Intrathecal antagonism of microglial TLR₄ reduces inflammatory damage to blood–spinal cord barrier following ischemia/reperfusion injury in rats

Spinal cord ischemia/reperfusion (I/R) injury is the most devastating complications encountered in many pathophysiological situations, such as hypotension, surgical procedures on thoracic, thoracoabdominal aneurysms and the spine [1]. It remains as a widespread and persistent problem, because its debilitating injuries to the central nervous system results in high incidence of paraplegia posing a serious threat to patients. However, the underlie mechanism of spinal cord I/R injury is not well understood. This is probably due to the induction of spinal cord I/R injury is multifactorial [2‐4]. Blood–spinal cord barrier (BSCB), surrounded by astrocytes and perivascular microglia, consists of a continuous capillary endothelium with tight junctions between the cells. As shown in our previous studies, BSCB disruption and inflammatory reactions play an important role in the evolution of spinal cord I/R injury and in promotion of neuronal damage [5, 6]. Research has expanded into the glial/neuronal transmission and immune responses of resident glial cells to spinal cord injury [7, 8]. Existing evidence shows that spinal glial activation involves important components of the immune system and triggers rapid signal transduction cascades of the transcriptionfactor nuclear factor κB (NF-κB), driving gene expression of proinflammatory cytokines (e.g. IL-1β) in the course of pathophysiological changes that occur after brain injury [9]. Nonetheless, the specific cellular source within microglia which is responsible for transferring immune stimuli into the nervous system responses is unknown. During the pathogenic cascade after central nervous system (CNS) injury, microglia are thought to be the first nonneuronal cells to express a plethora of growth factors, chemokines, and regulatory cytokines as well as free radicals and other toxic mediators [7, 10].

In microglia, Toll-like receptors (TLRs), especially TLR₄, have been shown to recognize various microbial products and to initiate innate immune responses upon interaction with infectious agents or endogenous ligands present in the spinal cord in vivo and in vitro[10, 11]. The general understanding is that TLR₄ can be specifically activated to initiate immune responses by presenting a pathogen-derived antigen to naïve T cells when sensing lipopolysaccharide (LPS), a common constituent in the cell wall of Gram-negative bacteria [12].Studies have demonstrated that TLR₄ is upregulated in different I/R-injured organs, including heart, brain, liver, and kidney [13, 14]. Nevertheless, the relationship between TLR₄ and microglial activation in spinal cord I/R injury remains unknown. Thus, we hypothesized that there might be a link between TLR₄ of microglia to the stressors that damaged neurons: endogenous antibodies or cytokines that leaked through disrupted BSCB, which, in turn, contributes to further activation of microglia and BSCB disruption. Moreover, recent studies suggest that microglia activation which could be inhibited by minocycline is a double-edged sword in various neurological models and under different conditions [8, 15]. Ischemia was regarded as a powerful stimulus that disabled the endogenous inhibitory signaling and triggered microglial activation [7]. Upon activation, microglia could exhibit plenty of phenotypes and release both pro- and anti-inflammatory mediators to either exacerbate ischemic injury or help repair. A few studies, however, have conducted on the spinal cord model of I/R injury and state that whether activation of microglia is deleterious and/or beneficial for spinal cord recovery is still a controversial topic. To testify our hypothesis, we first explore whether microglia are activated in a rat model of spinal cord I/R injury. Next, we evaluate the roles of TLR₄ and NF-κB during I/R-induced BSCB disruption with and without the involvement of microglia.

Ischemia/reperfusion (I/R) injury of the spinal cord after an operation on the thoracic aorta is an unpredictable, however, disastrous complication. To the best of our knowledge, the current study is the first to demonstrate activation of TLR₄ in microglia and we show TLR₄ is participates in inflammatory reactions in the blood–spinal cord barrier (BSCB) after I/R injury. Early and sustained microglial activation can be determined by the mRNA and protein expression of the microglial surface markers, Iba-1. The membrane-bound receptor TLR₄ is also overexpressed in the spinal cord but not in sham-operated rats from 12 to 36 h after I/R injury. In addition, intrathecal administration of minocycline for 3 days before ischemia showed obvious protective effects in the form of a decrease in I/R-induced microglial activation, in BSCB disruptions, and in the production of proinflammatory cytokines. Results from this study also demonstrated that when administered in this preemptive manner, intrathecal infusion of a TLR₄ receptor inhibitor, TAK-242, exhibits protective effects similar to minocycline pretreatment results, whereas intrathecal injection of LPS, a TLR₄ agonist, exacerbates deleterious effects. Given that NF-κB is known to be activated in the presence of TLR₄, it is expected that the NF-κB inhibitor,PDTC inhibit I/R-induced microglial activation via upregulating TLR₄.

The I/R model used and the period observed were referred to previous studies, in which the evolution of inflammatory cytokine activation in the spinal cord peaks between 24 and 48 h after reperfusion [5, 6, 16]. This model is a reliable and stable animal model for studying neuroprotective manipulations and molecular mechanisms in the spinal cord. BSCB consists of a continuous capillary endothelium with tight junctions between its cells, surrounded by astrocytes and perivascular microglia. The intact barrier can prevent vasogenic edema and pathological effects on CNS by restricting access of molecules and cells to the spinal cord under conditions of stroke and I/R [17, 18]. It is generally believed that inflammatory factors play a critical role in leakage of BSCB as a result of aberrant vascular permeability due to dissociation of zonula occludens-1 (ZO-1) from the cytoskeletal complex and due to an increased level of matrix metalloproteinases (MMPs) and tumor necrosis factor α (TNF-α) [5, 6, 17‐19]. As demonstrated in our previous study, BSCB permeability is altered in the course of spinal cord I/R injury, and these changes could be measured using extravasation of EB dye [5, 6]. This damage may caused by inflammatory processes and it may further exacerbate inflammation. Recent evidence support the notion that microglia, components of the immune system are activated and play an important role in the initiation phase of I/R-induced neurodegenerative and inflammatory processes. When triggered by peripheral inflammation or nerve injury, spinal microglia can be rapidly activated and can respond to the neurotransmitters released by central terminals of primary sensory neurons, such as glutamate, substance P, and adenosine triphosphate. After that, the release from activated glial cells of a series of growth factors, chemokines, regulatory cytokines as well as free radicals and other toxic mediators such as IL-1β, TNF-α, prostaglandin E_2, and reactive oxygen species (ROS), results in activation of rapid signal transduction cascades leading to either survival or death of neurons [9, 17]. Representative micrographs of ionized calcium–binding adaptor molecule 1 (Iba-1) are commonly used to quantify activated microglia. Upon activation, microglial cells transform from the ramified shape to rounded (amoeboid) macrophage-like morphology [20]. There are significantly greater numbers of Iba-1–positive cells in the I/R group both at 12 and 36 h in comparison to the lower numbers in the sham group. Furthermore, the colocalization with TLR₄ according to double-immunofluorescence analysis confirmed that TLR₄ is indeed upregulated in activated microglial cells in injured regions of the spinal cord.

There are substantial protective effects of minocycline (a member of the tetracycline antibiotic family), which prevent microglial activation and generation of glutamate, IL-1β, and nitric oxide (NO) [21]. To explore the role of microglia in BSCB disruption after I/R injury, minocycline was infused intrathecally during 3 days before the surgical procedure in the present study. We also found that inhibition of microglial activation by minocycline is accompanied by a decreased number of Iba-1–positive cells.

Blamire et al. [22] and van Vliet et al. [23] examined the role of proinflammatory cytokines in BSCB leakage. These investigators reported that increased plasma levels of inflammatory cytokines (such as IL-1β and IL-6) in residual microglia (where disruption of the blood–brain barrier occurred) might also be responsible for BSCB leakage. At the same time, we observed another protective role of minocycline: it seems to attenuate (1} the BSCB disruption as measured by EB extravasation and (2} an increase in spinal water content. Our data show that minocycline decreases the number of Iba-1–positive cells at 12 and 36 h after reperfusion and attenuates upregulation of the proinflammatory molecules IL-1β and NF-κB in the spinal cord. These findings were corroborated by previous studies not only in experiments with ischemia [3, 4] but also in studies of systemic proinflammatory states [9, 23, 24]. These observations suggest that activated microglia may potentiate damage to BSCB components, which is caused at least in part by proinflammatory cytokines.

Previously, we elucidated the molecular mechanisms underlying inflammatory and immunological processes of microglial activation after I/R. Upon activation, microglial cells start to express TLR_2–4 on their surface [11, 14]. Among diverse TLRs, it has been reported that TLR₄ enables microglia to induce immune and inflammatory responses and to release massive amounts of proinflammatory cytokines via activation of NF-κB, once TLR₄ binds to its endogenous or exogenous ligands. Studies show that TLR₄-deficient mice display significantly attenuated behavioral hypersensitivity and are characterized by weaker spinal glial activation and lowered release of proinflammatory cytokines [8, 12, 14]. There is usually no viral or bacterial infection in I/R models; recent research supports the notion that upregulated expression of TLR₄ is implicated in activation of microglia in cerebral ischemia models [8, 16]. Nonetheless, there is still debate and controversy regarding the role of microglial TLR₄ in the spinal cord during the earliest stage (<3 days) after I/R, where TLR₄ must be involved in various signal transduction pathways. Our results show that early, robust, and sustained microglial activation after I/R injury is characterized by a marked long-term induction of the TLR₄ expression at protein and mRNA levels, matching the pattern of immunostaining with Iba-1. In parallel, cytokines IL-1β are released in the spinal cord between 12 and 36 h after reperfusion, the finding that is consistent with the above reports. We also confirmed the specificity and the role of TLR₄ in the microglial activation and in inflammatory reactions induced by intrathecal infusion of TLR₄ receptor antagonists or agonists. We found that downregulation of TLR₄ receptor by TAK-242 lowers the amount of resident microglial activated cells, decreases levels of NF-κB translocation, and consequently downregulates the cytokine IL-1β in the spinal cord between 12 and 36 h after reperfusion. Because TLR₄ is a known specific receptor for Gram-negative bacterial components (LPS), the rats treated with LPS showed aggravated inflammatory processes, and BSCB disruption occurred in the I/R region, in keeping with the pattern of upregulated TLR₄.

In view of the pivotal role of TLR₄ in microglial activation in the initial phase of I/R injury, the question arises how TLR₄ mediates microglial activation in spinal cord I/R injury. After activation of the TLR₄ signaling pathway, NF-κB relocates to the nucleus and regulates expression of target inflammatory genes via modulation of both MyD₈₈ or MyD₈₈ adaptor–like adaptor protein and TIR domain–containing adaptor [12, 16, 25]. With double immunofluorescence in present study, we provided evidence that NF-κB signaling pathway activated by TLR₄ receptor played important roles in regulating immune and inflammatory responses after I/R. We detected greatly upregulation of NF-κB and IL-1β (at the mRNA and protein level) in spinal cords limited to the ischemic region between 12 and 36 h after I/R injury and was accompanied with a selective increase in TLR₄ expression. Pyrrolidine dithiocarbamate (PDTC) is a low-molecular-weight thiol compound, was initially regarded as a potent inhibitor of NF-κB by inhibiting factor I-κB phosphorylating, thus preventing the dissociation of the NF-κB -IκB complex and interfering with the generation of proinflammatory cytokines [26‐28]. It has been reported that intrathecal PDTC can delay and reverse mechanical allodynia in several neuropathic pain conditions [27]. Similarly, in present study, intrathecal infusion of PDTC was observed to have suppressive effects on mechanical allodynia, BSCB dysfunction and microglial activation as well as up-regulated TLR₄ mRNA and protein expressions in spinal cord, suggesting that the activity of NF-κB pathway could regulate spinal cord I/R injury via regulating TLR₄.

Neurons play very important roles in the nervous system, involved in the processes of memory, sense and behavior. A result of I/R-induced neurological deficit was partly contributed to apoptosis of neurons in spinal cord as reported in our previous studies [2, 5, 6]. Our data of NeuN immunoreactivity sufficiently evidenced I/R lead to a decrease neuron number in ventral gray matter and increase percentage of double-labeled cells with cleaved capase3, which referred as a mark of apoptosis. Meanwhile, microglia may release various factors to support and guide the transfer of neurons, participate in neurons repair and regeneration [29, 30]. IL-1β is one of the final inflammatory molecules produced in TLR₄ signaling pathway, which elicits a cascade of activation of cytokines and multiple biological effects [22]. In a variety of inflammatory conditions, reasonable upregulation of IL-1β has been shown to limit extreme inflammatory responses, with a possibility of rapidly activated phagocytosis of dead or dying cells to prevent a release of a cascade of proinflammatory cytokines and to resolve inflammation. Nevertheless, excess IL-1β can significantly worsen inflammation and tissue injury [9, 13, 22]. Thus, these signals should be regulated very tightly to balance proinflammatory and anti-inflammatory pathways. Brikos and colleagues found that the cytoplasmic portion of TLRs, called the Toll/IL-1 receptor (TIR) domain is highly similar to that of the IL-1 receptor family [1]. Our study confirmed that IL-1β expression and loss and apoptosis of neurons triggered by TLR4/ NF-κB signal in spinal cord were strongly increased by intrathecal infusion of LPS; in contrast, the expression levels were much lower in rats treated with minocycline, TAK-242 or PDTC, indicating that there might be a positive feedback circuit. In other words, the expression of inflammatory cytokines is regulated by TLR₄ in microglia, and in turn, inflammatory cytokines can cause microglial activation by amplifying and maintaining inflammatory response via the TLR₄ pathway. This notion also offers a probable reason for the difficulty in developing an effective therapy for spinal cord injury–related complications. Interestingly, the similar findings have been reported by Bell at al in a mice model of aortic cross-clamping, recently [31]. That study showed that inhibition of TLR₄-mediated microglial activation may be a major mechanism of neuroprotection associated with the anti-inflammatory effects. Regarding the controversial effects of microglial activation via its plenty of membrane-bound receptors for spinal cord recovery under different stimuli, further studies would be required to clarify the complicated role of microglial TLR₄ signaling in models of I/R injury. As potential therapeutic modalities, minocycline, TAK-242 and PDTC would need further research, including safety testing; for example, their effects on learning and memory are unknown.

Based on our results, we conclude that inhibition of microglial activation and proliferation via the TLR₄–microglia–NF-κB/IL-1β pathway result in protective effects in some neurological deficits, hence, fortifying BSCB integrity, and reducing spinal cord swelling.

Taken together, our data provide evidence to support the involvement of TLR₄ signaling in modulation of microglia activation in spinal cord I/R injury. We also suggest that TLR₄–microglia– NF-κB/IL-1β pathway as a positive feedback loop in the spinal dorsal horn after I/R injury; this pathway is capable of exacerbating inflammatory reactions and BSCB dysfunction. It could offer new therapeutic targets for managing severe and persistent complications of spinal cord injury caused by I/R.