HIF-1α Activation Attenuates IL-6 and TNF-α Pathways in Hippocampus of Rats Following Transient Global Ischemia

Pro-inflammatory cytokines (PICs) i.e., interleukins, lymphokines and cell signaling molecules are released by numerous cells including leukocytes, myocytes, microglia and astrocytes cells [1]. In particular, as mediators of immune and inflammatory reactions IL-1β, IL-6 and TNF-α play an important role in responding to global ischemic stress [2,3]. These PICs modulate the responsiveness of many cell types in a number of diseases. During diseased states, PICs can recruit cells to inflammatory sites and modulate cell survival, division, proliferation and differentiation [4].

Hypoxia inducible factor-1 (HIF-1) as an important endogenous signaling protein contributes to pathophysiologic changes of homeostasis under conditions of oxygen deprivation [5,6,7,8,9,10]. Accumulated subunit HIF-1α in the cellular nucleus and cytoplasm modulates the expression of several target genes that involve in neuroprotection, erythropoiesis, and apoptosis modulation [11,12]. Additionally, inadequate oxygen supply appears in the brain regions during global ischemia, which is associated with ischemia-evoked pathophysiologic changes [13,14]. Recent studies have demonstrated that HIF-1α is expressed in the brain tissues including hippocampus, indicating that HIF-1α is engaged in hippocampal apoptosis after induction of global ischemia [6,13,14,15]. Using a rat model of asphyxial cardiac arrest (CA), our recent study has further demonstrated that HIF-1α is increased in the hippocampus of rats with CA [16]. Stabilization of HIF-1α by systemic administration of ML228, an activator of HIF-1α [17], enhances vascular endothelial growth factor (VEGF) thereby leading to upregulated protein expression of its receptor VEGFR-2 [16]. This is beneficial to brain ischemic injuries after CA followed by cardiopulmonary resuscitation (CPR).

In light of the precise mechanisms responsible for HIF-1α in response to lacking of oxygen following CA, we examined the role played by HIF-1α in regulating PIC pathways (namely, IL-1β, IL-6 and TNF-α; and their respective receptors IL-1R, IL-6R and TNFR1) in the rat hippocampus after CA induced- transient global ischemia followed CPR. We hypothesized that CA increases IL-1β, IL-6 and TNF-α and upregulates their receptors IL-1R, IL-6R and TNFR1, and that systemic administration of ML228 attenuates those exaggerated PIC pathways.

In addition, nucleus transcription factor such as Caspase-3 is essential signaling pathways engaged in cell survival and hypoxia-induced apoptosis [18,19]. i.e., it has been reported that Caspase-3 expression was amplified in cortical neurons after brain ischemic injuries and inhibition of Caspase-3 reduces the neuronal loss and brain edema [20,21]. Our recent study also demonstrated that Caspase-3 is upregulated in in the rat hippocampus after CA [16]. Thus, we further hypothesized that ML228 lessens exaggerated expression of Caspase-3 in the hippocampus of rats with CA by attenuating PIC signal pathways.

All the animal procedures were approved by the Institutional Animal Care & Use Committee of Jilin University, which are in compliance with the Guideline for the Care and Use of laboratory Animals of the U.S. National Health Institute. Male Sprague-Dawley rats (200 - 300g) were used in our experiments.

The ischemia was produced by the CA model induced by asphyxia as described previously in our publication [16]. Rats were anesthetized with an isoflurane-oxygen mixture (2 - 5% isoflurane in 100% oxygen). An endotracheal tube was first inserted and attached to a ventilator. The ventral tail artery was cannulated to monitor systemic arterial pressure. The right jugular vein was cannulated for a continuous infusion of saline (at a rate of 0.1 ml/hour) to maintain baseline blood pressure and fluid balance. Body temperature was continuously monitored and maintained at 37°C with a heating pad and external heating lamps. Asphyxia was induced by stopping mechanical ventilation and clamping the tracheal tubes at the end of expiration. Resuscitation efforts began 6 min following CA was induced. For this purpose, rats were orotracheally intubated for mechanical ventilation accompanied by chest compression delivered by a mechanical compressor at a rate of 200/min for 5 min. Once spontaneous heartbeat returned, epinephrine (2 µg) was administered to achieve a mean arterial blood pressure of >80 mmHg. Ventilation was adjusted for animals to regain spontaneous respiration and achieve normoxia. The animals that survived from this procedure were used for further interventions. For pain relief those rats received analgesic buprenorphine (0.02 mg/kg) subcutaneously immediately and every 6 hours for a 24-hour period after the asphyxial procedures. In the sham operated animals, the same surgical procedures were performed without cardiac arrest and resuscitation.

The first group of rats was used to examine the time courses of HIF-1α and PICs responses to CA followed by CPR. The rats were sacrificed 0, 3, 6 and 24 hours after CPR, respectively (n = 8-15 in each group). To examine the role of HIF-1α the second group of rats was divided randomly (n = 10-15 in each group) as follow. Sham-operated group: the same surgical procedures and endotracheal intubation were performed with no asphyxia and CPR. Saline control group: CA and CPR were performed and 1 ml of saline (i.p., twice) was administered after CA. ML228 group: CA and CPR were carried out and 2 mg/kg of ML228 (i.p., twice) was injected after CA [16]. Note that the second injection was given 12 hours after CA in order to enhance the effects of this drug. At the end of each experiment, animals were sacrificed by overdose isoflurance followed by decapitation. Then, the brains were taken out to determine expression of HIF-1α, PICs and their receptors as well as Caspase-3 in the hippocampus of rats. Note that the hippocampus CA1 region was used in this study.

In the third groups (n = 10-12 in each group), following CA and CPR procedures rats were continued with an isoflurane-oxygen mixture and immobilized in a stereotaxic apparatus (David Kopf, USA). After making a midline incision, the skull was exposed and one burr hole was drilled. Following this, animals were cannulated with an L-shaped stainless steel cannula aimed at the lateral ventricle according to the coordinates: 3.7 mm posterior to the bregma, 4.1 mm lateral to the midline, and 3.5 mm under the dura. The guide cannula was fixed to the skull using dental zinc cement and jewelers' screw. Then, the cannula was connected to an osmotic minipump (Alzet pump brain infusion kit, DURECT Inc., Cupertino, CA) with polycarbonate tubing. The pumps were placed subcutaneously between the scapulae, and loaded with vehicle (aCSF) as control or 10μg of SC144 (gp130 inhibitor used to attenuate IL-6R activation) and 10 µM of etanercept [22] (ETAN, TNF-α receptor antagonist, Tocris Co.), respectively. SC144 and ETAN were delivered at 0.25 μl per hour. This intervention allowed animals to receive continuous intracerebroventricular (ICV) infusion via the osmotic minipumps before brain tissues were removed.

All the tissues from individual rats were sampled for the analysis. In brief, the hippocampus of the rats was removed. Total protein was then extracted by homogenizing hippocampus sample in ice-cold radioimmunoprecipitation assay buffer with protease inhibitor cocktail kit. The lysates were centrifuged and the supernatants were collected for measurements of protein concentrations using a bicinchoninic acid assay reagent kit.

The levels of HIF-1α were determined using an ELISA assay kit (Abcam Co.) according to the provided description and modification. Briefly, polystyrene 96-well microtitel immunoplates were coated with affinity-purified polyclonal rabbit anti-HIF-1α antibody. Parallel wells were coated with purified rabbit IgG for evaluation of nonspecific signal. After overnight incubation, plates were washed. Then, the diluted samples and the HIF-1α standard solutions were distributed in each plate. The plates were washed and incubated with anti-HIF-1α galactosidase. Then, the plates were washed and incubated with substrate solution. After incubation, the optical density was measured using an ELISA reader. This method was also employed to examine the levels of IL-1β, IL-6 and TNF-α according to the provided description and modification (Promega Corp. Madison, WI).

After being denatured by heating at 95°C in an SDS sample buffer, the supernatant samples was loaded onto 4-20% Mini-Protean TGX Precast gels and then electrically transferred to a polyvinylidene fluoride membrane. The membrane was then incubated overnight with primary antibodies: rabbit anti-IL-1R, anti-IL-6R, anti-TNFR1 and anti-Caspase-3/anti-cleaved Caspase-3 (Abcam Co., diluted 1:200 - 1:500). After being fully washed, the membrane was incubated with horseradish peroxidase-linked anti-rabbit secondary antibody and visualized for immunoreactivity. The membrane was stripped and incubated with mouse anti-β-actin to show equal loading of the protein. The bands recognized by the primary antibody were visualized by exposure of the membrane onto an X-ray film. Then, the film was scanned and the optical densities of IL-1R, IL-6R, TNFR1, Caspase-3, cleaved Caspase-3 and β-actin bands were determined using the NIH Scion Image Software. Then, values for densities of immunoreactive bands/β-actin band from the same lane were determined. Each of the values was then normalized to a control sample.

After brains were removed, the hippocampus was postfixed in 4% paraformaldehyde for 2 days at room temperature. Coronal sections (5-μm thick) were cut from each block. In order to localize Caspase-3 and cleaved Caspase-3, the sections were washed in 0.5% hydrogen peroxide and placed in PBS containing 1% normal goat serum and 0.1% Triton X-100 (PNT). They were then incubated in a primary rabbit anti-Caspase-3 (diluted 1:1000) and anti- cleaved Caspase-3 (diluted 1:500) antibodies. After sections were rinsed in PBS and in PNT, they were incubated in biotinylated goat anti-rabbit immunoglobulin G (diluted 1:200), washed in PBS, and incubated with avidin-biotinylated horseradish peroxidase complex solution. After a serial rinse in PBS and Tris (hydroxymethyl) aminomethane buffer, immunostaining was made visible by incubating sections with hydrogen peroxide and 3,3'-diaminobenzidine.

Experimental data were analyzed using one-way repeated measures analysis of variance (ANOVA). As appropriate, Tukey's post hoc tests were used. All values were presented as mean ± SEM. For all analyses, differences were considered significant at P < 0.05. All statistical analyses were performed using SPSS for Windows version 20.0.

The survival rate of 24 hours was 71% (32/45rats) in CA; 76.4% (42/55) in CA + ML228 group; and 77.4% (24/31) in CA + SC144 and ETAN. Data obtained from those survival rats were included for the analysis in this report.

Figure 1 shows that HIF-1α, IL-1β, IL-6 and TNF-α were significantly increased in the hippocampus 3, 6 and 24 hours after CA (P < 0.05 vs. control, n = 8-12 in each group). There were no significant differences observed in HIF-1α and those PICs between 3 and 6 hours following CA. As compared with 3 and 6 hours, the levels of HIF-1α were less 24 hours after induction of CA. Nonetheless, administration of ML228 significantly restored the declined HIF-1α (P < 0.05 vs. no treatment, n = 8-15 in each group). In addition, ML228 significantly attenuated the amplified levels of IL-6 and TNF-α (P < 0.05 vs. no treatment, n = 8-15 in each group), but not IL-1β in the in the hippocampus 24 hours after CA.

Figure 2 illustrates that induction of CA significantly increased the protein expression of IL-1R, IL-6R and TNFR1 in the hippocampus (P < 0.05, control rats vs. CA rats; n = 10-12 in each group). When ML228 was injected to stabilize HIF-1α, enhancement in expression of IL-6R and TNFR1 was significantly attenuated in the hippocampus of CA rats. Nonetheless, amplified expression of IL-1R evoked by CA tended to be attenuated by ML228, but was not significantly altered (P > 0.05 vs. rats without treatment, n = 10-15 in each group).

Figure 3A illustrates that induction of CA significantly increased the protein expression of Caspase-3 and cleaved Caspase-3 in the hippocampus (P < 0.05, control rats vs. CA rats; n = 10-12 in each group). As ML228 was injected, expression of both Caspase-3 and cleaved Caspase-3 was significantly decreased. Note that there were no significant differences in the levels of Caspase-3 and cleaved Caspase-3 in control rats and rats that received ML228. Furthermore, Fig. 3B&C show that we localized immunostaining of Caspase-3 and cleaved Caspase-3 in the hippocampus of control rats and CA rats without and with ML228 treatment. This result further shows the inhibitory effects of ML228 on Caspase-3.

In order to elucidate the effects of IL-6 and TNF-α pathway on Caspase-3 in the process of transient global ischemia, SC144 and ETAN were infused into the brain via ICV. Figure 4 illustrates that SC144 (n = 12) and ETAN (n = 12) significantly attenuated the amplified protein expression of Caspase-3 and cleaved Caspase-3 in the hippocampus of CA rats (P < 0.05 vs. control animals; n = 10). Insignificant differences were observed in Caspase-3 and cleaved Caspase-3 expression between control animals and animals infused with respective SC144 and ETAN.

Using a rat model we showed that CA increased HIF-1α and PICs including IL-1β, IL-6 and TNF-α in the hippocampus CA1 region. CA also augmented a representative nucleus transcription factor indicating cell apoptosis, namely Caspase-3 as well as a cleaved form of Caspase-3. Notably, systemic administration of ML228 attenuated enhancement of PIC receptors, IL-6R and TNFR1, and Caspase-3 evoked by CA. Blocking individual IL-6R and TNFR1 activities also attenuated Caspase-3 expression in the hippocampus of CA rats. Overall, we suggest that activated HIF-1α is likely to play a beneficial role in improving neuronal cell injuries induced by CA via engagement of PICs and Caspase-3 mechanisms.

Apoptosis represents a prominent form of cell death and is generally observed in the brain tissues after transient global ischemia [14]. Caspases, a family of thiol proteases, play an important role in regulating the apoptotic cascade [23], and brain ischemic stress activates neuronal caspases. The processing of pro-caspase-3 to its active form is considered a key processing in the death-signaling cascade [24]. Caspase-3 is a predominant target involved in the reactive oxygen species-mediated ischemia induced apoptosis in neuronal cells, and neuronal apoptosis results in blood brain barrier dysfunction, inflammation and oxidative cascades and thereby leading to brain damage [25,26]. It has been reported that Caspase-3 expression was exaggerated in cortical neurons after brain ischemic injuries and inhibition of Caspase-3 reduces the neuronal loss and brain edema [20,26]. In addition, a greater number of neurons that are stained with terminal deoxynucleotidyl transferase dUTP Nick End Labeling (TUNEL) are found in hippocampus as apoptosis is engaged in the pathological process of global ischemia-induced neuronal loss due to CA [14]. Thus, Caspase-3 expression is considered as an indicator of neuronal apoptosis with respect to global ischemia.

During CA occurs, blood flow and oxygen delivery are abruptly halted, which leads to systemic ischemic injury in various organs including brain [27]. Although CPR is applied inadequate blood flow and tissue oxygen delivery still persist due to myocardial dysfunction, hemodynamic instability and microvascular dysfunction. In response to hypoxic stress, HIF-1α was synthesized and the expression of its downstream product such as VEGF is upregulated and remains elevated [15,28]. This contributes to neuroprotection against brain ischemic conditions by improving the permeability of blood brain barrier, reducing brain edema formation and promoting the recovery of brain injuries [29]. Our recent study showed that stabilizing HIF-1α significantly attenuated increases of cleaved Caspase-3 and TUNEL evoked by induction of CA as ML228 was given [16]. As a result, this process improved neurological deficits and neuronal edema observed in rats after CA.

PICs, IL-1β, IL-6 and TNF-α are responsive to global ischemic stress [3], which is likely to modulate the responsiveness of many cell types in a number of diseases. For instance, during diseased states, PICs can recruit cells to inflammatory sites and modulate cell survival, division, proliferation and differentiation [4]. In the current study, we examined the role played by HIF-1α in regulating pathophysiological process via PIC signal pathways. The data of our current study demonstrated IL-1β, IL-6 and TNF-α are elevated in the hippocampus of animals 3-24 hours after CA followed by CPR. The time courses for increases of HIF-1α and PICs were similar. Interestingly, stabilization of HIF-1α by ML228 attenuated IL-6 and TNF-α and their receptor expression as well as Caspase-3. Our prior study suggests that attenuation of the hippocampal Caspase-3 improves impaired neurological functions and neuronal edema caused by CA [16]. While HIF-1α is synthesized during hypoxic stress, it is degraded [30]. Thus, maintaining HIF-1α in hypoxic and ischemic tissues has a potential significance to improve tissues injuries due to low oxygen supplies likely by attenuating upregulated PIC pathways. Data of our current study further provide evidence suggesting that stabilization of HIF-1 α and its target signal pathways is likely involved in neuroprotective effects and attenuating damages evoked by global cerebral ischemia.

IL-6 complexes with membrane-bound or soluble IL-6R to activate cells expressing the signal transducer glycoprotein (gp130) [31,32]. Most cells are devoid of membrane-bound IL-6R and are thus unresponsive to IL-6; however, they can still react to IL-6 complexed with a soluble form of the IL-6R (sIL-6R) to activate gp130, a pathway called “trans-signaling” [31]. Thus, in the current study we used SC144, a gp130 inhibitor, to block IL-6-mediated signal transduction in order to examine engagement of the IL-6R in Caspase-3 expression in the hippocampus of CA rats.

TNF-α produces its effects via activation of two TNF-α receptor subtypes, TNFR1 and TNFR2 [33]. TNFR1 is expressed exclusively on neuronal cells and plays a functional role, whereas TNFR2 is located predominantly on macrophages and/or monocytes in response to inflammation. Thus, in our current study application of ETAN attenuates Caspase-3 induced by CA, it is likely via TNFR1. In addition, we observed distinct expression of TNFR1 receptors in the hippocampus of rats after CA. Nonetheless, we have observed that blocking IL-6R or TNFR1 pathways lessened amplified Caspase-3 expression in the hippocampus evoked by CA as ML228 did.

Transient global ischemia induced by CA significantly increases the levels of HIF-1α, PICs and their receptors, and Caspase-3 in the hippocampus. HIF-1α appears to be greater at least 3 hours after CA and persists. Stabilization of HIF-1α can attenuate the protein expression of PIC receptors, IL-6R and TNFR1, and Caspase-3 in the hippocampus of rats 24 hours following CA. Blocking IL-6R and TNFR1 also attenuates Caspase-3 expression in the hippocampus of CA animals. Our data suggest that HIF-1α play an important role in alleviating cerebral ischemia reperfusion injury by attenuating neuronal apoptosis likely via PIC pathways. Our data further indicate that stimulation of HIF-1 α is likely a part of the intrinsic neuroprotective effects aimed at attenuating damages evoked by global cerebral ischemia. Accordingly, targeting one or more of these signaling molecules has clinical significance for treatment and management of CA-evoked global cerebral ischemia often observed in clinics.

None.

J. Xing and J. Lu equally contributed to this work