Hong-Liu Sun

Low-frequency stimulation of the hippocampal CA3 subfield is anti-epileptogenic and anti-ictogenic in rat amygdaloid kindling model of epilepsy.

Neuromodulation with low-frequency stimulation (LFS), of brain structures other than epileptic foci, is effective in inhibiting seizures in animals and patients, whereas selection of targets for LFS requires further investigation. The hippocampal CA(3) subfield is a key site in the circuit of seizure generation and propagation. The present study aimed to illustrate the effects of LFS of the CA(3) region on seizure acquisition and generalization in the rat amygdaloid kindling model of epilepsy. We found that LFS (monophasic square-wave pulses, 1Hz, 100 microA and 0.1ms per pulse) of the CA(3) region significantly depressed the duration of epileptiform activity and seizure acquisition by retarding progression from focal to generalized seizures (GS). Moreover, GS duration was significantly shortened and its latency was significantly increased in the LFS group demonstrating an inhibition of the severity of GS and the spread of epileptiform activity. Furthermore, LFS prevented the decline of afterdischarge threshold (ADT) and elevated GS threshold indicating an inhibition of susceptibility to GS. These results suggest that LFS of the hippocampal CA(3) subfield is anti-epileptogenic and anti-ictogenic. Neuromodulation of CA(3) activity using LFS may be an alternative potential approach for temporal lobe epilepsy treatment.

Time-dependent effect of low-frequency stimulation on amygdaloid-kindling seizures in rats

Low-frequency stimulation (LFS) has been considered as a new option for the treatment of intractable epilepsy. The present study was designed to determine whether LFS of the kindling focus given at different time points after seizures exert different roles on kindling seizures. Our results showed that: (i) In kindling animals, LFS delivered immediately after cessation of the kindling stimulus inhibited the seizure stage during kindling acquisition, whereas LFS delivered after the cessation of afterdischarge accelerated the kindling progression to stages 1 and 2. (ii) In fully kindled animals, when using the generalized seizure threshold current as the kindling stimulus, immediate LFS decreased the incidence of generalized seizures and the average seizure stage as well as shortened the cumulative generalized seizure duration (GSD). However, delayed LFS prolonged the cumulative GSD and afterdischarge duration. Our study indicates that there is a time-dependent aspect of LFS treatment, and immediate LFS has anti-epileptogenic action.

Therapeutic time window of low‐frequency stimulation at entorhinal cortex for amygdaloid‐kindling seizures in rats

The present study was designed to determine whether low‐frequency stimulation (LFS) of the entorhinal cortex (EC) has an anticonvulsive effect, and whether LFS delivered at different times plays different roles. We found that LFS of the EC immediately or 4 s after kindling stimulation had an anticonvulsive effect, and that the latter had a better effect on both kindling and kindled seizures. However, LFS delivered after the cessation of afterdischarge or 10 s after the kindling stimulation, augmented the epileptic activity. So the EC is a potential target for LFS to interfere with epilepsy. Our findings suggest that even in the duration of afterdischarge, there exists a “time window” for LFS treatment, indicating that the time delay of closed‐loop stimulation is crucial for LFS treatment.

Celastrol suppresses obesity process via increasing antioxidant capacity and improving lipid metabolism.

High fat diet, as an important risk factor, plays a pivotal role in atherosclerotic process. Celastrol is one of the active triterpenoid compounds with antioxidative and anti-inflammatory characters. The aims of this study were to evaluate the effect of celastrol on weight, blood lipid and oxidative injury induced by high fat emulsion, and investigate its potential pharmacological mechanisms. Male Sprague-Dawley rats were fed with high fat emulsion for 6 wk to mimic high fat mediated oxidative injury. The effects of celastrol on weight and blood lipid were evaluated, and its mechanisms were disclosed by applying western blot, ELISA and assay kits. Long-term consumption of high fat emulsion could significantly increase weight by enhancing total cholesterol (TC), triacylglycerol (TG), apolipoprotein B (Apo B), low-density lipoprotein cholesterol (LDL-c) levels, attenuating ATP-binding cassette transporter A1 (ABCA1) expression, and decreasing the levels of high-density lipoprotein cholesterol (HDL-c) and apolipoprotein A-I (Apo A-I), and inhibit antioxidant enzymes activities, improve nicotinamide adenine dinucleotide phosphate (NADPH) oxidase activity. Comparing with model group, celastrol was able to effectively suppress weight and attenuate high fat mediated oxidative injury by improving ABCA1 expression, reducing the levels of TC, TG, LDL-c and Apo B in plasma, and increasing antioxidant enzymes activities and inhibiting NADPH oxidase activity, and decreasing the serum levels of Malondialdehyde (MDA) and reactive oxygen species in dose-dependent way. These data demonstrated that celastrol was able to effectively suppress weight and alleviate high-fat mediated cardiovascular injury via mitigating oxidative stress and improving lipid metabolism.

Mode-dependent effect of low-frequency stimulation targeting the hippocampal CA3 subfield on amygdala-kindled seizures in rats

Brain stimulation with low-frequency stimulation (LFS) is emerging as an alternative treatment for refractory epilepsy. The present study aimed to investigate the effects of LFS targeting the hippocampal CA3 subfield in different modes on amygdala-kindled seizures in Sprague-Dawley rats. When fully kindled seizures were achieved by daily electrical stimulation of the amygdala, LFS (15 min train of 0.1 ms pulses at 1 Hz and 100 microA) of the CA3 was applied in several modes. Post-treatment with LFS significantly reduced the severity of and susceptibility to evoked seizures, whereas pre-treatment with LFS resulted in a similar but much weaker inhibition of seizures. Interestingly, prior consecutive daily application of LFS in the absence of kindling stimulation did not reduce subsequent evoked seizures, but abolished the anti-epileptic effect of post-treatment. These results indicated that LFS of the CA3 is able to reduce kindled seizures in a mode-dependent manner without cumulative feature. The hippocampal CA3 subfield could be considered as a potential target for epilepsy treatment using LFS, and should be delivered in an appropriate stimulation mode.

A Transient Upregulation of Glutamine Synthetase in the Dentate Gyrus Is Involved in Epileptogenesis Induced by Amygdala Kindling in the Rat

Reduction of glutamine synthetase (GS) function is closely related to established epilepsy, but little is known regarding its role in epileptogenesis. The present study aimed to elucidate the functional changes of GS in the brain and its involvement in epileptogenesis using the amygdala kindling model of epilepsy induced by daily electrical stimulation of basolateral amygdala in rats. Both expression and activity of GS in the ipsilateral dentate gyrus (DG) were upregulated when kindled seizures progressed to stage 4. A single dose of L-methionine sulfoximine (MSO, in 2 µl), a selective GS inhibitor, was administered into the ipsilateral DG on the third day following the first stage 3 seizure (just before GS was upregulated). It was found that low doses of MSO (5 or 10 µg) significantly and dose-dependently reduced the severity of and susceptibility to evoked seizures, whereas MSO at a high dose (20 µg) aggravated kindled seizures. In animals that seizure acquisition had been successfully suppressed with 10 µg MSO, GS upregulation reoccurred when seizures re-progressed to stage 4 and re-administration of 10 µg MSO consistently reduced the seizures. GLN at a dose of 1.5 µg abolished the alleviative effect of 10 µg MSO and deleterious effect of 20 µg MSO on kindled seizures. Moreover, appropriate artificial microRNA interference (1 and 1.5×106 TU/2 µl) of GS expression in the ipsilateral DG also inhibited seizure progression. In addition, a transient increase of GS expression and activity in the cortex was also observed during epileptogenesis evoked by pentylenetetrazole kindling. These results strongly suggest that a transient and region-specific upregulation of GS function occurs when epilepsy develops into a certain stage and eventually promotes the process of epileptogenesis. Inhibition of GS to an adequate degree and at an appropriate timing may be a potential therapeutic approach to interrupting epileptogenesis.

Hydroxysafflor yellow A of Carthamus tinctorius attenuates lung injury of aged rats exposed to gasoline engine exhaust by down-regulating platelet activation.

Long-term inhalation of gasoline engine exhaust (GEE) increases the risk of respiratory disease. Studies have suggested involvement of platelets in the development of some lung diseases. Hydroxysafflor yellow A (HSYA), a flavonoid compound, prevents hemostasis. Therefore, we investigated its effects on GEE-induced lung injury, and role of platelets in injury. Sixty-week-old male Sprague-Dawley rats were exposed to GEE for 4h/day for 6 weeks, and then grouped as follows: control, GEE, GEE+HSYA, GEE+HSYA+GW9662, and GEE+GW9662. Arterial oxygen tension (PaO2), carbon dioxide tension (PaCO2), pH, and the PaO2/fraction of inspired oxygen ratio (PaO2/FiO2) in the blood were detected using a blood gas analyzer. Wet/dry lung weight ratio, total protein in bronchoalveolar lavage fluid (BALF), and cytokine concentrations in serum and BALF were determined. Furthermore, cyclic adenosine monophosphate (cAMP) level and expression levels of target proteins were analyzed. Platelets were counted and their state was evaluated. HSYA attenuated GEE-mediated decreases in PaO2, PaO2/FiO2, platelet cAMP level, protein kinase A (PKA) activity, and peroxisome proliferator-activated receptor γ (PPARγ) expression. HSYA also attenuated GEE-mediated increases in lung permeability, cytokine levels in serum and BALF, plasma platelet count, and ADP-mediated platelet aggregation. Moreover, it suppressed GEE-induced increases in the expression of adhesion molecules and proinflammatory cytokines in platelets and lung tissue. Therefore, HSYA is therapeutically effective for GEE-mediated lung injury and acts by enhancing PKA activity and inhibiting platelet activation.

Safflor yellow B suppresses angiotensin II-mediated human umbilical vein cell injury via regulation of Bcl-2/p22phox expression

Intracellular reactive oxygen species (ROS) are derived from nicotinamide adenine dinucleotide phosphate (NADPH) oxidase. Angiotensin II (Ang II) can cause endothelial dysfunction by promoting intracellular ROS generation. Safflor yellow B (SYB) effectively inhibits ROS generation by upregulating Bcl-2 expression. In this study, we examined the effects of SYB on Ang II-induced injury to human umbilical vein endothelial cells (HUVECs), and elucidated the roles of NADPH oxidase and Bcl-2. We treated cultured HUVECs with Ang II, SYB, and Bcl-2 siRNA, and determined NADPH oxidase activity and ROS levels. Furthermore, cellular and mitochondrial physiological states were evaluated, and the expression levels of target proteins were analyzed. Ang II significantly enhanced intracellular ROS levels, caused mitochondrial membrane dysfunction, and decreased cell viability, leading to apoptosis. This was associated with increased expression of AT1R and p22(phox), increased NADPH oxidase activity, and an increased ratio of Bax/Bcl-2, leading to decreases in antioxidant enzyme activities, which were further strengthened after blocking Bcl-2. Compared to Ang II treatment alone, co-treatment with SYB significantly reversed HUVEC injury. Taken together, these results demonstrate that SYB could significantly protect endothelial cells from Ang II-induced cell damage, and that it does so by upregulating Bcl-2 expression and inhibiting ROS generation.

Reactive astrocytes contribute to increased epileptic susceptibility induced by subthreshold dose of pilocarpine

Seizures may influence epileptogenesis, but it is not yet clearly established whether subthreshold stimulations that are not sufficient to induce visible behavioral seizures change epileptic susceptibility, and the possible underlying mechanisms have not been completely understood. We assessed the susceptibility to epilepsy after subthreshold dose of pilocarpine, as well as glial fibrillary acidic protein (GFAP) expression using immunohistochemistry. An increase in the susceptibility to pentylenetetrazole (PTZ)-induced seizures was observed in rats previously subjected to subthreshold dose of pilocarpine. The immunoreactivity of GFAP was also increased, indicating that astrocytes became reactive in some brain subfields. The increased epileptic susceptibility was significantly reduced by L-alpha-aminoadipic acid (L-AAA), an inhibitor of astrocytic function. Our results suggest that subthreshold stimulation may increase the susceptibility to subsequent development of epilepsy, and reactive astrocytes might be an important contributor to this process. Adequate inhibition of astrocytic function may be a potential preventive approach against epileptogenesis.

Altered glutamate metabolism contributes to antiepileptogenic effects in the progression from focal seizure to generalized seizure by low-frequency stimulation in the ventral hippocampus

As a promising method for treating intractable epilepsy, the inhibitory effect of low-frequency stimulation (LFS) is well known, although its mechanisms remain unclear. Excessive levels of cerebral glutamate are considered a crucial factor for epilepsy. Therefore, we designed experiments to investigate the crucial parts of the glutamate cycle. We evaluated glutamine synthetase (GS, metabolizes glutamate), glutaminase (synthesizes glutamate), and glutamic acid decarboxylase (GAD, a γ-aminobutyric acid [GABA] synthetase) in different regions of the brain, including the dentate gyrus (DG), CA3, and CA1 subregions of the hippocampus, and the cortex, using western blots, immunohistochemistry, and enzyme activity assays. Additionally, the concentrations of glutamate, GABA, and glutamine (a product of GS) were measured using high-performance liquid chromatography (HPLC) in the same subregions. The results indicated that a transiently promoted glutamate cycle was closely involved in the progression from focal to generalized seizure. Low-frequency stimulation (LFS) delivered to the ventral hippocampus had an antiepileptogenic effect in rats exposed to amygdaloid-kindling stimulation. Simultaneously, LFS could partly reverse the effects of the promoted glutamate cycle, including increased GS function, accelerated glutamate-glutamine cycling, and an unbalanced glutamate/GABA ratio, all of which were induced by amygdaloid kindling in the DG when seizures progressed to stage 4. Moreover, glutamine treatment reversed the antiepileptic effect of LFS with regard to both epileptic severity and susceptibility. Our results suggest that the effects of LFS on the glutamate cycle may contribute to the antiepileptogenic role of LFS in the progression from focal to generalized seizure.