Role of microRNA‐155 in modifying neuroinflammation and γ‐aminobutyric acid transporters in specific central regions after post‐ischaemic seizures

Abstract In the central nervous system, interleukin (IL)‐1β, IL‐6 and tumour necrosis factor (TNF)‐α have a regulatory role in pathophysiological processes of epilepsy. In addition, γ‐aminobutyric acid (GABA) transporter type 1 and type 3 (GAT‐1 and GAT‐3) modulate the levels of extracellular GABA in involvement in the neuroinflammation on epileptogenesis. Thus, in the current report we examined the effects of inhibiting microRNA‐155 (miR‐155) on the levels of IL‐1β, IL‐6 and TNF‐α, and expression of GAT‐1 and GAT‐3 in the parietal cortex, hippocampus and amygdala of rats with nonconvulsive seizure (NCS) following cerebral ischaemia. Real time RT‐PCR, ELISA and Western blot analysis were used to examine the miR‐155, proinflammatory cytokines (PICs) and GAT‐1/GAT‐3 respectively. With induction of NCS, the levels of miR‐155 were amplified in the parietal cortex, hippocampus and amygdala and this was accompanied with increases of IL‐1β, IL‐6 and TNF‐α. In those central areas, expression of GAT‐1 and GAT‐3 was upregulated; and GABA was reduced in rats following NCS. Intracerebroventricular infusion of miR‐155 inhibitor attenuated the elevation of PICs, amplification of GAT‐1 and GAT‐3 and impairment of GABA. Furthermore, inhibition of miR‐155 decreased the number of NCS events following cerebral ischaemia. Inhibition of miR‐155 further improved post‐ischaemia‐evoked NCS by altering neuroinflammation‐GABA signal pathways in the parietal cortex, hippocampus and amygdala. Results suggest the role of miR‐155 in regulating post‐ischaemic seizures via PICs‐GABA mechanisms.

Nevertheless, the underlying mechanisms leading to provoked postischaemic seizures remain to be determined.
Two main subtypes of γ-aminobutyric acid (GABA) transports (GATs), namely GAT-1 and GAT-3, are responsible for the control of central extracellular GABA levels. 5,6 In the central nervous system (CNS), these transporters appear in neuronal cells (predominantly GAT-1) and glial cells (predominantly , and the prior reports have revealed the role of GATs in modifying GABA receptor-mediated postsynaptic tonic and phasic inhibition in the cerebral cortex, hippocampus, etc. 5,6 It is well known that imbalanced inhibitory (GABA) and excitatory (glutamate) synaptic neurotransmissions are associated with adjustment of ion channel activity in contribution to regulation of brain functions. [7][8][9][10] In order to determine the basic role of central GABAergic transmission in the process of epileptic activity following cerebral ischaemia, 11,12 in the current study, we performed  13,14 This result is in agreement with the effects of PICs on the pathophysiological responses of epilepsy and/ or seizure-induced cerebral damages. Additionally, a prior study has revealed that the protein levels of IL-1β, IL-6 and TNF-α in the parietal cortex, hippocampus and amygdala are considerably elevated during epilepsy evoked by cerebral injection of kainic acid. 15 Results of this prior study also indicate that GAT-1 and GAT-3 are upregulated and epilepsy-increased IL-1β, IL-6 and TNF-α results in enhanced GAT-1 and GAT-3 in those specific brain regions. 15 Thus, in the current study, we postulated that elevation of IL-1β, IL-6 and TNF-α is accompanied with the greater levels of GAT-1 and GAT-3 in the parietal cortex, hippocampus and amygdala of NCS rats after cerebral ischaemia.
MicroRNAs (miRNAs) are small noncoding endogenous RNA molecules that can alter their target mRNA through binding in the message 3′-UTR. 16 MicroRNAs have been shown to have important contributions to multiple pathophysiological processes: cellular death and survival, cellular response to stress, stem cell division, pluripotency, etc. 17 MicroRNAs also play a role in regulating disease processes including cancer, cardiovascular and neurological diseases. [18][19][20] As a result of their small size, relative ease of delivery and sequence specificity in recognizing their targets, miRNAs have been considered as promising therapeutic targets with respect to drug development. 21 Among various miRNAs, microRNA-155 (miR-155) plays a role in various physiological and pathological processes. [22][23][24][25] is involved in chronic immune response by amplifying the proliferative response of T cells via the downregulation of lymphocyte-associated antigens. 26 In autoimmune disorders, a higher expression of miR-155 is observed in patients' tissues and synovial fibroblasts. 24 In multiple sclerosis, upregulation of miR-155 has been observed in peripheral nerve and CNS-resident myeloid cells, blood monocytes in the circulation and stimulated microglia. 27 Also, a prior study has suggested that miR-155 is involved in inflammation and upregulation of miR-155 results in chronic inflammation in human beings. 25 Nonetheless, it remains unknown for the role of miR-155 in engagement of post-ischaemic seizures. Therefore, in the current study, we determined the modulating effects of miR-155 on IL-1β, IL-6 and TNF-α and GAT-1 and GAT-3 expression along with GABA concentrations in the parietal cortex, hippocampus and amygdala of rats after induction of cerebral ischaemia. We hypothesized that inhibition of miR-155 decreases ischaemia-activated PICs, and this attenuates upregulation of GABA transporters and thereby stabilizes GABA in the parietal cortex, hippocampus and amygdala. We also hypothesized that inhibition of miRNA-155 improves NCS following MCAO through PIC-GABA mechanisms.

| Animal
All animal experimental procedures were performed in accordance with the guidelines of the International Association for the Study of

| Lateral ventricle cannulation and administration of miR-155 inhibitor
The rats were anaesthetized by using sodium pentobarbital (45 mg/ kg bodyweight, ip) and after this they were immobilized in a stereotaxic apparatus (David Kopf, USA). Midline incision was made and the skull was exposed and one burr hole was drilled. Following this procedure, cannulation was made in the lateral ventricle with an L-shaped stainless steel in rats (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 secured to the skull using dental zinc cement. 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 the pumps were loaded with miR-155 inhibitor (5′AAU UAC GAU UAG CAC UAU CCC CA-3′) and its corresponding scramble for negative controls (5 μg in artificial cerebrospinal fluid, Biomics Biotech, Nantong, China) respectively. The inhibitor and scramble were delivered for a period of 24 hours at a rate at 0.25 μL per hour.
This intervention allowed us to give continuously drugs via intracerebroventricular (ICV) infusion.

| A model of MCAO and experimental groups
One day after ICV infusion of miR-155 inhibitor and its scramble, NCS was induced by the MCAO. Sodium pentobarbital (45 mg/kg bodyweight, ip) was used to anaesthetize the rats, the right common carotid artery was exposed at the level of external and internal carotid artery bifurcation. The external carotid artery and its branches were closed and cut at the lingual and maxillary artery branches.
Through the stump of the external carotid artery, nylon suture (3-0 monofilament) was inserted into the internal carotid artery. The filament was advanced for ~20 mm into the anterior cerebral artery.
During this procedure, a laser-doppler flowmeter was used to continuously monitor cortical cerebral blood flow. Once a constant decrease in blood flow was seen at ipsilateral side to the occlusion, the filament was secured to the vessel by ligation. Generally, the animals showing a reduction in cerebral blood flow of >70% are considered ischaemia and only those rats were included for data analysis in this report. In sham control rats, the same surgical procedures were conducted without arterial occlusion.
During 24 hours following MCAO, electroencephalogram (EEG) data were collected. 28 Through burr holes over bilateral frontal and parietal regions of the cortex four electrodes were implanted on skull. A reference electrode was implanted posterior to lambda over the transverse sinus. The electrodes were connected to a multi-pin connector. EEG data were obtained via a Grass polygraph amplifier and digitizing system (Grass Lab). The NCSs were examined via stainless steel electrodes implanted on the skull of rats. The off-line EEG traces were analyzed according to NCS criteria. 28 The number of NCS events over time was considered as the frequency of NCS in each animal.
At the end of recordings the brains were taken out and the levels of PICs, expression of GAT-1 and GAT-3 in the parietal cortex, hippocampus and amygdala were assessed. Accordingly, the rats were included in sham control group (n = 12) and MCAO group with scramble (n = 15) and MCAO group with miR-155 inhibitor (n = 15).
In addition groups, 25 rats were used to examine time course of miR-155 changes after induction of MCAO.

| Real-time PCR
The brain was taken out and the tissues of the parietal cortex, hippocampus and amygdala were obtained under an anatomical microscope. The tissues were processed for the extraction of total RNA

| ELISA measurement
All the tissues from individual rats were sampled for the analysis. In brief, the parietal cortex, hippocampus and amygdala were removed.
Total protein was then extracted by homogenizing the hippocampus sample in ice-cold immunoprecipitation 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.
Interleukin-1β, IL-6 and TNF-α were determined by using a two-site immunoenzymatic assay (Wuhan Fine Biotech Co). After this, the plates were washed and incubated with substrate solution. After an incubation of 2 hours, the optical density was determined using an ELISA reader with wavelength of 575 nm.
Likewise, the levels of GABA were determined by the ELISA methods. with an alkaline phosphatase (at 1:1000), and examined by enhanced chemiluminescence. Then the membrane was exposed onto an x-ray film to examine the recognized bands of the primary antibodies. After this, the film was scanned and the optical density of GAT-1/GAT-3 bands was determined by using the NIH Scion Image software.

| Statistical analysis
One-way measures analysis of variance was employed for comparison, followed by Tukey's post hoc test as appropriate. All data were shown as mean ± SEM. For all analyses, statistical significance was set at P < 0.05. All statistical analyses were performed by using spss for Windows (version 13, SPSS).

| The levels of miR-155 after MCAO
In this study, we first examined the time course for changes of miR-155 after MCAO. Figure 1 Figure 3B reveals that the levels of GABA were less in the parietal cortex, hippocampus and amygdala of MCAO rats (P < 0.05 vs control rats; n = 15 in MCAO group; and n = 12 in control group) in comparison with control group. As miR-155 inhibitor was infused in MCAO rats (n = 15), downregulation of GABA levels was largely restored (P < 0.05 vs rats with ICV miR-155 scramble).

| Incidence of NCS
Nonconvulsive seizure was not observed in control rats (n = 12). Rats

| D ISCUSS I ON
In the current study, we showed that inhibition of miR-155 decreases Increases of PICs in the CNS are related to seizure vulnerability and seizure-induced pathological changes, [30][31][32] indicating that neuroinflammation is engaged in the process of epileptogenesis. In epilepsy, neural injuries in the CNS are observed in rats and this is augmented by upregulation of PICs (IL-1β, IL-6 and TNF-α) in glial cells. 29,33 Additionally, neuronal degeneration is detected as a result of increases of PICs. 34,35 For example, IL-1β increases seizure susceptibility in rat brains. 36 Intracerebral injection of IL-1β leads to limbic seizures in wild-type mice, but not in transgenic mice with deficient IL-1β receptors. 37 Furthermore, after thalidomide is used In conclusion, our evidence shows that cerebral ischaemia increases incidence of NCS, and during this process, IL-1β, IL-6 and TNF-α are amplified in the parietal cortex, hippocampus and amygdala. Cerebral ischaemia further regulates GAT-1 and GAT-3 and thus reduces GABA in those central areas. These abnormalities augment neuronal excitability in the CNS of animals. Notably, ICV administration of miR-155 reduces PICs, attenuates expression of GAT-1 and GAT-3 and alleviates impairment of GABA. This also alleviates NCS incidence induced by cerebral ischaemia. Data may provide evidence for treatment of symptoms in patients with epilepsy after cerebral ischaemia.