International Journal of Neuroscience, 2014; 124(8): 585–592 Copyright © 2014 Informa Healthcare USA, Inc. ISSN: 0020-7454 print / 1543-5245 online DOI: 10.3109/00207454.2013.864289

RESEARCH ARTICLE

Lipopolysaccharide preconditioning attenuates apoptotic processes and improves neuropathologic changes after spinal cord injury in rats Wei-Chao Li,1 Rong Jiang,2 Dian-Ming Jiang,1 Feng-Chen Zhu,3 Bao Su,1 Bo Qiao,1 and Xiao-Tong Qi1 1

Department of Orthopedic Surgery, The First Affiliated Hospital of Chongqing Medical University, Chongqing, China; Laboratory of Stem Cell and Tissue Engineering, Chongqing Medical University, Chongqing, China; 3 Department of Orthopedic Surgery, Yongchuan Hospital of Chongqing Medical University, Chongqing, China

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We have shown earlier that administration of low-dose lipopolysaccharide (LPS) significantly contributed to recovery of motor function after traumatic spinal cord injury in the adult female rat. Using the same standardized animal model, we have now designed a set of experiments to test the hypothesis that LPS preconditioning attenuates stress-related apoptotic processes early after spinal cord trauma. The lower thoracic spinal cord injury in adult female Sprague-Dawley rats was caused by a 10 g weight rod drop from 25 mm on the dural surface of the exposed spinal cord at T10. The rats were randomly assigned to three groups: Sham injury, control (received normal saline alone), and LPS preconditioning (0.2 mg/kg, ip; 72 h prior to the injury). The animals were euthanized at 72 h postinjury. Neuropathologic changes were assessed using hematoxylin and eosin staining. SCI-induced apoptosis were observed by transmission electron microscopy. Caspase-3, cleaved caspase-3, Bax, and Bcl-2 were examined with immunohistochemistry or Western blotting. Compared with the control group, LPS preconditioning group showed significant improvement in the SCI-induced morphology changes. Furthermore, LPS preconditioning reduced the expressions of apoptotic markers caspase-3, cleaved caspase-3, and Bax, upregulated the expression of antiapoptotic marker Bcl-2 in the samples of spinal cord. Low-dose LPS attenuated the recruitment of inflammatory cells and the proliferation of glial cells in the site of injury. LPS preconditioning has neuroprotective effects against TSCI in rats due to its antiapoptosis properties as shown by the inhibition of caspase pathway and the upregulation of antiapoptotic protein. KEYWORDS: lipopolysaccharide, preconditioning, neurotrauma, apoptosis, rat

Introduction Spinal cord injury (SCI) is an often fatal injury in the central nervous system (CNS) that leads to loss of sensory and motor functions below the level of the lesion. In spite of many promising experimental studies, so far there is no effective treatment dramatically eliminating paraplegia from SCI in the clinic. Trauma to the spinal cord initiates primary mechanical damage and a cascade of secondary damage [1]. The primary mechanical injury to the cord cannot be reversed. Secondary Received 15 July 2013; revised 6 November 2013; accepted 6 November 2013 Correspondence: Dr Dian-Ming Jiang, Department of Orthopedic Surgery, The First Affiliated Hospital of Chongqing Medical University, 1 Youyi Road, Yuzhong District, Chongqing 400016, China. Tel. +86 02389011012, Fax: +86 02389011217. E-mail: [email protected]

damage is caused by a complex of pathological processes, which include a massive inflammatory response, ischemia, apoptosis, oxidative stress, and tissue necrosis [2]. Studies have demonstrated that proinflammatory cytokines produced by inflammatory cells are gathered widely at the site of the lesion [1,3]. Previous studies have shown that apoptosis caused by a massive inflammatory response, which is regulated primarily through the caspase and Bcl-2 families [4,5], is involved in glial scar formation and finally in the functional disability of the spinal cord [6,7]. Based on the current understanding of the pathogenesis of SCI, reducing neuroapoptosis may have a key role in the prognosis of SCI. Previously, Guth et al. [8] and Davis et al. [9] have demonstrated that LPS has a potential neuroprotective role in the treatment of injured brain and spinal cord. More recently, Popovich et al. [10] suggest that the 585

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combination therapy (LPS/pregnenolone/indomethacin) has more benefits for spinal cord injured rats. In earlier experiments, we have shown that LPS preconditioning contributes significantly to an improved recovery of motor function through inhibition of neuroapoptosis in an animal model of traumatic spinal cord injury (TSCI) [11]. Although a variety of studies have been reported, the mechanisms underlying low-dose LPS retarding apoptotic processes against TSCI are not fully understood. On the basis of these observations, we have designed a set of experiments to test the hypothesis that LPS preconditioning attenuates apoptotic processes in the early phase after acute TSCI.

nification ×10,000 with Hitachi-7500 electron microscope (Hitachi High-Tech, Tokyo, Japan).

Tissue processing The rats were anaesthetized and perfused transcardially with saline, which was followed by 4% paraformaldehyde (PFA), 72 h after TSCI. An 10-mm longitudinal section was harvested around the epicenter of the lesion to include the entire spinal cord lesion. Then the samples were fixed in 4% PFA for overnight and then embedded in paraffin. The tissues were sectioned at a thickness of 5 μm, then deparaffinized and hydrated gradually for detecting HE staining and immunohistochemistry (IHC).

Materials and methods Animals model and experimental procedures

HE staining

All procedures complied with the guiding principles for the care and use of animals approved by the ethics committee of Chongqing Medical University. We used the model of the lower thoracic TSCI, which has been well established in our laboratory for several years. After each rat was adequately anesthetized, the region over the lower thoracic spine was shaved and disinfected with povidone iodine. Laminectomies were performed at the T9–T11 level with the duramater intact. A moderate injury was created when a 10-g-weight rod was dropped 25 mm using an NYU/MASCIS impactor [12]. The rod touched the exposed spinal cord at vertebra T10. Then, the muscles and skin were sutured. The animals were placed in a room with moderate temperature and humidity. The rats’ bladders were pressed twice daily until a reflex bladder was established. Food and water was provided ad libitum. A total of 54 adult female Sprague-Dawley rats, weighing between 250 and 300 g were used. Rats were randomly assigned to 1 of 3 groups: a sham group (n = 18) that received a laminectomy without weight drop; a LPS preconditioning group (n = 18) that received the administration of LPS from Escherichia coil 055:B5 (0.2 mg/kg, ip; Sigma, St Louis, MO, USA) 72 h before injury; a control group (n = 18) that received saline injection on same schedule.

To assess the histopathologic change, the sections were further subjected to hematoxylin and eosin (HE) staining. After being dewaxed and rehydrated, the spinal cord tissue sections were stained with HE, respectively. Then, the sections were rinsed with double distilled water, dehydrated in ethanol solutions, and cleared in xylene. Two independent and blinded pathologists counted the number of neurons and non-neuronal cells (inflammatory cells, glial cells, endothelial cells, and fibroblasts) in five randomly chosen fields within each slide at 400× with the light microscope (CH30, Olympus, Japan). The number of neurons and non-neuronal cells in each field was counted, respectively, and the average number for the section was then calculated.

Transmission electron microscope At 72 h postsurgery, spinal cord sections were first fixed in 2.5% glutaraldehyde at 4◦ C for 8 h and 1% osmium tetroxide, gradually dehydrated with ethanol and acetone, and embedded with epoxy resin. Ultrathin sections (50 nm) were double stained with 4% uranyl acetate and 0.1% lead citrate, dried and observed at original mag-

Immunohistochemistry staining Standard IHC was performed on sections, obtained as described above. Antigen retrieval was carried out in a microwave oven at 700 W for two cycles of 6 min in citrate buffer (10 mM, PH 6.0). Endogenous peroxidase activity and nonspecific antibody binding site was blocked with 3% hydrogen peroxide and 5% goat serum, respectively. Sections were then incubated overnight at 4◦ C with the following primary antibodies: rabbit anticaspase-3 (diluted 1:200, Santa Cruz Biotechnology), rabbit anti-Bax (diluted 1:100, Santa Cruz Biotechnology), and rabbit anti-Bcl-2 (diluted 1:200, Santa Cruz Biotechnology). Subsequently, the sections were incubated with goat-anti-rabbit secondary antibody (1:400; Santa Cruz Biotechnology). Finally, sections were then visualized using diaminobenzidine, washed, and counterstained with hematoxylin. The sections were independently evaluated under light microscopy (CH30, Olympus, Japan) by two experienced pathologists. The International Journal of Neuroscience

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IHC-positive cells were counted in five randomly chosen fields on each slide at 400×magnification.

Western blotting assays Western blotting was performed to detect the expression of caspase-3, Bax, and Bcl-2 72 hours after TSCI. Protein was extracted as previously described [11]. Proteins were separated by 10% or 12% SDS–polyacrylamide gel electrophoresis and transferred to polyvinylidene difluoride membrane (Millipore, Bedford, MA, USA) which was blocked with 5% nonfat dry milk. Then it was incubated overnight at 4◦ C with the following antibodies: rabbit anti-caspase 3 (diluted 1:400, Santa Cruz Biotechnology), rabbit anticleaved caspase 3 (diluted 1:400, Santa Cruz Biotechnology), rabbit anti-Bax (diluted 1:300, Santa Cruz Biotechnology), rabbit anti-Bcl-2 (diluted 1:400, Santa Cruz Biotechnology), and mouse anti-β-actin (diluted 1:1000, Santa Cruz Biotechnology). The membranes were then incubated with appropriate horseradish peroxidase-conjugated secondary antibodies in blocking solution. Protein bands were detected using an enhanced chemiluminescence detection solution (ECL, Pierce Biotechnology). Protein levels were normalized to β-actin (as a loading control). The final data were subjected to grayscale scanning and quantitative analysis with Quantity One software (Bio-Rad).

Statistical analysis The experimental data were presented as the means ± standard deviation (SD). The SPSS 17.0 statisti-

cal software package (SPSS Inc., Chicago, IL, USA) was used for statistical analysis. The differences between groups in HE staining data were analyzed by oneway ANOVA and Tukey’s test. The differences in IHCpositive cells and Western blot results were compared by using independent t test between two groups or oneway analysis of variance among multiple groups. A difference between means was considered significant if the p < 0.05.

Results LPS preconditioning ameliorated ultrastructure of neuron after TSCI Ultrastructural changes were clearly observed using transmission electron microscopy (Figure 1A–C). In the sham operation group, nuclear membranes were integrative and granules in chromatin were delicate and distributed. In addition, mitochondrial crista structure and membrane were intact in the neurons of spinal cord. Instead, a large number of apoptotic cells were found in the control group. In the control group, neurons shrunk and exhibited abnormal morphology with cytoplasm vacuolization, vague and disappeared mitochondrial ridges, discontinuous nuclear membranes and karyotin margination. In contrast to the control group, the ultrastructures of the neurons in the LPS preconditioning group had less damage. These results suggested that pretreatment with low-dose LPS could efficiently inhibit injury-induced apoptosis of spinal cord neurons.

Ultrastructural findings of neurons in spinal cord at 72 h after TSCI (n = 6 rats/group). N, nucleus; C, cytoplasm. (A) In the sham group, neurons displayed intact nuclear membranes and mitochondrial structure, evenly distributed chromatin. (B) In the control group, neurons shrunk and exhibited abnormal morphology with vacuolized cytoplasm, karyotin margination (arrowheads), vague mitochondrial ridges, and discontinuous nuclear membranes (arrows). (C) However, neurons in the LPS preconditioning group showed less abnormal morphology and clear nuclear structure.

Figure 1.

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LPS preconditioning improves neuropathologic damage after TSCI We compared neuropathological alterations in spinal cord after injury. HE staining illustrated in Figure 2A showed that spinal cord in the sham group had integrated infrastructures and clear neuronal morphology. Blood vessels and central canal also showed normal morphology. No neuronal apoptosis, inflammatory cells infiltration and glial proliferation were observed in the sham group. Instead, in the control group (Figure 2B), patches of necrosis and a large number of the infiltration of red blood cells and inflammatory cells were seen in the gray matter. In addition, a portion of neurons were found with condensed nucleus and red stained cytoplasm. Reactive glial cells were found surrounding neurons like “satellites.” However, compared with the control group, the extent of neuronal damage and histological changes in LPS preconditioning group were marked improvement (Figure 2C). Quantitative data of neurons and non-neuronal cells staining were shown in Figure 2D.

LPS preconditioning inhibited the expression of caspase-3, cleaved caspase-3, and Bax and upregulated the expression of Bcl-2 after TSCI Seventy-two hours after TSCI, caspase-3, Bax, and Bcl2 were measured by immunohistochemical analysis in the spinal cord sections (Figure 3A-I). Spinal cord sections from the sham group showed negative staining for caspase-3, Bax, and Bcl-2, whereas spinal cord sections obtained from the control group exhibited strong positive staining of caspase-3 and Bax and moderate staining of Bcl-2, mainly localized in various cells in the gray matter. However, after LPS preconditioning, we observed that nerve cells apoptosis relieved demonstrated as reduced immunostaining of caspase-3 and Bax and increased immunostaining of Bcl-2. Compared with the control group, the number of positive cells of caspase-3 and Bax in the spinal cord was significantly decreased in the LPS preconditioning group (24.23 ± 0.81 vs. 17.93 ± 0.86; 22.03 ± 1.08 vs. 11.93 ± 0.95, Figure 3J, p < 0.01). Correspondingly, there was a significant

HE staining identified neuropathological changes 72 h after TSCI (n = 6 rats/group). (A) HE staining showed normal neural (arrows) and glial morphology in the sham group. (B) In the control group, spinal cord tissues displayed typical necrosis showing as broad hemorrhage, reactive gliosis (arrowheads), and neuronal apoptosis. (C) In contrast, tissues obtained from the LPS preconditioning group showed significantly more neurons and less non-neuronal cells. The data are presented as mean ± SEM in (D). ∗ p < 0.01, vs. sham group; ∗∗ p < 0.01, vs. control group. Figure 2.

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Immunohistochemistry of caspase-3 (A, B, and C), Bax (D, E, and F), and Bcl-2 (G, H, and I) were performed at 72 h after TSCI (n = 6 rats/group). There were no obvious positive cells in the sham group (A, D, and G). In the control group, dramatic increases of caspase-3, Bax, and Bcl-2 were observed at the lesion site (B, E, and H). Caspase-3 and Bax decreased significantly, whereas Bcl-2 production increased remarkably in the LPS preconditioning group compared with the control group (C, F, and I). The data are presented as means ± SEM in (J). ∗ p < 0.01, vs. sham group; ∗∗ p < 0.01, vs. control group.

Figure 3.

elevation in the number of Bcl-2-positive cells in the LPS preconditioning group (9.10 ± 0.93 vs. 16.20 ± 1.04, Figure 3J, p < 0.01). Furthermore, The concentrations of caspase-3, cleaved caspase-3, Bax, and Bcl-2 in the spinal cord  C

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72 h after TSCI was quantified by using Western blot (Figure 4A). Densitometric analysis showed the relative expression of caspase-3 in the sham group, the control group, LPS preconditioning group to be 0.32 ± 0.08, 0.86 ± 0.11, and 0.72 ± 0.10, respectively (Figure 4B,

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Expression level of apoptosis relevant proteins (n = 6 rats/group). (A) The expression levels of caspase-3, cleaved caspase-3, Bax, and Bcl-2 in the spinal cord at 72 h postinjury were identified by western blot. (B) Compared with the sham group, the control group showed a significant increase of caspase-3, cleaved caspase-3, and Bax but no obvious change in Bcl-2. Compared with the control group, the LPS preconditioning group showed a significant decreased of caspase-3, cleaved caspase-3, and Bax but increase of Bcl-2. Data are presented as means ± SEM. ∗ p < 0.01, vs. sham group; # p < 0.05, ∗∗ p < 0.01, vs. control group.

Figure 4.

p < 0.05). Western blot analysis of caspase-3 and cleaved caspase-3 revealed a consistent effect of LPS intervention in Figure 4B. Moreover, the relative expression of Bax in the sham group, the control group, LPS preconditioning group to be 0.40 ± 0.09, 0.78 ± 0.11, and 0.42 ± 0.10, respectively (Figure 4B, p < 0.01). Correspondingly, the antiapoptotic protein Bcl-2 has a low level expression in both sham group (0.51 ± 0.09) and control group (0.45 ± 0.10), while LPS preconditioning significantly elevated Bcl-2 expression after TSCI as compared with the control group (0.45 ± 0.10 vs. 0.94 ± 0.11, Figure 4B, p < 0.01).

Discussion The results of the present study clearly show that LPS preconditioning can protect the spinal cord from traumatic injury in rats in term of reduction of neurons death resulting from secondary injury. Moreover, the present study indicates that the protection was correlative with the neuroapoptosis-suppressive effect of low-dose LPS because low-dose LPS inhibited Caspase pathway and increased antiapoptotic protein Bcl-2. Apoptosis is the process of programmed cell death that involves a series of biochemical and molecular events, which is critical for optimizing synaptic connections and removing unnecessary neurons during nervous system development. Apoptosis occurs not only during the development but also after brain and SCI [13]. Over the last several years, numerous studies have suggested that neuronal death in the CNS after ischemia and trauma is profoundly associated with the activation of the caspase-dependent pathway [14,15]. It has reported that the markers of caspase-3, Bax, and Bcl-2 are essentially involved in the regulation of cell apop-

tosis [14–17]. Bax has distinct homology with Bcl-2, but the function of Bax is counteracted by Bcl-2 antiapoptotic members. As a consequence of apoptotic stimuli, Bax causes MOMP (mitochondrial outer membrane permeabilization) by destabilizing the lipid bilayer, creating pores or interacting with channels [16]. Furthermore, cytochrome c is released and activates executioner caspase-3 which is a key mediator of programmed cell death. Once activated, caspase-3 can lead to DNA fragmentation and other hallmarks of apoptotic cell death [14]. In our study, transmission electron microscopy showed that low-dose LPS remarkably reduced neuronal apoptosis and ameliorated neurons ultrastructure changes in spinal cord after TSCI. HE staining displayed that LPS preconditioning was the remarkable expansion of neurons survival and inhibited the proliferation of non-neuronal cells in spinal cord after TSCI. Western blot further showed that low-dose LPS elevated Bcl-2 expression while inhibiting caspase3, cleaved caspase-3, and Bax after TSCI, which retarded the initiation of apoptosis and interrupted the pathological process. The phenomenon of LPS-induced cross-tolerance is likely due to a host mechanism that limits inflammatory damage upon activation of the immune system [18,19]. Researchers have studied the mechanism of LPS-induced tolerance mostly by in vitro experiments. However, it is necessary to investigate endotoxic cross-tolerance in vivo because this phenomenon was initially found in animal experiments and clinical research on humans [20–22]. Some authors have described the global or organ-specific features of endotoxic cross-tolerance, as in the lung [23], in blood vessels [24], in the brain [25,26], and in the spinal cord [8,10]. However, LPS preconditioning does not always increase protection; some studies provide different findings International Journal of Neuroscience

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compared to the current study. It has been reported that wognin [27,28] and Scutellaria baicalensis [29] provide neuroprotection against ischemic brain damage and SCI by suppressing LPS-induced cell death and ROS production. These studies showed that LPS-induced protection effect may be highly dependent on the dose and timing of LPS administration. The window of protection is induced 24 h after LPS administration and extends out to 7 d [30,31]. The volume of the LPS dose which induces protection depends on the route of administration and on the animal model, and ranges from 0.02 to 1 mg/kg [32]. Our study show that low-dose LPS (0.2 mg/kg, ip.) preconditioning can induce a protective state in spinal cord after TSCI in rats in terms of reduction of neuroapoptosis. We believe the protection was correlative with the inhibition of caspase pathway and upregulation of Bcl-2. However, it is not known whether more apoptosis pathways and molecules are involved in LPS-induced cross-tolerance in spinal cord, and future studies are needed to address this issue. The experimental approach of LPS preconditioning has interesting clinical applications potentially. First, it allows the identification of endogenous protective/regenerative mechanisms that the spinal cord has evolved to tolerate noxious stimuli such as trauma, ischemia/reperfusion injury, infections, etc. It is conceivable that once these pathways have been clarified, their potentiation after the occurrence of SCI may be used as therapeutic tool. Interestingly, in the setting of SCI, all monodrugs approaches have failed. At present, highdose methylprednisolone therapy is the most widely used, but the treatment dose not significantly improved neurologic functions and can result in serious adverse effects [33]. It has been advocated that the combination therapies might represent a new promising strategy for SCI [34] and preconditioning-based strategies might have the potential advantage of modulating different aspects of the after injury cascades. An additional relevant application to the clinical practice is the implementation of preconditioning to induce protection in those people that can lead to SCI caused by automobile accidents, falls from heights, firearms, and contact sports [35].

Conclusion The results of our studies strongly suggest that lowdose LPS exerted potent neuroprotective mechanisms against TSCI by reducing the expression of apoptotic proteins caspase-3, cleaved caspase-3, and Bax, upregulating the expression of antiapoptotic protein Bcl-2. In addition, LPS preconditioning suppresses activation of non-neural cells to traumatic injury in the spinal cord. The diminished proliferation of non-neural cells that ordinarily exacerbate traumatic injury may contribute  C

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to neuroprotection induced by LPS preconditioning. Therefore, our findings may be helpful for recognizing LPS induced cross-tolerance mechanisms for the CNS injury. A better understanding of these mechanisms might lead to the introduction of LPS into clinical practice.

Acknowledgements The authors would like to thank Dr. Bo Liu and Dr. Xiao-ji Luo for their outstanding technical support.

Declaration of Interest The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper. This study was supported by a scholarship from the University Development Program, Chongqing.

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Lipopolysaccharide preconditioning attenuates apoptotic processes and improves neuropathologic changes after spinal cord injury in rats.

We have shown earlier that administration of low-dose lipopolysaccharide (LPS) significantly contributed to recovery of motor function after traumatic...
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