Toll-like receptor 4 is associated with seizures following ischemia with hyperglycemia.

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Research Report

Toll-like receptor 4 is associated with seizures following ischemia with hyperglycemia Q1 Q4

Yanling Lianga, Zhigang Leib, Hui Zhangc, Zhiqiang Xua, Qiliang Cuic, Zao C. Xub a

Department of Neurology, The Third Affiliated Hospital of Guangzhou Medical University, China Department of Anatomy and Cell Biology, Indiana University School of Medicine, USA c Department of Pediatrics, the Third Affiliated Hospital of Guangzhou Medical University, China b

art i cle i nfo

ab st rac t

Article history:

Seizures are a common sequel of cerebral ischemia, and hyperglycemia markedly

Accepted 6 September 2014

increases the onset of seizures following an ischemic insult. However, the underlying mechanism of seizures is unclear. The toll-like receptor 4 (TLR4) pathway is known to be

Keywords:

involved in temporal lobe epilepsy. The present study investigated the potential involve-

Toll-like receptor 4

ment of TLR4 in the pathogenesis of seizures following cerebral ischemia with hypergly-

HMGB1

cemia. Fifteen minutes of global ischemia was produced in adult Wistar rats using a

Diabetes

4-vessel occlusion method. Hyperglycemia was induced via an intraperitoneal injection of

Post-ischemic seizures

glucose 15 min prior to ischemia. We determined that 56.7% of the hyperglycemic rats, but

Inflammation

none of the normoglycemic rats, developed tonic-clonic seizures within 12 h after

Hyperactivity

ischemia. TLR4 was mildly expressed in a few cells in the control hippocampus, primarily in interneurons, and was localized in the cytoplasm. The TLR4-positive cells were significantly increased 3–12 h after ischemia. In the hyperglycemic ischemia group, TLR4-positive cells were further increased in quantity and intensity, with a peak at 3 h after ischemia relative to the normoglycemic group. There was no difference in the expression of TLR4 between the hyperglycemic ischemia and LPS groups or between the hyperglycemic non-ischemia and control groups. Western blot analysis consistently exhibited an increase in TLR4 protein levels in the CA3 region 3 h after hyperglycemic ischemia. High mobility group box 1 (HMGB1) (an endogenous ligand of TLR4) was localized in the nucleus of neuronal cells throughout the hippocampus in the control animals. We observed a dramatic decrease in HMGB1 immunostaining at 3 h after hyperglycemic ischemia that gradually returned to control levels. These results suggest that the TLR4 pathway is associated with seizures following global ischemia with hyperglycemia, which provides a new direction for the study of the pathogenesis of seizures that result from hyperglycemic ischemia. & 2014 Published by Elsevier B.V.

http://dx.doi.org/10.1016/j.brainres.2014.09.020 0006-8993/& 2014 Published by Elsevier B.V.

Please cite this article as: Liang, Y., et al., Toll-like receptor 4 is associated with seizures following ischemia with hyperglycemia. Brain Research (2014), http://dx.doi.org/10.1016/j.brainres.2014.09.020

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1.

Introduction

Pre-ischemic hyperglycemia has been associated with worse outcomes in numerous animal models of global ischemia, and these outcomes typically manifest as an increased incidence of post-ischemic seizures and death. Although numerous studies have been conducted, the underlying causes of seizures following cerebral ischemia with hyperglycemia are not fully understood. In the last decade, increasing clinical and experimental evidence has supported the involvement of inflammatory and immune processes in the etiopathogenesis of seizures and epilepsy (Vezzani and Granata, 2005; Vezzani et al., 2011a). Inflammatory responses induced by brain-damaging events, such as stroke, trauma, and infection, are associated with acute symptomatic seizures and a high risk of developing epilepsy (Bartfai et al., 2007; Ravizza et al., 2011; Vezzani et al., 2011b). The activation of specific proinflammatory signals, such as the Toll-like receptor 4 (TLR4) pathway, has been implicated in the precipitation and recurrence of seizures in experimental models (Maroso et al., 2010; Vezzani et al., 2011b). The toll-like receptors (TLRs) are evolutionarily conserved protein receptors that are fundamental to the activation of the innate immune system. TLRs can be transmembrane or intracellular protein receptors, and thirteen homologues have been identified in mammals (Medzhitov et al., 1997). TLRs were initially found to recognize microbial components, such as lipopolysaccharide (LPS), termed “pathogen-associated molecular patterns” (PAMPs) (Mollen et al., 2006). PAMPs trigger inflammation by inducing the transcription of genes that encode cytokines, including IL-1β. Furthermore, increasing evidence indicates that certain TLRs also respond to endogenous molecules released from stressed or damaged cells, termed “damage-associated molecular patterns” (DAMPs), including the endogenous ligand high-mobility group box-1 (HMGB1) (Tsan and Gao, 2004; Bianchi and Manfredi, 2009). The activation of specific TLR pathways in animal models has been demonstrated to play a vital role in the pathogenesis of critical conditions, including ischemic stroke, brain trauma, “sterile” inflammation and tissue injury in the absence of pathogens. Among these TLRs, TLR4 has been the most extensively studied.

It is increasingly clear that post-stroke neuroinflammation from TLR4 signaling worsens stroke outcomes, as measured by infarct volumes, neurological function and inflammatory markers (Caso et al., 2007; Hua et al., 2009). Studies using different models of cerebral ischemia have elucidated the role of TLR4 signaling in mediating neuroinflammation and exacerbating stroke injury. Neurological outcomes after cerebral infarction are improved in mice with a TLR4 deficiency (Cao et al., 2007; Hua et al., 2007). Exercise therapy, electroacupuncture and certain medicines may play a protective role against ischemic injury via the downregulation of TLR4 expression (Ajamieh et al., 2012a, b; Suzuki et al., 2012; Lan et al., 2013; Ma et al., 2013). Both in vivo and in vitro studies have demonstrated that hyperglycemia activates TLR4 expression and exacerbates the inflammatory response (Devaraj et al., 2009; Amir et al., 2011; Kaur et al., 2012). TLR4 gene silencing offers protection against hyperglycemiainduced cell apoptosis (Zhang et al., 2010). Based on the evidence that the TLR4 pathway plays an important role in the precipitation and recurrence of seizures, post-stroke neuroinflammation and hyperglycemia-induced inflammation response, it is possible that the TLR4 pathway is involved in seizures following ischemia with hyperglycemia. However, there is little information available regarding the role of TLR4 in seizures following cerebral ischemia with hyperglycemia. The present study investigates the potential involvement of TLR4 in the pathogenesis of seizures following cerebral ischemia with hyperglycemia. Furthermore, the changes in the endogenous ligand ofTLR4, HMGB1, were also investigated.

2.

Results

2.1.

Seizure rate and brain damage

More than half of the rats (17/30, 56.7%) in the hyperglycemic group developed tonic-clonic seizures within 12 h after 15 min of ischemia (Fig. 2A). Most seizures (15/17, 88.2%) occurred within 3 h after ischemia. Every animal with seizures died of status epilepticus within 2 h after the onset of seizures. In contrast, no rats in the normoglycemic group developed seizures during the 12-h period after ischemia (Fig. 2A).

Fig. 1 – Fields chosen for TLR4 cell quantification analysis and HMGB1 immunoreactivity quantification analysis. (A) Fields chosen for TLR4 cell quantification analysis; each rectangular frame is 850 μm 640 μm for 200 . (B) Fields chosen for HMGB1 immunoreactivity quantification analysis; each rectangular frame is 425 μm 320 μm for 400 . Please cite this article as: Liang, Y., et al., Toll-like receptor 4 is associated with seizures following ischemia with hyperglycemia. Brain Research (2014), http://dx.doi.org/10.1016/j.brainres.2014.09.020

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Fig. 2 – Seizure rate and H&E staining. (A) The majority rats in the hyperglycemic group developed tonic-clonic seizures after ischemia (56.7% vs. 0% in the control group). (B, C, D) No apparent cell death was observed under H&E staining in any of the groups at 12 h after ischemia.

We used H&E staining to compare the brain damage in the hippocampus at 12 h after ischemia among the different groups. No apparent cell death was observed in any group at 12 h after ischemia (Fig. 2B-D). This result indicates that cell death has a minimal effect on the alterations in TLR4, HMGB1 and c-fos expression.

2.2. TLR4 expression was significantly increased following ischemia with hyperglycemia In the control hippocampus, a few cells were lightly stained with TLR4 staining. The number of TLR4-positive cells were significantly increased 3–12 h after ischemia (At 3 h, Control: CA1, 3.2571.031; CA3, 1.5671.15; and DG, 0.2571.19, n ¼8. Isch: CA1, 19.9571.67; CA3, 24.2873.2; and DG, 24.2574.94, n¼ 6. Isch vs. Control, all Po0.01, Fig. 3). In the hyperglycemic ischemia group, the number ofTLR4-positive cells and the staining intensity were increased, with a peak at 3 h after ischemia relative to the normoglycemic group (At 3 h, IschþHG: CA1, 34.5272.47, Po0.01; CA3, 54.0774.50, Po0.01; and DG, 36.7574.25, n¼7. IschþHG vs. Isch, all Po0.05; Fig. 3). No differences in TLR4 expression were observed between the hyperglycemic ischemia and LPS groups or between the hyperglycemic non-ischemia and control groups (Fig. 3). TLR4 was primarily expressed in the cytoplasm. Based on their location, the TLR4-positive cells were predominately identified as interneurons (Fig. 4). Western blotting analysis was performed to quantify TLR4 protein expression. The CA3 region was selected because it is one of the most susceptible brain regions to seizures. The TLR4 band recognized by the polyclonal anti-TLR4 antibody was at 95 kDa (Fig. 5). Consistent with the immunohistochemical results, TLR4 protein expression in the CA3 region in the hyperglycemic group was significantly increased at 3 h after ischemia compared with the normoglycemic group (IschþHG: 137.1715.4% of control, Isch: 118.97 8.7% of control, n¼ 6, Po0.01) (Fig. 5).

2.3. Expression of HMGB1 in the hippocampus following ischemia with hyperglycemia To further study the TLR4 pathway, the endogenous ligand HMGB1 was examined by immunostaining. As shown in Fig. 3, HMGB1 was localized in the nucleus of neuronal cells

throughout the hippocampus in the control group. HMGB1 expression was increased at 3 h and returned to the control level 6–12 h after ischemia (At 3 h, relative OD of the normoglycemic ischemia group: CA1, 1.27870.063; CA3, 1.38870.105, n¼4, Po0.05 vs. control; DG, 1.11670.065, n¼4, P40.05 vs. control). In contrast, in the hyperglycemic ischemia group, a dramatic decrease of HMGB1 expression was observed 3 h after hyperglycemic ischemia, and the expression then gradually returned to control levels (At 3 h, relative OD: CA1, 0.32070.045; CA3, 0.53370.119; and DG, 0.45370.107; n¼3, all Po0.01 vs. the normoglycemic ischemia group) (Fig. 6). No difference was observed between the hyperglycemia non-ischemia and control groups (data not shown).

2.4. C-fos expression was enhanced after ischemia with hyperglycemia The c-fos protein, a product generated by an immediate-early gene, was used to indicate neural activities. C-fos immunostaining was light in the hippocampus of the control group. Following ischemia, c-fos immunostaining was significantly increased 3–6 h after reperfusion. In the hyperglycemic ischemia group, c-fos immunostaining was further increased 3– 12 h after reperfusion, especially in the dentate granule cells. These data suggested a greater increase of neuronal activities in the hyperglycemic ischemia group than the normoglycemic ischemia and control groups (Fig. 7).

3.

Discussion and conclusions

Hyperglycemia is a common complication in stroke patients. Strokes are common in diabetic patients. Furthermore, hyperglycemia is always occurs induced by an ischemic insult. Hyperglycemia has adverse effects on the outcome of strokes and remarkably increases the onset of seizures following an ischemic insult. Previous studies in animal models have uniformly demonstrated that pre-ischemic hyperglycemia worsens brain damage after ischemia and leads to post-ischemic seizures. Eighteen to 24 h after 10 min of forebrain ischemia, no animal with a plasma glucose levelo13 mM developed seizures, and all animals with a plasma glucose level416 mM died in status epilepticus. Half of the animals with plasma glucose in the range of


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Fig. 3 – Expression of TLR4 in the hippocampus was significantly increased following ischemia with hyperglycemia. (A) TLR4 immunostaining in the CA1 region, the CA3 region and the dentate gyrus in each group at 3 h after ischemia. (B) Change in the number of TLR4-positive cells in the CA1 region, the CA3 region and the dentate gyrus at 3, 6 and 12 h after ischemia in the hyperglycemic and normoglycemic groups. In the control hippocampus, a few cells were lightly stained with TLR4 staining. The number of TLR4-positive cells were significantly increased 3–12 h after ischemia (in the Isch group). In the IschþHG group, the number and staining intensity of TLR4-positive cells were further increased with a peak at 3 h after ischemia compared with the Isch group. No difference in the expression of TLR4 was observed between the IschþHG and LPS groups or between the HG and control groups. The data are presented as the mean7SEM. Statistical analysis was performed using oneway ANOVA. Control: n ¼8. HG (hyperglycemic non-ischemia) group: n ¼ 3. Isch (normoglycemic ischemia) group: 3 h, n ¼6; 6 h, n ¼4; and 12 h, n ¼5. IschþHG (Hyperglycemic ischemia) group: total n ¼16, 9 with seizure; 3 h, n¼ 7; 6 h, n¼ 4; and 12 h, n¼ 5. LPS group: n ¼3. IschþHG group vs. Isch group, ** Po0.01, * Po0.05. IschþHG and Isch groups vs. control or HG groups, all Po0.01.

Fig. 4 – TLR4 expression in the hippocampus after ischemia with hyperglycemia. TLR4 was primarily expressed in the cytoplasm. Based on their location, the TLR4-positive cells were identified predominately as interneurons (A: CA1, B: CA3, C: DG).

13–16 mM developed seizures, and 50% of these animals died (Li et al., 1994). Streptozotocin-induced diabetic animals develop post-ischemic seizures with the same frequency

and results, and post-ischemic brain damage of the same density and distribution was observed in acutely glucoseinfused animals (Li et al., 1998). Clinically, hyperglycemia at


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Fig. 5 – Western blotting of TLR4 in the CA3 region of normoglycemic, hyperglycemic and control groups. Total TLR4 protein levels were significantly increased within the CA3 3 h after ischemia with hyperglycemia. β-actin functioned as a loading control. Blots are representative of 6 independent experiments. The data are presented as the mean7SEM. Statistical analysis was performed using oneway ANOVA. ** Po0.01 vs. control (CTL).

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the time of stroke is known to be an important risk factor with a more severe adverse outcome compared with a chronically stable diabetic state (Sahay and Sahay, 2001). In the present study, hyperglycemia (4 200 mg/dl, approximately 11 mM) was induced via an intraperitoneal injection of glucose (3 g/kg) 15 min prior to ischemia. We determined that most rats (56.7%) in the hyperglycemic group developed tonic-clonic seizures within 12 h after ischemia. Most seizures occurred within 3 h after ischemia. The animals with seizures died of status epilepticus within 2 h after the onset of seizure. No animals in the normoglycemic group developed seizures after ischemia. Our results are consistent with previously discussed studies. We subsequently used H&E staining to compare the brain damage in the CA1 of the hippocampus after ischemia among the different groups for the following reasons: 1. H&E staining is one of the most convenient and direct methods to observe the morphological signs of cell death. 2. The CA1 pyramidal neurons in the dorsal hippocampus are the neurons most

Fig. 6 – HMGB1 immunostaining in the hippocampus was dramatically decreased following ischemia with hyperglycemia. (A) HMGB1 immunostaining in the CA1 region, the CA3 region and dentate gyrus in each group 3 h after ischemia. (B) Changes in HMGB1 immunoreactivity in the CA1 region, the CA3 region and the dentate gyrus at 3, 6 and 12 h after ischemia in the hyperglycemic and normoglycemic groups. HMGB1 expression was increased at 3 h and returned to the control level 6–12 h after ischemia (in the Isch group). In contrast, in the IschþHG group, a dramatic decrease in HMGB1 expression was observed 3 h after hyperglycemic ischemia and gradually returned to control levels. The data are presented as the mean7SEM. Statistical analysis was performed using one-way ANOVA. Control: n ¼ 3, Isch (normoglycemic ischemia) group: 3 h, n ¼ 4; 6 h, n ¼ 4; and 12 h, n ¼ 3. IschþHG (Hyperglycemic ischemia) group: all time points, n ¼3. IschþHG group vs. Ischemia group, ** Po0.01, * Po0.05. IschþHG and Isch groups vs. control, # Po0.05. Please cite this article as: Liang, Y., et al., Toll-like receptor 4 is associated with seizures following ischemia with hyperglycemia. Brain Research (2014), http://dx.doi.org/10.1016/j.brainres.2014.09.020

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Fig. 7 – C-fos immunostaining in the hippocampus was significantly increased 6 h following ischemia with hyperglycemia. Light C-fos immunostaining was observed in the hippocampus of the control group. Following ischemia, c-fos immunostaining was significantly increased. In the hyperglycemic group, c-fos immunostaining was further increased, especially in the dentate granule cells (A: control, B: Isch, C: IschþHG).

vulnerable to ischemia. In the present study, we did not identify apparent cell death in any group at 12 h after ischemia. This result was inconsistent with previous reports that CA1 neurons begin to die approximately two days after 10–15 min of global ischemia and reach maximal cell death in seven days (Pulsinelli). This result indicates that cell death has minimal effects on alterations in TLR4, HMGB1 and c-fos expression. Many potential mechanisms of seizure generation after hyperglycemic ischemia have been proposed and investigated, including tissue acidosis (Katsura et al., 1994; Li et al., 1995), selective damage to GABAergic neurons in the SNPR (Smith et al., 1988; Li et al., 1998), greater brain edema and higher intracranial pressure (Warner et al., 1987; Gisselsson et al., 1992; Morimoto et al., 1996). To our knowledge, the present study is the first study to suggest the involvement of

TLR4 in the occurrence of seizures following ischemia with hyperglycemia. In the present study, a significant increase in TLR4 expression was detected after 15 min of ischemia in the hyperglycemic group compared with the normoglycemia group. Comparing the seizure rates between the normoglycemic and hyperglycemic groups (Fig. 1) strongly suggests that TLR4 is associated with seizures following ischemia with hyperglycemia. Hyperglycemia may activate or reinforce TLR4 expression after cerebral ischemia in two potential mechanisms: 1) Hyperglycemia may directly up-regulate TLR4 expression. In vitro studies have demonstrated that a high concentration of glucose induces TLR4 expression in human monocytes (THP-1) and mouse mesangial cells (MMC) (Dasu et al., 2008; Kaur et al., 2012). In an in vivo study, hyperglycemia exacerbated the inflammatory response via TLR4 in diabetic patients (Devaraj et al., 2009; Amir et al., 2011; Veloso et al., 2011). 2) Hyperglycemia may up-regulate TLR4 expression indirectly. Hyperglycemia may trigger the upstream cascades that can be further augmented by cerebral ischemia. Acute hyperglycemia induced by an intravenous infusion of a glucose solution significantly enhances the concentrations of malondialdehyde (MDA) and HMGB1 (an endogenous ligand of TLR4) in the brain and plasma and a soluble intercellular adhesion molecule-1 (ICAM-1) in the plasma after 10 min of forebrain ischemia (Tsuruta et al., 2010). In the present study, the TLR4 expression level did not increase at 3 h after glucose injection without cerebral ischemia (data not shown), although the levels did increase in hyperglycemic ischemia. This result supports the second possible mechanism, i.e., a transient increase of blood glucose triggers or augments an upstream cascade, such as HMGB1, which leads to increased TLR4 expression with cerebral ischemia. Hyperglycemia may up-regulate TLR4 expression with ischemia. Further studies are needed to clarify this issue. The present study indicated that TLR4 is predominately expressed in the cytoplasm of interneurons in the hippocampus after ischemia. Most interneurons release the inhibitory neurotransmitter γ-amino butyric acid (GABA). Elevated expression of TLR4 in interneurons may attenuate the inhibitive function of interneurons, which leads to the enhanced excitability of pyramidal neurons and subsequently triggers seizure. To evaluate the alteration of neuronal excitability, the level of c-fos protein expression, an indicator of neuronal activity (Kaczmarek and Robertson, 2002), was examined. We found that c-fos immunostaining was significantly increased in the hippocampus within 6 h after ischemia, especially in the hyperglycemic group (even up to 12 h), which suggests that the greater increase in neuronal activities in the hyperglycemic group may be responsible for the seizures. Furthermore, LPS was used as a positive control. LPS is an outer membrane component of Gram-negative bacteria and is generally considered the most common ligand of TLR4 in PAMPs. TLR4 activation by LPS mimics a bacterial infection, decreases the seizure threshold in adult and immature mice and rats and is associated with a chronic increase in hippocampal neuronal network excitability (Sayyah et al., 2003; Galic et al., 2008). Furthermore, a recent report demonstrated that LPS may cause epileptiform activity in rats, with rapid


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onset (Rodgers et al., 2009). In the present study, the quantity and localization pattern of TLR4 in the LPS group was similar to the results in the ischemia with hyperglycemia group. This indicates that ischemic injury after hyperglycemia may trigger upstream cascades that function similarly to LPS and initiate TLR4 and its downstream cascade. We subsequently examined the pathway upstream of TLR4, focusing on HMGB1, based on previous studies. HMGB1 is a ubiquitous, highly conserved DNA-binding protein with well-established functions in the maintenance of nuclear homeostasis (Czura et al., 2001). A substantial amount of the recent work regarding its signaling functions in the brain has focused on its proinflammatory properties and its relationship to known inflammatory receptors, such as TLR4. HMGB1 is a DAMP released by damaged or highly stressed cells and functions as an endogenous ligand of TLR4. HMGB1 is initially expressed in the nucleus of neurons and astrocytes. Following an ischemic insult, HMGB1 predominantly translocates into the cytoplasm of neurons and then rapidly disappears from neurons (Faraco et al., 2007; Kim et al., 2008; Qiu et al., 2008). Massive release of HMGB1 into the extracellular space subsequently induces neuroinflammation in the post-ischemic brain (Kim et al., 2006). Serum HMGB1 levels have been observed to be significantly elevated and correlated with the severity of neurologic impairment in acute cerebral infarct patients (Goldstein et al., 2006). HMGB1 may be released from neural cells into the blood. The downregulation or silencing of HMGB1 with an anti-HMGB1 antibody or HMGB1 siRNA results in neuroprotection in the postischemic brain, indicating that HMGB1 is associated with ischemic brain injury (Zhang et al., 2011; Kim et al., 2012). One study also suggested that HMGB1 mediates ischemiareperfusion injury via TLR4 signaling (Yang et al., 2011). However, a study by Maroso demonstrated that HMGB1 initiates TLR4-mediated effects in acute drug-induced seizures and likely plays a key role in seizure precipitation and recurrence (Maroso et al., 2010). Thus, HMGB1 may also contribute to ischemia-induced epilepsy. In the present study, a dramatic decrease in HMGB1 expression was observed in the hippocampus 3 h after hyperglycemic ischemia, followed by a gradual return to control levels. In the normoglycemic group, an increase of HMGB1 expression was observed at 3 h, followed by a gradual return to control levels at 12 h after ischemia. In the study of Huang, the expression of HMGB1 was significantly decreased at 1 d but higher at 3, 5, and 7 d post-reperfusion after 15 min of global cerebral ischemia (Huang et al., 2011). Thus, we hypothesize that ischemia plus hyperglycemia might induce substantially greater and more rapid HMGB1 release into the extracellular space and into the blood after an ischemic insult compared with ischemia alone. We could not identify the precise timepoint when HMGB1 was released into the cytoplasm. Thus, HMGB1 may activate the TLR4-mediated cascade in seizure generation following ischemia with hyperglycemia. Although lingering questions remain, such as the definitive role of the interaction between the HMGB1-TLR4 axis and inflammatory mediators (such as IL1-β and nuclear factor-κB (NF-κB)) in seizures following ischemia with hyperglycemia, sufficient evidence is available to warrant interventional studies. Because there are multiple known inhibitors of both

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TLR4 and HMGB1, including etitoran and TAK-242, which have completed phase 3 and phase 2 clinical trials, respectively for severe sepsis, HMGB1 and TLR4 may represent attractive targets for the pharmacological treatment of hyperglycemic ischemia-induced seizures.

4.

Experimental procedures

4.1.

Animals

Adult male Wistar rats (12–14 weeks old, weight 200–300 g) were used in this study. The rats were purchased from Charles River (Wilmington, MA) and housed in pairs with a 12 h light/dark cycle. All rats had access to food and water throughout the study. The rats were randomly allocated to five treatment groups: 1. Control group (Control); 2. Hyperglycemic non-ischemia group (HG): The rats were sacrificed 3 h after a intraperitoneal injection of glucose without ischemia; 3.Normoglycemic ischemia group (Isch): The rats were sacrificed 3, 6, or 12 h after ischemia; 4. Hyperglycemic ischemia group (IschþHG): The rats without seizures were sacrificed 3, 6, or 12 h after ischemia, and the rats with seizures within 3, 3–6, and 6–12 h after ischemia were sacrificed immediately after the seizure attack and were subgrouped as 3, 6, or 12 h, respectively, like the non-seizure rats; 5. LPS group (LPS): The rats injected with LPS were used as the positive control group. The rats were sacrificed 3 h after the LPS injection. The experimental protocols were approved by the Institutional Animal Care and Use Committee of Indiana University School of Medicine in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals.

4.2.

Transient global cerebral ischemia

Transient global ischemia was induced using the 4-vessel occlusion method with modifications (Xu et al., 1999). Briefly, on day 1, the animals were anesthetized with 1–2% isoflurane mixed with 33% O2 and 66% N2. An occlusion device (a silicone tube) was placed loosely around each common carotid artery to enable the subsequent occlusion of the vessels. The animals were then placed in a stereotaxic frame, and the vertebral arteries were electrocauterized. On day 2, both common carotid arteries were occluded by the occlusion device while the animals were awake. Following the occlusion of both common carotid arteries, complete ischemia was defined as unresponsiveness and loss of the righting reflex within 1 min; animals that met these conditions underwent the subsequent procedures. After 15 min, the animals were reperfused by releasing the occlusion of both common carotid arteries. The body temperature was maintained at 37 1C during the occlusion via a rectal probe coupled to a heating lamp, which was connected to a temperature control system (Physitemp BAT-10, Clifton, NJ).

4.3.

Hyperglycemia and LPS injection

Hyperglycemia (4 200 mg/dl) was induced via an intraperitoneal injection of glucose (3 g/kg) 15 min prior to ischemia. The LPS group was induced via an intraperitoneal injection of


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20 mg/kg LPS (lipopolysaccharides from Escherichia coli, Sigma, St. Louis, MO).

4.4.

Immunohistochemical staining

The rats were deeply anesthetized and perfused through the ascending aorta with a solution of phosphate-buffered saline (PBS) (0.01 M, pH 7.4) for 5 min, followed by 4% paraformaldehyde in PBS for 20–30 min. The brains were removed and postfixed in 4% paraformaldehyde at 41C overnight. Sets of coronal sections that contained the hippocampus were cut (40 μm) with a vibratome (VT 1000S, Leica, Nussloch, Germany) and collected in PBS. One set of sections was stained with hematoxylin and eosin (H&E, Fisher Scientific, Pittsburgh, PA). All groups were stained together in each immunohistochemical session. After rinsing 3 5 min in PBS, the sections were incubated for 30 min in 0.3% H2O2 diluted in PBS to quench the endogenous peroxidase activity. The sections were subsequently blocked and permeabilized in a permeabilization solution (10% goat serum, 0.1% Triton X-100 in PBS) for 1 h at room temperature. Thereafter, the sections were incubated with a rabbit polyclonal anti-TLR4 antibody (1:200; Santa Cruz Biotechnology, Santa Cruz, CA), rabbit polyclonal anti-HMGB1 antibody (1:5,000; Abcam, Cambridge, MA), or rabbit polyclonal anti-c-fos antibody (1: 20,000; EMD Millipore, Philadelphia, PA) in permeabilization solution overnight at 41C. Following the overnight incubation in the primary antibody, the sections were washed in PBS for 3 10 min and incubated with biotinylated goat anti-rabbit IgG (1:200; Vector Laboratories, Temecula, CA) in a blocking solution (10% goat serum in PBS) for 2 h at room temperature. Following incubation, the sections were washed 3 10 min in PBS and processed according to the avidin–biotin–peroxidase complex method using Vector kits (1:50; Vector Laboratories). After 3 10 min rinses in PBS, the immunoreactivity was visualized using 0.05% diaminobenzidine tetrahydrochloride (DAB; Sigma) in PBS containing 0.015% H2O2 for 1–2 min at room temperature. All sections within the reaction were exposed to DAB for the exact same time. The sections were mounted onto slides, air dried, dehydrated in graded series of ethanol, infiltrated in xylene, and embedded in paraffin. The slides were then examined with a microscope (BX50; Olympus, Tokyo, Japan). The images were acquired with a digital camera coupled to control software (DP70-BSW; Olympus) at 40 , 200 and 400 magnification. The settings were maintained throughout all experiments.

4.5.

TLR4 cell quantification analysis

TLR4 cell quantification in rat brains was performed in the CA1 region, the CA3 region and the dentate gyrus by an investigator who was blinded to the treatments. In each brain, three representative slides were selected. In each slide, two fields in the CA1 region (medial and lateral) and one field in the CA3 region and the dentate gyrus were captured using rectangular frames (850 μm 640 μm for 200 , Fig. 1A) and digitized using a digital camera coupled to control software (DP70-BSW; Olympus).

4.6.

HMGB1 immunoreactivity quantification analysis

HMGB1 immunoreactivity quantification analysis was performed using the software Image J in the CA1 region, the CA3 region and the dentate gyrus by an investigator who was blind to the treatments (n¼ 4 rats per group). In each brain, three representative slides that contained the hippocampus were selected. In each slide, one field in the CA1 and CA3 regions and two fields in the dentate gyrus (upper band and the lower band) of interest were captured using rectangular frames (425 μm 320 μm for 400 , Fig. 1B) and digitized using a digital camera coupled to control software (DP70-BSW; Olympus). To analyze the optical density (OD) of HMGB1 immunostaining, the images were captured in black and white 8-bit grayscale under 400 using a CCD camera. The optical density was measured in a fixed region (3500 μm2) across all images. The density in the neuropile regions without positive staining (625 μm2) in the same slide served as the background. The OD was obtained by subtracting the background value from the measuring value. The relative OD (experiment group OD/control OD) was used for the normoglycemic ischemia and hyperglycemic ischemia groups.

4.7.

Western blotting

Brain slices were prepared using procedures similar to those previously described (Lei et al., 2012). Briefly, the animals were anesthetized with ketamine-HCl (80 mg/kg, intraperitoneally) and decapitated. The brains were quickly removed and immersed in ice-cold artificial cerebrospinal fluid (ACSF) that contained the following: 130 mM NaCl, 3 mM KCl, 2 mM CaCl2, 2 mM MgCl2, 1.25 mM NaH2PO4, 26 mM NaHCO3, and 10 mM glucose (pH 7.4, 295–305 mOsm/L). Transverse 400-μmthick hippocampus slices were cut using a vibratome (VT 1000; Leica, Nussloch, Germany). The regions of CA3 were subsequently microdissected under a surgical microscope (Bausch & Lomb, Rochester, NY) and frozen in liquid nitrogen. The tissues were lysed with an ice-cold radioimmunoprecipitation assay (RIPA) buffer (50 mM Tris, pH 7.4, 150 mM NaCl, 1% NP-40, 0.5% sodium deoxycholate, 0.1% sodium dodecyl sulfate [SDS]; Boston BioProducts, Worcester, MA), supplemented with a protease inhibitor cocktail (Roche, Indianapolis, IN) and incubated an additional 30 min on ice. After brief sonication on ice, the cell lysates were centrifuged at 12,000 x g for 20 min at 41C to pellet nuclei and debris, and the resulting supernatants were collected for analysis. The protein concentration was determined by BCA protein assay (Bio-Rad, Hercules, CA). The protein samples were boiled in 2 SDS gel-loading buffer (Invitrogen, Carlsbad, CA) prior to SDS-polyacrylamide gel electrophoresis (SDS-PAGE). The proteins (20 μg) were separated on 10% SDS-PAGE gels and transferred to nitrocellulose membranes (Millipore, Bedford, MA). The membranes were rinsed with distilled water, blocked with 1% bovine serum albumin (BSA; Sigma) in TBS 0.1% Tween 20 (TBST) for 1 h, and then incubated with primary antibodies overnight in blocking buffer at 41C. We used rabbit polyclonal anti-TLR4 (1:1,000, Santa Cruz Biotechnology). The membranes were washed with TBST and incubated at room temperature for 1 h with horseradish peroxidase (HRP)-conjugated anti-rabbit (1:10,000; Chemicon).


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Bands were detected using enhanced chemiluminescence (ECL; Amersham, Piscataway, NJ) and visualized by exposing the membrane to X-ray films (Fuji, Tokyo, Japan). Band densitometry analysis of the membrane was performed using scanned images of unsaturated immunoblot films and the NIH ImageJ 1.37 analysis software. The western analyses were performed as described in our previous studies (Lei et al., 2008; Lei et al., 2010). The following protocol was used for the experimental treatments. Each gel that we used in the experiments had 10 wells. The first well was loaded with protein marker. The remaining nine wells were loaded in sequence with control (sample 1), normoglycemic ischemia (sample 1), hyperglycemic ischemia (sample 1), control (sample 2), normoglycemic ischemia (sample 2), hyperglycemic ischemia (sample 2), control (sample 3), normoglycemic ischemia (sample 3), and hyperglycemic ischemia (sample 3). It was not possible to analyze all six different samples of each group on one western blot; therefore, we analyzed the samples using two blots (three samples per blot). We normalized all western signals of TLR4 to β-actin and subsequently expressed all values as the “percent of control” using the controls from a specific blot to normalize only those signals from the same blot. This approach provided us with a series of “percent of control” values for the control, normoglycemic ischemia, and hyperglycemic ischemia groups.

4.8.

Data analysis

The values are presented as the means7SEM. The results were analyzed using one-way ANOVA followed by post-hoc Fisher PLSD test (StatView 5.0; Abacus Concepts, Berkeley, CA). The changes were considered significant if Po0.05.

Acknowledgments The work was supported by grants from the NIH (NS071238), the AHA (GRNT4500000, 13SDG17140056) and the Guangdong Provincial Department of Science and Technology (2009B030801352).

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Hyperglycemia presenting with occipital seizures.

Magnetic resonance imaging features in seizures associated with nonketotic hyperglycemia.

Pulmonary edema following generalized tonic clonic seizures is directly associated with seizure duration.

Occipital lobe seizures and subcortical T2 and T2* hypointensity associated with nonketotic hyperglycemia: a case report.

Admission hyperglycemia is associated with failed reperfusion following fibrinolytic therapy in patients with STEMI: results of a retrospective study.

Chorea-Ballismus Associated with Hyperglycemia.

Seizures associated with meningitis.

MicroRNA-29b is a therapeutic target in cerebral ischemia associated with aquaporin 4.

reperfusion in mice is associated with altered microRNA expression.

Nonketotic hyperglycemia-related epileptic seizures.

Recurrent seizures following focal motor status epilepticus in a patient with non-ketotic hyperglycemia and acute cerebral infarction.

Chorea-ballism associated with ketotic hyperglycemia.

Acute Hyperglycemia Associated with Anti-Cancer Medication.

Hyperglycemia is associated with lower levels of urokinase-type plasminogen activator and urokinase-type plasminogen activator receptor in wound fluid.

4) is associated with severe asthma.

Toll-like receptor 4 gene polymorphism is associated with chronic periodontitis.

Fibroblast growth factor receptor 4 polymorphism is associated with liver cirrhosis in hepatocarcinoma.

A toll-like receptor 4 (TLR4) variant is associated with asthma severity.

Serum ischemia-modified albumin levels in children with epileptic seizures.

Gaze-evoked visual seizures in nonketotic hyperglycemia.

Treatment of serous retinal detachment associated with choroidal ischemia with intravitreal bevacizumab following brain surgery.

SLC7A11 expression is associated with seizures and predicts poor survival in patients with malignant glioma.

IDH1 mutation is associated with seizures and protoplasmic subtype in patients with low-grade gliomas.

Seizures associated with Lupus during pregnancy.