ceRNA network of lncRNA MIR210HG/miR-377-3p/LMX1A in malignant proliferation of glioma cells.

BACKGROUND: Glioma represents the most heterogeneous and malignant form of brain tumor with a poor prognosis. The long non-coding RNA (LncRNA)-mediated competing endogenous RNA (ceRNA) network plays a regulatory role in cancer progression. OBJECTIVES: The present study was conducted to expound on the role of lncRNA MIR210 host gene (MIR210HG)-mediated ceRNA mechanism in the malignant proliferation of glioma cells and provide a novel theoretical basis for the treatment of glioma. METHODS: Expression levels of lncRNA MIR210HG, microRNA (miR)-377-3p, and LIM homeobox transcription factor 1 alpha (LMX1A) in glioma tissues and cells were determined by reverse-transcription quantitative polymerase chain reaction. Then, cell proliferation was assessed by cell counting kit-8 and colony formation assays. After that, the subcellular localization of lncRNA MIR210HG was analyzed by subcellular fractionation assay and the bindings of miR-377-3p to lncRNA MIR210HG and LMX1A were analyzed by the dual-luciferase assay. Glioma cells were transfected with si-MIR210HG, miR-377-3p inhibitor, or overexpressed-LMX1A vectors to evaluate their effects on the malignant proliferation of glioma cells. RESULTS: LncRNA MIR210HG was elevated in glioma tissues and cells and inhibition of lncRNA MIR210HG reduced the proliferation potential of glioma cells. LncRNA MIR210HG targeted and inhibited miR-377-3p and miR-377-3p targeted and inhibited LMX1A transcription. miR-377-3p downregulation or LMX1A overexpression reversed the inhibition of silencing lncRNA MIR210HG on glioma cell proliferation. CONCLUSION: LncRNA MIR210HG was upregulated in glioma tissues and cells and inhibition of lncRNA MIR210HG suppressed glioma cell proliferation through promoting miR-377-3p and repressing LMX1A.

The role of Toll-like receptor 4 in apoptosis of brain tissue after induction of intracerebral hemorrhage.

BACKGROUND: Inflammation and apoptosis caused by intracerebral hemorrhage (ICH) are two important factors that affect patient prognosis and survival. Toll-like receptor 4 (TLR4) triggers activation of the inflammatory pathway, causing synthesis and release of inflammatory factors. The inflammatory environment also causes neuronal apoptosis. However, no studies have reported the role of TLR4 in inflammation and apoptosis. METHODS: We performed survival curve analysis and behavioral scores on TLR4 knockout mice and wild-type mice after inducing ICH. We used TLR4 knockout mice and wild-type mice to make ICH models with type VII collagenase and explored the link between TLR4 in inflammation and apoptosis. We used Western blot to detect the expression of apoptosis-related proteins, inflammatory factors, and their receptors at different time points after ICH induction. The effects of TLR4 on apoptosis were observed by TUNEL, Hoechst, and HE staining techniques. The association with TLR4 in inflammation and apoptosis was explored using IL-1ß and TNF-α antagonists. Data conforming to a normal distribution are expressed as mean ± standard deviation. Grade and quantitative data were compared with rank sum test and t test between two groups. P < 0.05 was considered statistically significant. RESULTS: TLR4 knockout significantly increased the survival rate of ICH mice. The scores of TLR4 knockout mice were significantly lower than those of wild-type mice. We found that TLR4 knockout mice significantly inhibited apoptosis and the expression of inflammatory factors after the induction of ICH. The apoptosis of ICH-induced mice was significantly improved after injecting IL-1ß and TNF-α antagonists. Moreover, the anti-apoptotic effect of the antagonist in wild-type mice is more pronounced. A single injection of the antagonist failed to improve apoptosis in TLR4 knockout mice. CONCLUSIONS: We conclude that TLR4-induced inflammation after ICH promotes neuronal apoptosis. IL-1ß and TNF-α antagonists attenuate this apoptotic effect. Therefore, targeting TLR4 in patients with clinical ICH may attenuate inflammatory response, thereby attenuating apoptosis and improving prognosis.

AKR1B10 (Aldo-keto reductase family 1 B10) promotes brain metastasis of lung cancer cells in a multi-organ microfluidic chip model.

Brain metastasis (BM) is a leading cause of mortality in patients with non-small cell lung cancer (NSCLC). However, the molecular mechanisms underlying BM of NSCLC remain largely unknown because of the lack of models to accurately investigate such a dynamic and complex process. Here we developed a multi-organ microfluidic chip as a new methodological platform to study BM. The chip consisted of two bionic organ units - an upstream "lung" and a downstream "brain" characterized by a functional "blood-brain barrier (BBB)" structure, allowing real-time visual monitoring of the entire BM process, from the growth of primary tumor to its breaking through the BBB, and finally reaching the brain parenchyma. The chip was verified by lung cancer cell lines with differing metastatic abilities and then applied for the BM research where we first demonstrated that the protein expression of Aldo-keto reductase family 1 B10 (AKR1B10) was significantly elevated in lung cancer BM. Silencing AKR1B10 in brain metastatic tumor cells suppressed their extravasation through the BBB in the in vitro Transwell model, in our ex vivo microfluidic chip, as well as the in vivo model of brain metastasis in nude mice. Moreover, AKR1B10 downregulated the expression of matrix metalloproteinase (MMP)-2 and MMP-9 via MEK/ERK signaling in metastatic lung cancers. These data suggest that our multi-organ microfluidic chip is a practical alternative to study BM pathogenesis, and AKR1B10 is a diagnostic biomarker and a prospective therapeutic target for NSCLC BM. STATEMENT OF SIGNIFICANCE: Brain metastasis (BM) of non-small cell lung cancer (NSCLC) is a complex cascade, and in particular, the process of lung cancer cells penetrating the blood-brain barrier (BBB) is very unique. However, due to the lack of reliable models that can faithfully mimic the dynamic process of BBB breaking, its molecular mechanisms have not well elucidated so far. In addition, although Aldo-keto reductase family 1 B10 (AKR1B10) has been implicated to the tumor development of liver cancer and many other cancers, little is known on its roles in the BM. Here, we established a multi-organ microfluidic bionic chip platform to recapitulate the entire BM process, and applied it to the BM pathology research, especially BBB extravasation. By using the chip and traditional models synergistically, we first demonstrated that AKR1B10 was significantly elevated in lung cancer BM, and defined the value of AKR1B10 as a diagnostic serum biomarker for lung cancer patients suffering from BM. Further, we investigated the role and mechanisms of AKR1B10 in BM that it promotes the extravasation of cancer cells through the BBB.

Limitations of Local Brain Cooling on Generalized Motor Seizures from Unknown Foci in Awake Rats.

Local brain cooling of an epileptic focus at 15°C reduces the number of spikes on an electrocorticogram (ECoG), terminates seizures, and maintains neurological functions. In this study, we attempted to suppress generalized motor seizures (GMSs) by cooling a unilateral sensorimotor area. GMSs were induced in rats by intraperitoneal injection of bicuculline methiodide, an antagonist of gamma-aminobutyric acid. While monitoring the ECoG and behavior, the right sensorimotor cortex was cooled for 10 min using an implanted device. The number of spikes recorded from the cooled cortex significantly decreased to 71.2% and 62.5% compared with the control group at temperatures of 15 and 5°C (both P <0.01), respectively. The number of spikes recorded from the contralateral mirror cortex reduced to 61.7% and 62.7% (both P <0.05), respectively. The ECoG power also declined to 85% and 50% in the cooled cortex, and to 94% and 49% in the mirror cortex by the cooling at 15 and 5°C, respectively. The spikes regained in the middle of the cooling period at 15°C and in the late period at 5°C. Seizure-free durations during the 10-min periods of cooling at 15 and 5°C lasted for 4.1 ± 2.2 and 5.9 ± 1.1 min, respectively. Although temperature-dependent seizure alleviation was observed, the effect of local cortical cooling on GMSs was limited compared with the effect of local cooling of the epileptic focus on GSMs.

Epidural focal brain cooling abolishes neocortical seizures in cats and non-human primates.

Focal brain cooling (FBC) is under investigation in preclinical trials of intractable epilepsy (IE), including status epilepticus (SE). This method has been studied in rodents as a possible treatment for epileptic disorders, but more evidence from large animal studies is required. To provide evidence that FBC is a safe and effective therapy for IE, we investigated if FBC using a titanium cooling plate can reduce or terminate focal neocortical seizures without having a significant impact on brain tissue. Two cats and two macaque monkeys were chronically implanted with an epidural FBC device over the somatosensory and motor cortex. Penicillin G was delivered via the intracranial cannula for induction of local seizures. Repetitive FBC was performed using a cooling device implanted for a medium-term period (FBC for 30min at least twice every week; 3 months total) in three of the four animals. The animals exhibited seizures with repetitive epileptiform discharges (EDs) after administration of penicillin G, and these discharges decreased at less than 20°C cooling with no adverse histological effects. The results of this study suggest that epidural FBC is a safe and effective potential treatment for IE and SE.

Changes in glutamate concentration, glucose metabolism, and cerebral blood flow during focal brain cooling of the epileptogenic cortex in humans.

OBJECTIVE: Recently, focal brain cooling (FBC) was proposed as a method for treating refractory epilepsy. However, the precise influence of cooling on the molecular basis of epilepsy has not been elucidated. Thus the aim of this study was to assess the effect of FBC on glutamate (Glu) concentration, cerebral blood flow (CBF), and glucose metabolism in patients with intractable epilepsy. METHODS: Nine patients underwent FBC at 15°C for 30 min prior to cortical resection (n = 6) or hippocampectomy (n = 3). Measurement of metabolites and CBF, as well as electrocorticography (ECoG), was performed. RESULTS: Epileptic discharge (ED), as observed by ECoG, disappeared in the cooling period and reappeared in the rewarming period. Glu concentrations were high during the precooling period and were reduced to 51.2% during the cooling period (p = 0.025). Glycerol levels showed a similar decrease (p = 0.028). Lactate concentration was high during the precooling period and was reduced during the cooling period (21.3% decrease; p = 0.005). Glucose and pyruvate levels were maintained throughout the procedure. Changes in CBF were parallel to those observed by ECoG. SIGNIFICANCE: FBC reduced EDs and concentrations of Glu and glycerol. This demonstrates the neuroprotective effect of FBC. Our findings confirm that FBC is a reasonable and optimal treatment option for patients with intractable epilepsy.

Development of an implantable flexible probe for simultaneous near-infrared spectroscopy and electrocorticography.

A combination of near-infrared spectroscopy (NIRS) and electrocorticography (ECoG) provides beneficial information on cortical activity from different aspects. Integration of such multimodal measurement capability into a single apparatus and the direct measurement of cortical activity during chronic subdural implantation may be a powerful means for clinical diagnosis and neuroscience. However, an optical fiber-based NIRS probe cannot be miniaturized for implantation into the brain, and the light-scattering effect of ECoG electrodes in NIRS measurements is unknown. We describe here the development of a flexible probe, small enough for chronic subdural implantation, for simultaneous NIRS and ECoG. Two light-emitting diodes of different wavelengths and two photodiodes were mounted on a polyimide-based flexible substrate, and ECoG electrodes were formed with a design minimizing artifacts in NIRS recording. The fabricated probe measured ECoGs at sufficient spatial resolution and submicromolar changes in hemoglobin concentrations in in vivo experiments with acute implantation into a rat. Comparison of measured changes in hemoglobin concentrations for different source-detector distances reveals the reliability of the measured values and the practicality of the simulation model. The proposed intracranial multimodality probe may provide beneficial evidence for pre- and intrasurgical assessment of neurosurgery and reveal the interaction of electrophysiology and hemodynamics at high spatial resolution without artifacts due to scalp blood flow.

Cooling treatment transiently increases the permeability of brain capillary endothelial cells through translocation of claudin-5.

The blood-brain-barrier (BBB) is formed by different cell types, of which brain microvascular endothelial cells are major structural constituents. The goal of this study was to examine the effects of cooling on the permeability of the BBB with reference to tight junction formation of brain microendothelial cells. The sensorimotor cortex above the dura mater in adult male Wistar rats was focally cooled to a temperature of 5 °C for 1 h, then immunostaining for immunoglobulin G (IgG) was performed to evaluate the permeability of the BBB. Permeability produced by cooling was also evaluated in cultured murine brain endothelial cells (bEnd3) based on measurement of trans-epithelial electric resistance (TEER). Immunocytochemistry and Western blotting of proteins associated with tight junctions in bEnd3 were performed to determine protein distribution before and after cooling. After focal cooling of the rat brain cortex, diffuse immunostaining for IgG was observed primarily around the small vasculature and in the extracellular spaces of parenchyma of the cortex. In cultured bEnd3, TEER significantly decreased during cooling (15 °C) and recovered to normal levels after rewarming to 37 °C. Immunocytochemistry and Western blotting showed that claudin-5, a critical regulatory protein for tight junctions, was translocated from the membrane to the cytoplasm after cooling in cultured bEnd3 cells. These results suggest that focal brain cooling may open the BBB transiently through an effect on tight junctions of brain microendothelial cells, and that therapeutically this approach may allow control of BBB function and drug delivery through the BBB.

Neuroprotective effects of focal brain cooling on photochemically-induced cerebral infarction in rats: analysis from a neurophysiological perspective.

Although systemic hypothermia provides favorable outcomes in stroke patients, it has only been adopted in a limited number of patients because of fatal complications. To resolve these issues, focal brain cooling (FBC) has recently drawn attention as a less-invasive treatment for brain injuries. Therefore, we investigated whether FBC has a favorable effect on focal cerebral ischemia (FCI). Male-adult-Wistar rats were used. Under general anesthesia, a small burr hole was made and FCI was induced in the primary sensorimotor area (SI-MI) using photothrombosis. An additional craniotomy was made over the SI-MI and FBC was performed at a temperature of 15°C for 5h. Electrocorticograms (ECoG) were recorded on the border cortex of the ischemic focus. Thereafter, rats were sacrificed and the infarct area was measured. In another experiment, rats were allowed to recover for 5 days after cooling and neurobehavioral function was evaluated. FBC suppressed all ECoG frequency bands during and after cooling (p<0.05), except for the delta frequency band in the precooling versus rewarming periods. The injured areas in the cooling and non-cooling groups were 0.99±0.30 and 1.71±0.54 mm(2), respectively (p<0.03). The grip strength at 2 days after surgery was preserved in the cooling group (p<0.05). We report the novel finding that epileptiform discharges were suppressed in the ischemic border, the infarct area was reduced and neurobehaviour was preserved by FBC. These results indicate that FBC is neuroprotective in the ischemic brain and has demonstrated therapeutic potential for cerebral infarction.

Focal brain cooling terminates the faster frequency components of epileptic discharges induced by penicillin G in anesthetized rats.

OBJECTIVE: The goal of the study was to investigate the effects of focal brain cooling on epileptic discharges (EDs) and background rhythms in the sensorimotor cortex of anesthetized rats using spectral analysis of electroencephalography (EEG). METHODS: Penicillin G was administered intracortically into superficial layers of the left sensorimotor cortex and EDs were induced. Focal brain cooling was achieved using a cooling device attached to the cortical surface. The cortical surface was cooled to 25°C, 20°C and 15°C, and EEG was continuously recorded just beneath the cooling device. EEG spectral powers were determined using fast Fourier transform before and during cooling. RESULTS: Penicillin G induced EDs and increased the Alpha and Beta power spectra. Cooling suppressed EDs with an effect that depended on the brain temperature. Cooling to 25°C attenuated Beta powers, cooling to 20°C attenuated Alpha and Beta powers, and cooling to 15°C suppressed spectral powers ranging from Delta to Beta bands. CONCLUSIONS: These results suggest that focal brain cooling can terminate EDs in the cortex and suppress spectral powers with a temperature-dependent effect. SIGNIFICANCE: These findings may contribute to development of a new clinical treatment for patients with epilepsy.

Cooling of the epileptic focus suppresses seizures with minimal influence on neurologic functions.

PURPOSE: Focal brain cooling is effective for suppression of epileptic seizures, but it is unclear if seizures can be suppressed without a substantial influence on normal neurologic function. To address the issue, a thermoelectrically driven cooling system was developed and applied in free-moving rat models of focal seizure and epilepsy. METHODS: Focal seizures limited to the unilateral forelimb were induced by local application of a penicillin G solution or cobalt powder to the unilateral sensorimotor cortex. A proportional integration and differentiation (PID)-controlled, thermoelectrically driven cooling device (weight of 11 g) and bipolar electrodes were chronically implanted on the eloquent area (on the epileptic focus) and the effects of cooling (20, 15, and 10°C) on electrocorticography, seizure frequency, and neurologic changes were investigated. KEY FINDINGS: Cooling was associated with a distinct reduction of the epileptic discharges. In both models, cooling of epileptic foci significantly improved both seizure frequency and neurologic functions from 20°C down to 15°C. Cooling to 10°C also suppressed seizures, but with no further improvement in neurologic function. Subsequent investigation of sensorimotor function revealed significant deterioration in foot-fault tests and the receptive field size at 15°C. SIGNIFICANCE: Despite the beneficial effects in ictal rats, sensorimotor functions deteriorated at 15°C, thereby suggesting a lower limit for the therapeutic temperature. These results provide important evidence of a therapeutic effect of temperatures from 20 to 15°C using an implantable, hypothermal device for focal epilepsy.