as infiltration of blood immune cells [6–8]. The overactivated astrocytes and microglia cells release plenty of inflammatory cytokines such as interleukin-1β (IL1β), tumor necrosis factor-α (TNF-α) and macrophage inflammatory protein-1, etc. [9–11]. Furthermore, the inflammatory cytokines promote the activation of astrocytes and microglia cells in return [12], and then facilitate the ischemic injury. It is known that IL-1β is secreted immediately after the ischemia, directly induces apoptosis of neuronal cells and enhances the expression of other inflammatory cytokines and neural excitotoxicity [13–15]. Therefore, IL-1β becomes a marker for 657
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predicting the inflammatory level after stroke [16]. Moreover, the recent study has shown that neuroprotection by CTX pretreatment may be mediated via inhibition of the inflammatory response in the animal models of traumatic brain injury [17]. We hypothesized that CTX pretreatment can exert neuroprotective effects in ischemic stroke by partially reducing the overactivation of microglia cells and inhibiting the expression of IL-1β. In present study, we tested this hypothesis in the middle cerebral artery occlusion (MCAO) model in rats.
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Materials and methods Animal care and experimental protocol The experimental protocol used in this study was approved by the Ethics Committee for Animal Experimentation of the Fourth Military Medical University and was conducted according to the Guidelines for Animal Experimentation of the Fourth Military Medical University (Xi’an, China). Male Sprague Dawley rats, weighing 230–250 g, were provided by the Experimental Animal Center of the Fourth Military Medical University. They were housed with 12-h light/dark cycle, with food and water ad libitum, temperature of 22 ± 2◦ C for at least one week before they received CTX pretreatment or surgery. To determine the CTX pretreatment effect, 72 rats were randomly assigned to the following experimental groups: Sham group (n = 12), control group (n = 30) and CTX pretreatment group (n = 30). There is no difference in weight between the above three groups. Rats in CTX pretreatment group underwent injection with CTX (200 mg/kg dissolved in 0.5 ml PBS, i.p.) (Shanghai Roche Pharmaceutical Ltd., China) once a day, for 5 consecutive days before the MCAO surgery [18]. Rats in Sham group were given injection of PBS (0.5 ml, i.p.) once a day, for 5 consecutive days and then received identical surgery without MCAO. The rats in control group were given injection of PBS (0.5 ml, i.p.) once a day, for 5 consecutive days before they received the MCAO surgery. The numbers of animals per group for various experiments are summarized in Table 1.
Transient middle cerebral artery occlusion with reperfusion We used the traditional transient MCAO model to induce focal cerebral ischemia [19, 20]. Under general anesthesia with intraperitoneal injection of pentobarbital 40 mg/kg, the right cervical carotid bifurcation was exposed through a midline neck incision. A 3–0 monofilament nylon suture (Ethicon, Inc., Osaka, Japan) was
gently inserted in the common carotid artery and advanced into the internal carotid artery up to middle cerebral artery. After 120 min, the filament was withdrawn from the internal carotid artery to allow reperfusion. Sham-operated rats underwent the same surgical procedures except that the filament was not inserted. Cerebral blood flow (CBF) was monitored during surgical intervention with a probe attached to the skull above the supply territory of the MCA (2 mm caudal to bregma and 4 mm lateral) by using Laser Doppler flowmetry (PeriFlux 5000, Perimed AB, Sweden). The monitoring time points were as followed: pre-MCAO, 1 min after occlusion, 120 min after occlusion and reperfusion. The cerebral blood flow reduced to less than 20% of the baseline after the filament inserted and rapidly restored to 70% of the preoperative during reperfusion. Animals were excluded if they did not meet the above requirement. The temperature of the rats was maintained at 37–38◦ C with a heating pad.
Neurological deficit scores In order to observe the change of neurological function, the animals were assessed by an investigator who was unaware to those experimental groups 24 h after reperfusion. The neurological deficit scores (NDS) were first reported by Garcia [21]. There are: (1) spontaneous activity, (2) symmetry in the movement of four limbs, (3) forepaw outstretching, (4) climbing, (5) body proprioception, and (6) response to vibrissae touch. The final scores given to each rat at the completion of the evaluation are the summation of all six individual test score. The higher the scores, the better the neurobehavioral function.
Infarct volumes measurement We measured infarct volumes 24 h after reperfusion using 2,3,5-triphenyltetrazolium chloride (TTC; Sigma–Aldrich, USA) staining. The rats were sacrificed using an overdose of pentobarbital by intraperitoneal injection (60 mg/kg), and brains were rapidly removed, cooled in iced saline for 10 min. Each brain was traced into 2-mm slices with the aid of brain matrix. All six sections were immersed in 2% TTC at 37◦ C for 10 min. Afterward, an image analysis system (Adobe Photoshop CS5) was used to measure the infarct volumes. To compensate for the effect of brain edema, corrected infarct volumes were calculated as previously described using the formula for the corrected infarct area. The area of infarction in one section was calculated by subtracting the area of the nonlesioned ipsilateral hemisphere from the total area of the contralateral hemisphere. The total volumes of infarction were calculated by integration of International Journal of Neuroscience
Neuroprotective effect of CTX Table 1.
Numbers of animals per group for different experiments.
Protocol
Experiment
TTC staininga and neurological deficit scores Activation of astrocytes, microglia Immunofluorescence staining and IL-1β Activation of microglia, astrocytes Immunofluorescence staining and IL-1β Expression of and IL-1β Western blot and ELISA
Effect of CTX retreatment
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Time point
Number
24 h
Sham (n = 2), MCAO and CTX (n = 6)
3h
Sham (n = 2), MCAO and CTX (n = 3)
24 h
Sham (n = 2), MCAO and CTX (n = 3)
3 h, 24 h, 24 h Sham (n = 2), MCAO and CTX (n = 6) Sham (n = 2), MCAO and CTX (n = 6) Sham (n = 2), MCAO and CTX (n = 6)
TTC staining: 2,3,5-triphenyltetrazolium chloride staining.
the lesion areas from all six sections. Infarct volume was expressed as percentages of total hemispheric volume.
Double immunofluorescence for astrocytes and IL-1β, microglia cells and IL-1β At 3 and 24 h after reperfusion, rats were anesthetized using an overdose of pentobarbital (60 mg/kg, i.p.) and transcardially perfused with 4% paraformaldehyde. At 3 h after paraformaldehyde perfusion, the brains were transferred to 30% sucrose in PB solution at 4◦ C until it sank. To ensure that homologous areas of injury were sampled between different groups, parallel sets of sections from –3.0 to –5.0 mm from bregma (covering the infarct area) were used. All 10-μm thick coronal tissue sections were incubated for 16 h at 4◦ C with the following primary antibodies: mouse anti-GFAP antibody (1:500 dilution, abcam, ab4648, UK) to label activated astrocytes; mouse anti-CD11b antibody (OX42) (1:400 dilution, abcam, ab78457, UK) to label activated microglia cells; and rabbit anti-IL1 beta antibody (1:200 dilution, abcam, ab9787, UK) to label IL-1β. After washed three times by PBS, sections were incubated for 2 h at room temperature in a solution containing an appropriate mixture of the corresponding secondary antibodies: goat anti-rabbit IgG FITC (1:200 dilution, cwbio, cw0114, China) and donkey anti-mouse IgG FITC (1:200 dilution, cwbio, cw0224, China). Six slices were selected in each group, and then counted the positive numbers of glial cells in two different fields of each slice. The sections were mounted on gelatin-coated slides. The images were captured by Olympus BX-60 fluorescence microscope (Olympus Corporation, Tokyo, Japan).
Western blot analysis of IL-1β After 3 and 24 h of reperfusion, rats were anesthetized by pentobarbital (60 mg/kg, i.p.) and the ischemic penumbras were microdissected according to the protocols in rodent models of unilateral proximal MCAO [22]. The C
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penumbra cortex was divided and homogenized into icecold lysis buffer for 20 min and then was centrifuged at 12 000 rpm at 4◦ C for 10 min. After separation, the electrophoretic blotting procedures were as previously described for 2 h. PVDF membrane was blocked for 1 h at room temperature in Tris-buffered saline (TBS) containing 0.1% Tween-20 and 5% dry milk, then incubated with the primary rabbit anti-IL-1β antibody (1:200 dilution, abcam, ab9787, UK) at 4◦ C overnight. After washed three times by TBST, the membranes were incubated with secondary horseradish peroxidaseconjugated goat anti-rabbit secondary antibody (1:5000 dilution, Pierce Biotechnology Inc., France) for 1 h at room temperature. The intensity of the band was visualized with the NIH image program (NIH Image Version 1.61, Bethesda, MD) and each sample calculated three times.
ELISA analysis of IL-1β At 24 h after reperfusion, rats were anesthetized by overdose pentobarbital as previously described. To determine whether there were differences between penumbra and the infarct core area, we rapidly removed brains and separated the ischemic penumbra cortex and the infarct core area of striatum. We used double antibody sandwich ABC-ELISA method by rat IL-1β kit (Westang biological science and technology company, Shanghai, China). Added 100 μl sample for 120 min at 37◦ C, then washed the plate five times. First antibody working solution (100 μl) was added and reacted for about 1 h at 37◦ C. Washed the plate and added HRP antibody solution 100 μl. Repeated the washing steps and added 100 μl substrate solution for 15 min at 37◦ C in the dark. We used enzyme-labeled instrument (Thermo, DENLEY DRAGON Wellscan MK 3) to measure the absorbance values at 450 nm.
Statistical analysis SPSS 13.0 for Windows (SPSS Inc., Chicago, IL) was used to conduct statistical analyses. All the data,
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except neurological deficit scores and the quantity of glial cells, were presented as mean ± SD and analyzed by Single-factor analysis of variance (ANOVA). To compare differences between groups, Tukey’s post hoc tests were used. The neurological deficit scores and the quantity of glial cells were expressed as median and tested by the Kruskal–Wallis test, if there were differences between the two groups, Mann–Whitney U-test with Bonferroni was used to correct the data. p Value of less than 0.05 was considered as statistically significant.
Results Figure 2. Exclusion of rats during the MCAO surgery. There
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Cerebral blood flow During the MCAO surgery, we monitored cerebral blood flow among three groups at four time points. After reperfusion, there was no difference between control and CTX pretreatment groups (p = 0.908) (Figure 1). Animals were excluded if they did not meet the reperfusion requirement (Figure 2). However, we used additional rats with similar weight to supplement the animal number to meet the scheduled design.
were five rats died of intracranial hemorrhage and three rats were eliminated due to bad reperfusion and one rat died of carotid artery rupture and bleeding.
in control and CTX pretreatment groups (Figure 4A). Compared with the control group, the percentage of infarct volumes in CTX pretreatment group was significantly smaller (p = 0.012). Moreover, the difference of infarct volume was most in the cortex, not in the striatum (Figure 4B).
Neurological deficit scores At 24 h after reperfusion, we observed the neurological behavior changes among three groups. After the MCAO surgery, the NDS decreased both in control and CTX groups. However, compared with the control group, the NDS of CTX pretreatment group were much higher (p = 0.001) (Figure 3).
Double immunofluorescence of glia cells and IL-1β We observed the activation of astrocytes and microglia cells at 3 h after reperfusion. It showed that in the early stage of acute ischemia, astrocytes were rapidly activated by the ischemic insult both in the control and CTX pretreatment groups. The number of astrocytes-positive
Infarct volumes At 24 h after reperfusion, the TTC staining results showed that the brain tissues were seriously injured
Figure 1. Cerebral blood flow during the MCAO surgery. Cere-
Figure 3. Neurological deficit scores at 24 h after ischemic-
bral blood flow (n = 12 in Sham group, n = 30 in MCAO and CTX group). No difference between MCAO and CTX group at 1 min (33.4 ± 11.5 vs. 36.5 ± 12.3), 120 min (35.2 ± 13.5 vs. 38.9 ± 10.1) and reperfusion (136.0 ± 18.4 vs. 146.4 ± 17.2).
reperfusion. Neurological deficit scores (n = 8 in Sham group, n = 21 in MCAO and CTX group). CTX pretreatment group (12.0 ± 0.4) was higher than control group (8.4 ± 0.3). ∗ p < 0.01 vs. MCAO group.
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Figure 4. Neuroprotective effect induced by CTX pretreatment at 24 h after ischemic-reperfusion. (A) TTC stain-
ing. (B) Percentage of infarct volumes. (n = 2 in Sham group, n = 6 in MCAO and CTX group). The percentage of infarct volumes in CTX pretreatment group (18.92 ± 1.977)% was smaller than control group (32.748 ± 1.802)%. ∗ p < 0.01 vs. MCAO group.
cells was less in CTX pretreatment group than that in control group (p = 0.028) (Figure 5B). In addition, IL1β and astrocytes showed little colocalization, as shown in Figure 4A. The quantity of microglia cells in the control group was not different from the CTX group (p = 0.762) (Figure 6B). At 24 h after reperfusion, the quantity of astrocytes showed no difference between control and CTX groups (p = 0.825) (Figure 7B). However, microglia cells were largely activated, as shown in Figure 8. The number of microglia-positive cells was significantly decreased in the CTX pretreatment group than that in the control group (p = 0.034) (Figure 8B). IL-1β positive cells were colocalized with microglia cells, as shown in double immunofluorescence images (Figure 8A).
The changes in expression of IL-1β We separately tested the changes in the expression of IL-1β at 3 and 24 h after reperfusion by western blot analysis. As shown in Figure 9, the expression of IL-1β was sharply increased both in control and CTX pretreatment groups at 3 h after reperfusion, but there was no difference between the two groups (p = 0.820). However, in the late period after ischemia, the expression of IL-1β in the CTX pretreatment group was lower than that in the control group at 24 h after reperfusion (p = 0.036). C
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In order to determine the difference of IL-1β in the cortex and striatum, we separately tested the expression of IL-1β in penumbra cortex and striatum by ELISA. The result (Figure 10) showed that the expression level of IL-1β in cortex in the CTX pretreatment group was less than the MCAO group but the difference was not statistically significant (p = 0.183). There was no difference between the two groups in the striatum either (p = 0.910).
Discussion Focal cerebral ischemia evokes a robust inflammatory response that begins within a few minutes after ischemia and can last couple of hours or even few days [23–26]. The inflammatory response is a composite process that involves various kinds of cell types, inflammatory mediators, and extracellular receptors. Among them, glia cells play important role in the process of inflammation. The activated astrocytes and microglia cells express various kinds of interleukins, such as IL-1β, TNF-α, to mediate further damage [27]. Innate immune response is a consequence of stroke but contributes to ischemic brain injury. Among all inflammatory signals, glia-interleukins pathway is one of the crucial targets to suppress the innate inflammation responses [28]. However, the role of inflammation in neuroprotection induced by ceftriaxone pretreatment is still unclear.
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Figure 5. Immunofluorescent double-labeling of astrocytes and IL-1β at 3 h after reperfusion. The arrowhead indicates doublelabeled cell. (A) Red color represents astrocytes and green color represents IL-1β in the penumbra at 3 h after reperfusion. (B) Quantitative analysis of the number of astrocytes-positive cells in the penumbra. (n = 2 in Sham group, n = 3 in MCAO and CTX group) ∗ p < 0.05 vs. MCAO group. Scale bars = 50 μm.
In the present study, we found that with CTX pretreatment for 5 consecutive days before the MCAO surgery, the brain damage of rats could be partially reversed. At 24 h after reperfusion, the neurological deficit scores shown that the CTX pretreatment group had less neurological deficit compared with the control group, which demonstrates the interventions by CTX could attenuate the damage of behavior after the MCAO surgery. The TTC-staining results demonstrated that the infarct volumes of CTX pretreatment group were smaller than control group and the infarct area was mainly in the striatum. This result indicates that CTX pretreatment could partially reverse the damage in the penumbra cortex. In the early period of ischemia, astrocytes can be immediately activated to mediate neurotoxicity and release large amount of inflammatory factors [29, 30]. Microglia cells are activated subsequently with the increasing expression of different kinds of interleukins, such as
Figure 6. Immunofluorescent double-labeling of microglia cells and IL-1β at 3 h after reperfusion. The arrowhead indicates double-labeled cell. (A) Red color represents microglia cells while green color represents IL-1β in the penumbra at 3 h after reperfusion. (B) Quantitative analysis of the number of microglia-positive cells in the penumbra. (n = 2 in Sham group, n = 3 in MCAO and CTX group). No difference between MCAO and CTX group. Scale bars = 50 μm.
IL-1β. Our immunofluorescent staining shown that astrocytes and microglia cells were involved in the expression of IL-1β and the activation of glia cells could be partially inhibited by CTX pretreatment. In addition, the expression level of IL-1β was significantly decreased after 24 h of reperfusion according to the western blot. These results are consistent with the possibility that the activation of glia cells and interleukins both play important role in inflammation response and the CTX pretreatment may attenuate the damage by preventing this inflammatory process. IL-1β is known elevated by ischemia and contributes to the apoptosis after stroke [31]. As a neurotoxic mediator, IL-1β is reported one of the effective targets to reduce the infarct size of brain [32]. Some studies have reported that IL-1β is produced from activation-microglia cells and astrocytes [33], whereas another data show that IL-1β is mostly produced from monocytes, which are International Journal of Neuroscience
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Figure 7. Immunofluorescent double-labeling of astrocytes and
Figure 8. Immunofluorescent double-labeling of microglia cells
IL-1β at 24 h after reperfusion. The arrowhead indicates doublelabeled cell. (A) Red color represents astrocytes and green color represents IL-1β in the penumbra at 24 h after reperfusion. (B) Quantitative analysis of the number of astrocytes-positive cells in the penumbra. (n = 2 in Sham group, n = 3 in MCAO and CTX group). No difference was observed in MCAO and CTX group. Scale bars = 50 μm.
and IL-1β at 24 h after reperfusion. The arrowhead indicates double-labeled cell. (A) Red color represents microglia cells while green color represents IL-1β in the penumbra at 24 h after reperfusion. (B) Quantitative analysis of the number of microgliapositive cells in the penumbra. (n = 2 in Sham group, n = 3 in MCAO and CTX group). ∗ p < 0.05 vs. MCAO group. Scale bars = 50 μm.
activated by endogenous Toll-like receptor (TLR) ligands [34]. Our findings indicated that both microglia cells and astrocytes could express IL-1β. Moreover, the expression of IL-1β could be suppressed by CTX. CTX pretreatment could partially attenuate the activation of microglia cells and downregulate the expression level of IL-1β at 24 h after the MCAO surgery. These results indicate that at the early phase of reperfusion, there may be other different mechanisms contribute to the expression of IL-1β rather than astrocytes-IL-1β pathway. But in the late phase of reperfusion, the activation of microglia may be the main source of the IL-1β. Thus, these findings confirm the role of microglia cells in IL-1β releasing, and suggest that controlling the expression of IL1β is one of the effective ways to decrease inflammatory factors in late phase after brain ischemia-reperfusion injury.
There is compelling evidence that CTX could provide a neuroprotective effect against central nervous system diseases, such as stroke, epilepsy and amyotrophic lateral sclerosis [35]. Some studies show that ceftriaxone could increase the expression of glutamate transporter (GLT-1), reduce the glutamate excitotoxicity in the ischemic area and inhibit glial glutamate transporters on astrocytes [18]. But the relation of glutamate transporter and inflammation is not fully understood. Activation astrocytes can express inflammatory factors and secrete proapoptotic mediators and downregulate GLT-1 [36]. In addition, it has been reported that microglia cells also contribute to the secretion of glutamate [37]. Therefore, we assume that the inhibition of astrocytes and microglia cells activation might result in a decrease in the glutamate release and the excitotoxicity damage, as well as downregulating the preapoptotic mediators.
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However, the mechanism of the microglia cells responding to CTX still need to be further explored. Clinically, CTX is used against bacterial infections, and administered in the perioperative period. Our findings indicate that CTX also play a neuroprotective role in the central nervous system. In the future, those with high risk of ischemic stroke situations, like cardiopulmonary bypass surgery, may choose CTX as rational antibiotic, which may induce ischemic tolerance in the perioperative period.
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Conclusion In summary, our results suggest that CTX pretreatment may provide a neuroprotective effect against cerebral ischemia injury and reduce the expression of IL-1β after the MCAO surgery. Our results also demonstrate that CTX pretreatment can partially inhibit the activation of astrocytes and microglia cells. Although further research is needed to elucidate how CTX affects the glia cells, our finding may represent a novel mechanism of CTX pretreatment-induced ischemia tolerance in rats.
Declaration of Interest
Figure 9. The expression changes of IL-1β at 3 and 24 h after
reperfusion. Western blot showing representative IL-1β protein expression in the penumbra cortex at 3 and 24 h after reperfusion. The density represents IL-1β expression relative to β-actin protein expression. (n = 2 in Sham group, n = 6 in MCAO and CTX group) ∗ p < 0.05 vs. MCAO group.
Figure 10. The expression level of IL-1β in different parts of the brain at 24 h after reperfusion. The result of ELISA represents the expression levels of IL-1β in the brain. But the difference was not significant in statistics. (n = 2 in Sham group, n = 6 in MCAO and CTX group).
The authors declare that they have no competing interests. The authors alone are responsible for the content and writing of this paper.
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