Microwell-based flow culture increases viability and restores drug response in prostate cancer spheroids.

3D cancer spheroids represent a highly promising model for study of cancer progression and therapeutic development. Wide-scale adoption of cancer spheroids, however, remains a challenge due to the lack of control over hypoxic gradients that may cloud the assessment of cell morphology and drug response. Here, we present a Microwell Flow Device (MFD) that generates in-well laminar flow around 3D tissues via repetitive tissue sedimentation. Using a prostate cancer cell line, we demonstrate the spheroids in the MFD exhibit improved cell growth, reduced necrotic core formation, enhanced structural integrity, and downregulated expression of cell stress genes. The flow-cultured spheroids also exhibit an improved sensitivity to chemotherapy with greater transcriptional response. These results demonstrate how fluidic stimuli reveal the cellular phenotype previously masked by severe necrosis. Our platform advances 3D cellular models and enables study into hypoxia modulation, cancer metabolism, and drug screening within pathophysiological conditions.

The serum amyloid A3 promoter-driven luciferase reporter mice is a valuable tool to image early renal fibrosis development and shows the therapeutic effect of glucosyl-hesperidin treatment.

Tubulointerstitial fibrosis is a progressive process affecting the kidneys, causing renal failure that can be life-threatening. Thus, renal fibrosis has become a serious concern in the ageing population; however, fibrotic development cannot be diagnosed early and assessed noninvasively in both patients and experimental animal models. Here, we found that serum amyloid A3 (Saa3) expression is a potent indicator of early renal fibrosis; we also established in vivo Saa3/C/EBPß-promoter bioluminescence imaging as a sensitive and specific tool for early detection and visualization of tubulointerstitial fibrosis. Saa3 promoter activity is specifically upregulated in parallel with tumor necrosis factor α (TNF-α) and fibrotic marker collagen I in injured kidneys. C/EBPß, upregulated in injured kidneys and expressed in tubular epithelial cells, is essential for the increased Saa3 promoter activity in response to TNF-α, suggesting that C/EBPß plays a crucial role in renal fibrosis development. Our model successfully enabled visualization of the suppressive effects of a citrus flavonoid derivative, glucosyl-hesperidin, on inflammation and fibrosis in kidney disease, indicating that this model could be widely used in exploring therapeutic agents for fibrotic diseases.

Thalamic and cerebellar hypermetabolism and cortical hypometabolism during absence status epilepticus.

We report on a 17-year-old girl with absence status epilepticus who developed recurrent motionless confusional state and continuous generalised 3-4 Hz rhythmic delta waves on electroencephalogram (EEG). The patient had no history of absence, myoclonus or generalised convulsion. Her seizure was resistant to a combination of antiepileptic drugs including carbamazepine. Ictal positron emission tomography using [18F]fluorodeoxyglucose ([18F]FDG-PET) revealed hypermetabolism of the bilateral thalamus and cerebellum and hypometabolism of the frontal, parietal and posterior cingulate cortices. We diagnosed her seizure as absence status and obtained remission by changing medication. The findings of ictal metabolic alteration in previous studies and in our case confirm the pathogenic importance of the thalamus in absence status and that associated cortical deactivation and cerebellar activation may be related to the generation or maintenance of epileptic EEG discharges.

External pallidal stimulation improves parkinsonian motor signs and modulates neuronal activity throughout the basal ganglia thalamic network.

Deep brain stimulation (DBS) of the internal segment of the globus pallidus (GPi) and the subthalamic nucleus (STN) are effective for the treatment of advanced Parkinson's disease (PD). We have shown previously that DBS of the external segment of the globus pallidus (GPe) is associated with improvements in parkinsonian motor signs; however, the mechanism of this effect is not known. In this study, we extend our findings on the effect of STN and GPi DBS on neuronal activity in the basal ganglia thalamic network to include GPe DBS using the 1-methyl-4-phenyl-1.2.3.6-tetrahydropyridine (MPTP) monkey model. Stimulation parameters that improved bradykinesia were associated with changes in the pattern and mean discharge rate of neuronal activity in the GPi, STN, and the pallidal [ventralis lateralis pars oralis (VLo) and ventralis anterior (VA)] and cerebellar [ventralis lateralis posterior pars oralis (VPLo)] receiving areas of the motor thalamus. Population post-stimulation time histograms revealed a complex pattern of stimulation-related inhibition and excitation for the GPi and VA/VLo, with a more consistent pattern of inhibition in STN and excitation in VPLo. Mean discharge rate was reduced in the GPi and STN and increased in the VPLo. Effective GPe DBS also reduced bursting in the STN and GPi. These data support the hypothesis that therapeutic DBS activates output from the stimulated structure and changes the temporal pattern of neuronal activity throughout the basal ganglia thalamic network and provide further support for GPe as a potential therapeutic target for DBS in the treatment of PD.

Subthalamic nucleus stimulation modulates thalamic neuronal activity.

Deep brain stimulation (DBS) in the subthalamic nucleus (STN) is an effective tool for the treatment of advanced Parkinson's disease. The mechanism by which STN DBS elicits its beneficial effect, however, remains unclear. We previously reported STN stimulation increased the rate and produced a more regular and periodic pattern of neuronal activity in the internal segment of the globus pallidus (GPi). Here we extend our observations to neurons in the pallidal [ventralis lateralis pars oralis (VLo) and ventralis anterior (VA)] and cerebellar [ventralis lateralis posterior pars oralis (VPLo)] receiving areas of the motor thalamus during STN DBS. Stimulation parameters that produced improvement in rigidity and bradykinesia resulted in changes in the pattern and power of oscillatory activity of neuronal activity that were similar in both regions of the motor thalamus. Neurons in both VA/VLo and VPLo tended to become more periodic and regular with a shift in oscillatory activity from low to high frequencies. Burst activity was reduced in VA/VLo, but was not significantly changed in VPLo. There was also a significant shift in the population of VA/VLo neurons that were inhibited during STN DBS, whereas VPLo neurons tended to be activated. These data are consistent with the hypothesis that STN DBS increases output from the nucleus and produces a change in the pattern and periodicity of neuronal activity in the basal ganglia thalamic network, and that these changes include cerebellar pathways likely via activation of adjacent cerebello-thalamic fiber bundles.

Deep brain stimulation reduces neuronal entropy in the MPTP-primate model of Parkinson's disease.

High-frequency stimulation (HFS) of the subthalamic nucleus (STN) or internal segment of the globus pallidus is a clinically successful treatment for the motor symptoms of Parkinson's disease. However, the mechanisms by which HFS alleviates these symptoms are not understood. Whereas initial studies focused on HFS-induced changes in neuronal firing rates, recent studies suggest that changes in patterns of neuronal activity may correlate with symptom alleviation. We hypothesized that effective STN HFS reduces the disorder of neuronal firing patterns in the basal ganglia thalamic circuit, minimizing the pathological activity associated with parkinsonism. Stimulating leads were implanted in the STN of two rhesus monkeys rendered parkinsonian by 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). Action potentials were recorded from neurons of the internal and external globus pallidus and the motor thalamus (ventralis anterior, ventralis lateralis pars oralis, and ventralis posterior lateralis pars oralis) during HFS that reduced motor symptoms and during clinically ineffective low-frequency stimulation (LFS). Firing pattern entropy was calculated from the recorded spike times to quantify the disorder of the neuronal activity. The firing pattern entropy of neurons within each region of the pallidum and motor thalamus decreased in response to HFS (n > or = 18 and P < or = 0.02 in each region), whereas firing rate changes were specific to pallidal neurons only. In response to LFS, firing rates were unchanged, but firing pattern entropy increased throughout the circuit (n > or = 24 and P < or = 10(-4) in each region). These data suggest that the clinical effectiveness of HFS is correlated with, and potentially mediated by, a regularization of the pattern of neuronal activity throughout the basal ganglia thalamic circuit.

Acute stimulation in the external segment of the globus pallidus improves parkinsonian motor signs.

High frequency (>100Hz) electrical stimulation in both the external (GPe) and internal (GPi) segments of the globus pallidus was effective in improving parkinsonian motor signs. Improvement generally occurred at short latency (<5-10 seconds) in both GPe and GPi but was often (50% of the time) delayed in GPi. Dyskinetic movements were observed during stimulation within GPe and GPi but were more frequent in GPe (20% vs. 9%). These findings suggest that electrical stimulation in both GPe and GPi may ameliorate parkinsonian motor signs. The mechanisms responsible for these observations, however, may differ. The tendency for delayed responses with GPi stimulation suggests a more complex spatial-temporal profile of stimulation on the electrical activity of GPi neurons and/or its effect on network activity in pallido-thalamo-cortical circuitry. The rarity of delayed effects with GPe stimulation suggests a more direct role of synaptic inhibition or normalization of neuronal activity of GPi either directly by means of activation of striatopallidal fibers passing through GPe (direct pathway), by means of activation of GPe-->GPi or GPe-->subthalamic nucleus projections (indirect pathway) or indirectly by means of the tonic activation of adjacent fiber pathways. These data provide a rationale for the exploration of electrical stimulation in GPe in patients with medically intractable Parkinson's disease and provide a basis on which to develop further investigations into the use of chronic electrical stimulation for the treatment of Parkinson's disease and other movement disorders.

Stimulation of the subthalamic nucleus changes the firing pattern of pallidal neurons.

To clarify the mechanism underlying improvement of parkinsonian signs by high-frequency electrical stimulation (HFS) of the subthalamic nucleus (STN), we investigated the effects of STN HFS on neuronal activity of the internal and external segment of the globus pallidus (GPi and GPe, respectively) in two rhesus monkeys rendered parkinsonian by administration of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine. A scaled-down version of the chronic stimulating electrode used in humans, consisting of four metal contacts 0.50 mm in length each separated by 0.50 mm, was implanted through a cephalic chamber targeting the STN. Histological reconstruction revealed that the cathode was located in the STN in both monkeys. Extracellular recordings from a total of 110 pallidal neurons during STN stimulation were performed. Poststimulus time histograms of single neurons triggered by 2 Hz STN stimulation pulses at 2.4-3.0 V revealed short-latency excitations at 2.5-4.5 and 5.5-7.0 msec after stimulation onset and inhibitions at 1.0-2.5, 4.5-5.5, and 7.0-9.0 msec for both GPe and GPi neurons. These short-latency responses were present with 136 Hz stimulation, at voltages effective for alleviation of parkinsonian signs, resulting in a significant increase in mean discharge rate and a stimulus-synchronized regular firing pattern. These results indicate that activation of the STN efferent fibers and resultant changes in the temporal firing pattern of neurons in GPe and GPi underlie the beneficial effect of HFS in the STN in Parkinson's disease and further support the role of temporal firing patterns in the basal ganglia in the development of Parkinson's disease and other movement disorders.