Role of the TLR4 pathway in blood-spinal cord barrier dysfunction during the bimodal stage after ischemia/reperfusion injury in rats

Spinal cord ischemic/reperfusion (I/R) injury is a serious complication of thoracoabdominal aortic surgery. The injury occurs as a two-phase process that correlates with its onset and delayed clinical progression. The first phase includes metabolic and inflammatory processes following ischemia, and the second one is a set of amplified inflammatory responses mediated by biochemical cytokines after restoration of spinal cord blood flow [1, 2]. Clinically, this is observed as the bimodal distribution of paraplegia, in which lower limb movement is observed immediately after operation but later deteriorates [3‐5]. This phenomenon was reported to occur in 2% to 18% of patients, and a total of 50% of patients displayed delayed neurological deficits due to multifactorial causes [5]. The blood-spinal cord barrier (BSCB), consisting of continuous capillary endothelial cells surrounded by astrocytes and pericytes as well as perivascular microglia, is critical to maintain spinal cord homeostasis and neurologic function [6, 7]. Impairment of the BSCB may induce spinal cord edema and progressive breakdown of BSCB integrity, which may aggravate injury, resulting in paraplegia or even death, thus highlighting the complexity of the injury [1, 3, 8, 9]. Previous studies have demonstrated that Toll-like receptors 4 (TLR₄), a class of transmembrane proteins that can recognize specific ligands extracellularly, are closely associated with the inflammation that occurs after I/R injury and mediate the pathogenesis of hind-limb function and neuronal viability [10‐12]. The myeloid differentiation factor 88 (MyD₈₈) and TIR domain-containing adaptor-inducing IFN-β (TRIF), which function via the up regulation of TLR₄, are the two most important adaptor proteins that provoke the transduction of common downstream signaling molecules, such as NF-κB, into the nucleus and regulate the expression of target inflammatory genes [13‐15]. In the MyD₈₈-dependent pathway, previous studies have demonstrated that TLR₄ directly activates MyD₈₈ and thus facilitates further activation of NF-kB and downstream inflammatory cytokine interleukin (IL)-1β in the early phase [10, 13, 16]. However, recent studies suggest that TLR₃ is not indispensable for the TRIF-dependent pathway [17]. In the TLR₄ signaling pathway, TRIF is recruited upon receiving stimuli from TICAM-2, leading to rapid activation of interferon regulatory factor 3 and beta interferon, which in turn activates TRIF and MyD₈₈[15‐17] and induces delayed NF-kB activation [18]. In our previous studies, we have reported the breakdown of BSCB integrity in the spinal cord after I/R injury [8, 9]. Therefore, procedures that can preserve the intact function of the BSCB and attenuate the inflammatory responses after I/R injury would improve prognosis. However, not much is known about the underlying roles of TLR₄ signaling and the transduction of its downstream adaptor receptors. The aim of this study is to investigate the role of TLR₄ in increased BSCB leakage using a specific cell population during a post-injury bimodal stage. We established a functional deletion of TLR₄, MyD₈₈, and TRIF receptor by intrathecal treatment with TAK-242 [19], MyD₈₈ inhibitory peptide (MIP) [16], or Resveratrol [20] to prevent TLR₄ from interacting with its downstream receptors, namely, MyD₈₈ and TRIF. In addition, we explored the underlying relevance of TLR₄/MyD₈₈-dependent and TLR₄/TRIF-dependent pathways in the bimodal phase of inflammatory responses after I/R injury by determining the activation of NF-kB and subsequent products of the proinflammatory cytokine, IL-1β.

Spinal cord I/R injury after thoracic aortic surgery is invariably associated with dysfunction of the BSCB and plays a fundamental role in the progression of several unpredictable and disastrous complications, such as spinal cord swelling and secondary nerve injury, which account for much of the morbidity and mortality of this condition [4, 5]. Spinal cord edema is often long lasting and resistant to therapeutic intervention. Recently, TLRs, especially TLR₄, have gained extensive attention for their important roles in various models of I/R injury [1, 18]. In our previous study, we demonstrated that increased activation of TLR₄ in the spinal cord was associated with increases of BSCB leakage after I/R injury. Our present study is the first attempt to further characterize the inflammation that occurs in a specific cell population after spinal cord I/R injury and suggests the functional relevance of TLR₄-mediated downstream signal transduction during the bimodal stage after injury.

With an improved understanding of spinal cord I/R injury, the damage sustained during thoracoabdominal aortic occlusion has been classically defined as an initial ischemic event and a delayed reperfusion injury, corresponding with the clinical presentation of bimodal distribution of spinal cord injury [1‐3]. Clinically, the manifestation of immediate severe spinal cord injury is rare. Usually, patients experience delayed neurological deficits and develop paraplegia in the several hours to days after surgery, which highlights the pathological mechanisms defined by the responses in the ischemic phase, and an ongoing amplification of molecular insults during the reperfusion phase [23]. After I/R injury, the cytokines released by local cellular populations in the early phase may cause increased accumulation of inflammatory cells, and the chemokines released amplify inflammatory responses, leading to the clinical presentation of functional deficits [24, 25]. As demonstrated in our previous study, increased BSCB leakage closely associated with invasion of exogenous ligands inducing immune and inflammatory responses were found during the course of spinal cord I/R injury, as evaluated by extravasation of EB dye [8, 9]. Similarly, in this study, the bimodal distribution of EB extravasations was clearly observed as two phases, one stage from 6 to 18 h after surgery which continued for several hours, and the later one began at 36 h and peaked at 48 h (Figure 1), paralleling the clinical manifestation of an early ischemic phase with great risks of delayed and exacerbated injury, such as paralysis, two days after surgery [4, 5].

A growing body of evidence indicates the important role of TLR₄ in the evolution of an inflammatory reaction after I/R injury [16, 19]. The stimulation from ischemic or necrotic cells causes TLR₄ activation, subsequent translocation of NF-κB to the nucleus, and the production of proinflammatory cytokines and chemokines [10, 16]. Similarly, the immunohistochemistry data in our study confirm the essential role of TLR₄ in the inflammatory processes associated with increased BSCB leakage. Significantly, our study lies in the understanding of the correlation between the TLR₄-mediated MyD₈₈-dependent and TRIF-dependent signaling pathways during the bimodal stage after I/R injury. Therefore, we chose 12 and 48 h time points at which maximal changes were reached after I/R injury to further explore the role of TLR₄-mediated downstream signaling. It is essential to develop potential therapies targeting these points to attenuate and avoid subsequent injury. Some investigations have already reported the effects of TAK-242, MIP, and Resveratrol on the inhibiting functions of TLR₄ and its downstream adaptor receptors, MyD₈₈ and TRIF, which receive the stress stimulation [16, 19, 20]. In this study, we depleted the function of the TLR₄ signaling pathway by continuous intrathecal injection with TAK-242, MIP, or Resveratrol at a 12-h interval 3 days before surgery. The effects of the inhibitors were detected by Western blot. Based on our results, the protein levels (Figure 3A-C) and integrated density values (Figure 3D-F) of TLR₄, MyD₈₈, and TRIF in the I/R group were obviously higher than those in rats intrathecally pretreated with TAK-242, MIP, or Resveratrol, as well as those in the Sham group at both observed time points. Therefore, TAK-242, MIP, and Resveratrol employed before reperfusion significantly prevented their functions from receiving the reperfusion-induced stimulation. However, pretreatment with only one of the inhibitors lead to changes in three indicators (TLR₄, MyD₈₈, and TRIF) in each group simultaneously. Therefore, it is important to further assure specificity of each inhibitor by detecting whether there is cross-talk in signal transduction. As tested herein, TAK-242 specifically binds the TLR₄ receptor without much impact on the functions and expressions of adaptor receptors MyD₈₈ and TRIF downstream. Likewise, MIP and Resveratrol were proven to solely decrease the protein expression of MyD₈₈ and TRIF, respectively, demonstrating the reliability of intrathecal injection with specific binding proteins to explore the role of the TLR₄ pathway, as performed in our study (Figure 3).

Our study showed that I/R injury caused vascular events of BSCB in segments most vulnerable to ischemia and exhibited more pronounced vascular disruptions at 48 h than those at 12 h after surgery. The BSCB act as a metabolic barrier to strictly regulate molecular exchange between the circulating blood and spinal cord, and strictly controls the spinal cord microenvironment required for normal neuronal function and micro environmental homeostasis [6, 9, 26]. Increased BSCB leakage would expose the spinal cord to endogenous ligands and exogenous invading pathogens in the circulation and aggravate inflammatory responses and neurotoxic effects in the development and recovery associated with spinal cord injury, resulting in irreversible damage to the physiological functions of aesthesia and kinesis, as well as to bladder, bowel, and sexual functions following the injured plane. Moreover, the BSCB may determine the innate immune reaction [26]. Therefore, the exploration of the cellular compositions of the BSCB implicated in TLR₄ activation will greatly contribute to the better understanding the physiological function or pathological development in the early and late phases of inflammation after I/R injury. As reported in previous studies, glial activation occurred in response to ischemia and the responses were very rapid, especially in microglia, which changed in morphology and increased in number within the first few minutes to hours after reperfusion [1, 3, 7, 26], while astrocytes were observed to be mildly increased over 8 h after reperfusion [26]. Similarly, the microglia increased in number and showed intense staining in the spinal cord after surgery and peaked at 12 and 48 h, whereas the GFAP staining became intense 24 h after reperfusion and peaked at 48 h, suggesting that increased BSCB leakage may be attributed to glial activation through their membrane-bound receptor TLR₄ which is closely associated with inflammatory reactions (Figure 4). Increasing evidence has shown that there is an important and complicated signal transmission within activated neuralgia in response to spinal cord injury [6, 7, 18]. To explain the different activation phases of glial cells, one can easily consider the different degrees of BSCB leakage and the different roles of glial cells involved. Perivascular microglia surrounding the BSCB are considered as the first actors to remove tissue, cells debris, or macromolecular proteins penetrating through functional leakage of the BSCB, as suggested in the model of spinal cord ischemia and compressive injury [7, 26, 27]. Astrocytes have been demonstrated to play a major role in homeostasis of the BSCB and recovery of neurological function after ischemia [7]. In the present study, compared with the widespread activation of microglia both in the early (12 h) and late phase (48 h) after surgery, the increases in GFAP immunoreactivity were mainly distributed in spinal gray matter, where the capillaries were more numerous and formed a dense capillary bed in comparison with white matter. Thus, astrocytes were considered to be hyper-responsive states in cases of organic damage of BSCB. In addition, the effects of the inhibitors on double-labeled glial cells were chosen to quantify at 48 h after surgery for the maximal activation based on micrographs. As shown in Figure 4B and C, compared with I/R group, the number of double-labeled microglia and astrocytes in groups pretreated with TAK-242, MIP, and Resveratrol, respectively, decreased the most in the TAK group, suggesting that the relatively intact microenvironment of the spinal cord contributed to the weak responses of glial cells in the BSCB lacking the functional TLR₄ receptor after surgery. Comparing the effects of MIP and Resveratrol treatment, immunofluorescence staining and quantification data demonstrated that I/R-induced elevated double-labeled microglia and astrocytes were significantly decreased in the Resveratrol group, whereas less so in the MIP group, indicating a major involvement of the TLR₄-TRIF-dependent pathway 48 h after surgery.

Up regulating TLR4 and NF-κB was accompanied with the production of proinflammatory cytokines. As expected, our results revealed increases in NF-κB and IL-1β with increasing time after I/R injury, signifying the amplified and aggravated inflammatory responses in pathogenesis in the spinal cord. Previous studies have demonstrated the capacity of TLR₃ to activate TRIF [15, 28] and that TLR₄ directly activated MyD₈₈[16, 19]. With recent advancements in this area, some studies have indicated that TLR₃is not indispensable for the TRIF-dependent pathway while the TLR₄ receptor has the potential to activate the TRIF-dependent pathway upon receiving a signal from MyD₈₈-dependent pathways [15]. The signals that bridged through TICAM-₂ to TICAM-₁ led to the recruitment of TRIF and TRAM in a cascade [15, 29], resulting in the rapid production of cytokines and the late phase activation of NF-κB and mitogen-activated protein kinases, thus exerting positive feedback on MyD₈₈-activation [30, 31]. Therefore, based on our data, the ongoing cascade of inflammatory responses involves the activation of both the MyD₈₈ and TRIF pathways.

IL-1β is the most commonly implicated protein in inflammatory responses and exhibits biphasic distribution in various models of I/R injury [2, 3, 32]. These studies revealed that IL-1β significantly increased and remained high in the reperfusion period, and that heightened IL-1β were associated with both early ischemic and late reperfusion injury [2, 3, 32, 33]. Based on our results, we found analogously biphasic IL-1β responses during the reperfusion period since the levels expressed at 12 and 48 h were much higher than those at 24 h and were in accordance with the activation of NF-κB. It was intriguing to explore the temporal relationship between TLR₄/MyD₈₈ and TLR₄/TRIF. Similar to the results in our study, O’Neill and Bowie suggested that the expression of NF-κB occurred in two phases after TLR₄ activation [17]: MyD88 mediated events in the early phase, while TRIF mediated the later phase. We found that there was a significant increase in NF-κB release and IL-1β production at 12 h. Nevertheless, contrasting results were observed at 48 h in the Resveratrol group (Figure 4), indicating that MyD₈₈ and TRIF dependence occurred in the early and late phases, respectively.

Of note, sequential activation of microglia and astrocytes were supported by immunofluorescence staining, but not necessarily in that order given the complexity of in vivo experiments. Nevertheless, a sustained majority of TLR₄ expression occurs in microglia throughout the inflammatory responses, and the increasing involvement of astrocytes with time in the late phase might be an explanation for the delayed neurological deficits [3, 7, 26, 27, 34]. Further in vitro studies still need to be conducted to identify the development and mode of action of these inflammatory mediators to better elucidate the mechanism underlying the pathogenesis.

Taken together, our data suggest that there could be different phases of spinal cord I/R injury. Inflammation is a subsequent event during the bimodal stage after injury. The early phase of I/R injury in the spinal cord was found to be largely TLR₄/MyD₈₈-dependent and microglia-dependent, and the following late phase was found to be mainly dependent on TLR₄/TRIF activation, which was amplified by MyD₈₈ signaling with the involvement of both microglia and astrocytes. These findings explain why several therapeutic treatments have failed in patients with I/R injury. A novel and ideal therapy that would address the different phases of injury and inflammation may be valuable in preventing delayed injury and its clinical manifestations.