Sensorimotor content of multi-unit activity recorded in the paramedian lobule of the cerebellum using carbon fiber microelectrode arrays.

The cerebellum takes in a great deal of sensory information from the periphery and descending signals from the cerebral cortices. It has been debated whether the paramedian lobule (PML) in the rat and its paravermal regions that project to the interpositus nucleus (IPN) are primarily involved in motor execution or motor planning. Studies that have relied on single spike recordings in behaving animals have led to conflicting conclusions regarding this issue. In this study, we tried a different approach and investigated the correlation of field potentials and multi-unit signals recorded with multi-electrode arrays from the PML cortex along with the forelimb electromyography (EMG) signals in rats during behavior. Linear regression was performed to predict the EMG signal envelopes using the PML activity for various time shifts (±25, ±50, ±100, and ± 400 ms) between the two signals to determine a causal relation. The highest correlations (~0.5 on average) between the neural and EMG envelopes were observed for zero and small (±25 ms) time shifts and decreased with larger time shifts in both directions, suggesting that paravermal PML is involved both in processing of sensory signals and motor execution in the context of forelimb reaching behavior. EMG envelopes were predicted with higher success rates when neural signals from multiple phases of the behavior were utilized for regression. The forelimb extension phase was the most difficult to predict while the releasing of the bar phase prediction was the most successful. The high frequency (>300 Hz) components of the neural signal, reflecting multi-unit activity, had a higher contribution to the EMG prediction than did the lower frequency components, corresponding to local field potentials. The results of this study suggest that the paravermal PML in the rat cerebellum is primarily involved in the execution of forelimb movements rather than the planning aspect and that the PML is more active at the initiation and termination of the behavior, rather than the progression.

Transsynaptic entrainment of cerebellar nuclear cells by alternating currents in a frequency dependent manner.

Transcranial alternating current stimulation (tACS) is a non-invasive neuromodulation technique that is being tested clinically for treatment of a variety of neural disorders. Animal studies investigating the underlying mechanisms of tACS are scarce, and nearly absent in the cerebellum. In the present study, we applied 10-400 Hz alternating currents (AC) to the cerebellar cortex in ketamine/xylazine anesthetized rats. The spiking activity of cerebellar nuclear (CN) cells was transsynaptically entrained to the frequency of AC stimulation in an intensity and frequency-dependent manner. Interestingly, there was a tuning curve for modulation where the frequencies in the midrange (100 and 150 Hz) were more effective, although the stimulation frequency for maximum modulation differed for each CN cell with slight dependence on the stimulation amplitude. CN spikes were entrained with latencies of a few milliseconds with respect to the AC stimulation cycle. These short latencies and that the transsynaptic modulation of the CN cells can occur at such high frequencies strongly suggests that PC simple spike synchrony at millisecond time scales is the underlying mechanism for CN cell entrainment. These results show that subthreshold AC stimulation can induce such PC spike synchrony without resorting to supra-threshold pulse stimulation for precise timing. Transsynaptic entrainment of deep CN cells via cortical stimulation could help keep stimulation currents within safety limits in tACS applications, allowing development of tACS as an alternative treatment to deep cerebellar stimulation. Our results also provide a possible explanation for human trials of cerebellar stimulation where the functional impacts of tACS were frequency dependent.

Modulation of cerebellar cortical, cerebellar nuclear and vestibular nuclear activity using alternating electric currents.

Introduction: Cerebellar transcranial alternating current stimulation (ctACS) has shown promise as a therapeutic modality for treating a variety of neurological disorders, and for affecting normal learning processes. Yet, little is known about how electric fields induced by applied currents affect cerebellar activity in the mammalian cerebellum under in vivo conditions. Methods: Alternating current (AC) stimulation with frequencies from 0.5 to 20 Hz was applied to the surface of the cerebellum in anesthetized rats. Extracellular recordings were obtained from Purkinje cells (PC), cerebellar and vestibular nuclear neurons, and other cerebellar cortical neurons. Results and discussion: AC stimulation modulated the activity of all classes of neurons. Cerebellar and vestibular nuclear neurons most often showed increased spike activity during the negative phase of the AC stimulation. Purkinje cell simple spike activity was also increased during the negative phase at most locations, except for the cortex directly below the stimulus electrode, where activity was most often increased during the positive phase of the AC cycle. Other cortical neurons showed a more mixed, generally weaker pattern of modulation. The patterns of Purkinje cell responses suggest that AC stimulation induces a complex electrical field with changes in amplitude and orientation between local regions that may reflect the folding of the cerebellar cortex. Direct measurements of the induced electric field show that it deviates significantly from the theoretically predicted radial field for an isotropic, homogeneous medium, in both its orientation and magnitude. These results have relevance for models of the electric field induced in the cerebellum by AC stimulation.

Selective neural stimulation by leveraging electrophysiological differentiation and using pre-pulsing and non-rectangular waveforms.

Efforts on selective neural stimulation have concentrated on segregating axons based on their size and geometry. Nonetheless, axons of the white matter or peripheral nerves may also differ in their electrophysiological properties. The primary objective of this study was to investigate the possibility of selective activation of axons by leveraging an assumed level of diversity in passive (Cm & Gleak) and active membrane properties (Ktemp & Gnamax). First, the stimulus waveforms with hyperpolarizing (HPP) and depolarizing pre-pulsing (DPP) were tested on selectivity in a local membrane model. The default value of membrane capacitance (Cm) was found to play a critical role in sensitivity of the chronaxie time (Chr) and rheobase (Rhe) to variations of all the four membrane parameters. Decreasing the default value of Cm, and thus the passive time constant of the membrane, amplified the sensitivity to the active parameters, Ktemp and GNamax, on Chr. The HPP waveform could selectively activate neurons even if they were diversified by membrane leakage (Gleak) only, and produced higher selectivity than DPP when parameters are varied in pairs. Selectivity measures were larger when the passive parameters (Cm & Gleak) were varied together, compared to the active parameters. Second, this novel mechanism of selectivity was investigated with non-rectangular waveforms for the stimulating phase (and HPP) in the same local membrane model. Simulation results suggest that Kt2 is the most selective waveform followed by Linear and Gaussian waveforms. Traditional rectangular pulse was among the least selective of all. Finally, a compartmental axon model confirmed the main findings of the local model that Kt2 is the most selective, but rank ordered the other waveforms differently. These results suggest a potentially novel mechanism of stimulation selectivity, leveraging electrophysiological variations in membrane properties, that can lead to various neural prosthetic applications.

The size of via holes influence the amplitude and selectivity of neural signals in Micro-ECoG arrays.

BACKGROUND: Electrocorticography (ECoG) arrays are commonly used to record the brain activity both in animal and human subjects. There is a lack of guidelines in the literature as to how the array geometry, particularly the via holes in the substrate, affects the recorded signals. A finite element (FE) model was developed to simulate the electric field generated by neurons located at different depths in the rat brain cortex and a micro ECoG array (µECoG) was placed on the pia surface for recording the neural signal. The array design chosen was a typical array of 8 × 8 circular (100 µm in diam.) contacts with 500 µm pitch. The size of the via holes between the recording contacts was varied to see the effect. RESULTS: The results showed that recorded signal amplitudes were reduced if the substrate was smaller than about four times the depth of the neuron in the gray matter. The signal amplitude profiles had dips around the via holes and the amplitudes were also lower at the contact sites as compared to the design without the holes; an effect that increased with the hole size. Another noteworthy result is that the spatial selectivity of the multi-contact recordings could be improved or reduced by the selection of the via hole sizes, and the effect depended on the distance between the neuron pair targeted for selective recording and its depth. CONCLUSIONS: The results suggest that the via-hole size clearly affects the recorded neural signal amplitudes and it can be leveraged as a parameter to reduce the inter-channel correlation and thus maximize the information content of neural signals with µECoG arrays.

A Wearable Ultrasonic Neurostimulator - Part I: A 1D CMUT Phased Array System for Chronic Implantation in Small Animals.

In this work, we present a wireless ultrasonic neurostimulator, aiming at a truly wearable device for brain stimulation in small behaving animals. A 1D 5-MHz capacitive micromachined ultrasonic transducer (CMUT) array is adopted to implement a head-mounted stimulation device. A companion ASIC with integrated 16-channel high-voltage (60-V) pulsers was designed to drive the 16-element CMUT array. The ASIC can generate excitation signals with element-wise programmable phases and amplitudes: 1) programmable sixteen phase delays enable electrical beam focusing and steering, and 2) four scalable amplitude levels, implemented with a symmetric pulse-width-modulation technique, are sufficient to suppress unwanted sidelobes (apodization). The ASIC was fabricated in the TSMC 0.18- µm HV BCD process within a die size of 2.5 × 2.5 mm2. To realize a completely wearable system, the system is partitioned into two parts for weight distribution: 1) a head unit (17 mg) with the CMUT array, 2) a backpack unit (19.7 g) that includes electronics such as the ASIC, a power management unit, a wireless module, and a battery. Hydrophone-based acoustic measurements were performed to demonstrate the focusing and beam steering capability of the proposed system. Also, we achieved a peak-to-peak pressure of 2.1 MPa, which corresponds to a spatial peak pulse average intensity ( ISPPA) of 33.5 W/cm2, with a lateral full width at half maximum (FWHM) of 0.6 mm at a depth of 3.5 mm.

Entrainment of cerebellar Purkinje cell spiking activity using pulsed ultrasound stimulation.

BACKGROUND: Focused ultrasound (FUS) has excellent characteristics over other non-invasive stimulation methods in terms of spatial resolution and steering capability of the target. FUS has not been tested in the cerebellar cortex and cellular effects of FUS are not fully understood. OBJECTIVE/HYPOTHESIS: To investigate how the activity of cerebellar Purkinje cells (PCs) is modulated by FUS with varying pulse durations and pulse repetition frequencies. METHODS: A glass microelectrode was inserted into the cerebellar vermis lobule 6 from the dorsal side to extracellularly record single unit activity of the PCs in anesthetized rats. Ultrasonic stimulation (500 kHz) was applied through a coupling cone, filled with degassed water, from the posterior side to target the recording area with varying pulse durations and frequencies. RESULTS: Simple spike (SS) activity of PCs was entrained by the FUS pattern where the probability of spike occurrences peaked at around 1 ms following the onset of the stimulus regardless of its duration (0.5, 1, or 2 ms). The level of entrainment was stronger with shorter pulse durations at 50-Hz pulse repetition frequency (PRF), however, peri-event histograms spread wider and the peaks delayed slightly at 100-Hz PRF, suggesting involvement of a long-lasting inhibitory mechanism. There was no significant difference between the average firing rates in the baseline and stimulation periods. CONCLUSION: FUS can entrain spiking activity of single cells on a spike-by-spike basis as demonstrated here in the rat cerebellar cortex. The observed modulation potentially results from the aggregate of excitatory and inhibitory effects of FUS on the entire cortical network rather than on the PCs alone.

Entrainment of cerebellar purkinje cells with directional AC electric fields in anesthetized rats.

BACKGROUND: Transcranial electrical stimulation (tES) shows promise to treat neurological disorders. Knowledge of how the orthogonal components of the electric field (E-field) alter neuronal activity is required for strategic placement of transcranial electrodes. Yet, essentially no information exists on this relationship for mammalian cerebellum in vivo, despite the cerebellum being a target for clinical tES studies. OBJECTIVE: To characterize how cerebellar Purkinje cell (PC) activity varies with the intensity, frequency, and direction of applied AC and DC E-fields. METHODS: Extracellular recordings were obtained from vermis lobule 7 PCs in anesthetized rats. AC (2-100 Hz) or DC E-fields were generated in a range of intensities (0.75-30 mV/mm) in three orthogonal directions. Field-evoked PC simple spike activity was characterized in terms of firing rate modulation and phase-locking as a function of these parameters. t-tests were used for statistical comparisons. RESULTS: The effect of applied E-fields was direction and intensity dependent, with rostrocaudally directed fields causing stronger modulations than dorsoventral fields and mediolaterally directed ones causing little to no effect, on average. The directionality dependent modulation suggests that PC is the primary cell type affected the most by electric stimulation, and this effect was probably given rise by a large dendritic tree and a soma. AC stimulation entrained activity in a frequency dependent manner, with stronger phase-locking to the stimulus cycle at higher frequencies. DC fields produced a modulation consisting of strong transients at current onset and offset with an intervening plateau. CONCLUSION: Orientation of the exogenous E-field critically determines the modulation depth of cerebellar cortical output. With properly oriented fields, PC simple spike activity can strongly be entrained by AC fields, overriding the spontaneous firing pattern.

The role of TNF-α in chordoma progression and inflammatory pathways.

PURPOSE: Chordomas are highly therapy-resistant primary bone tumors that exhibit high relapse rates and may induce local destruction. Here, we evaluated the effects of tumor necrosis factor-alpha (TNF-α) on chordoma progression and clinical outcome. METHODS: Chordoma cells were treated with TNF-α after which its short- and long-term effects were evaluated. Functional assays, qRT-PCR and microarray-based expression analyses were carried out to assess the effect of TNF-α on chemo-resistance, epithelial to mesenchymal transition (EMT), migration, invasion and cancer stem cell-like properties. Finally, relationships between TNF-α expression and clinicopathological features were assessed in a chordoma patient cohort. RESULTS: We found that TNF-α treatment increased the migration and invasion of chordoma cells. Also, NF-κB activation was observed along with increased EMT marker expression. In addition, enhanced tumor sphere formation and soft agar colony formation were observed, concomitantly with increased chemo-resistance and CD338 marker expression. The TNF-α and TNFR1 expression levels were found to be significantly correlated with LIF, PD-L1 and Ki67 expression levels, tumor volume and a short survival time in patients. In addition, a high neutrophil to lymphocyte ratio was found to be associated with recurrence and a decreased overall survival. CONCLUSIONS: From our data we conclude that TNF-α may serve as a prognostic marker for chordoma progression and that tumor-promoting inflammation may be a major factor in chordoma tumor progression.

Prediction of Forelimb EMGs and Movement Phases from Corticospinal Signals in the Rat During the Reach-to-Pull Task.

Brain-computer interfaces access the volitional command signals from various brain areas in order to substitute for the motor functions lost due to spinal cord injury or disease. As the final common pathway of the central nervous system (CNS) outputs, the descending tracts of the spinal cord offer an alternative site to extract movement-related command signals. Using flexible 2D microelectrode arrays, we have recorded the corticospinal tract (CST) signals in rats during a reach-to-pull task. The CST activity was then classified by the forelimb movement phases into two or three classes in a training dataset and cross validated in a test set. The average classification accuracies were 80 ± 10% (min: 62% to max: 97%) and 55 ± 8% (min: 43% to max: 71%) for two-class and three-class cases, respectively. The forelimb flexor and extensor EMG envelopes were also predicted from the CST signals using linear regression. The average correlation coefficient between the actual and predicted EMG signals was 0.5 ± 0.13 (n = 124), whereas the highest correlation was 0.81 for the biceps EMG. Although the forelimb motor function cannot be explained completely by the CST activity alone, the success rates obtained in reconstructing the EMG signals support the feasibility of a spinal-cord-computer interface as a concept.

Genioglossal response to mechanical vibrations of the mandible and the submandibular muscles.

The extrinsic tongue muscles are activated in coordination with pharyngeal muscles to dilate the airways as needed during breathing. The genioglossus (GG) activity is known to be modulated by several reflexes evoked via the mechanoreceptors of the upper airways. The primary objective of this paper was to investigate the effectiveness of activating these reflex pathways using mechanical stimulation of the mandible or the submandibular muscles. In eight healthy subjects, 3-s long, 5-mm vertical mechanical vibrations were delivered at 8 and 12 Hz to the lower jaw in a seated position, while the GG EMG was recorded using a custom-made sublingual electrode, along with the activity of the masseter (MS) and mylohyoid (MH). All three muscle activities were significantly higher during stimulation compared with the baseline (P < 0.02), and the increase was larger at 12 Hz versus 8 Hz (P < 0.02). All three muscle responses had components that synchronized with the mechanical stimuli, but those of MS were much more strongly phase-locked to the vibrational cycle. In 10 healthy subjects, we also applied mechanical vibrations to the submandibular muscles at three different stimulation intensities, while subjects were lying in a supine position. The GG activity increased significantly above the baseline (P = 0.026) in 9 out of 10 subjects, and the elevated activity persisted after termination of the stimulus for a few seconds. The results demonstrate that GG muscle responses can be evoked with mechanical vibrations applied to the lower jaw or the submandibular muscles in healthy subjects during wakefulness. NEW & NOTEWORTHY The evoked responses observed in the genioglossus (GG) activity during mechanical vibrations of the lower jaw or the submandibular muscles may lead to therapeutic applications for improving the patency of airways during sleep. The presence of these GG reflexes may also explain a mechanism by which the vibrations produced during snoring can help the airways stay open in individuals who may otherwise have obstructed airways in sleep.

Electrical fields induced inside the rat brain with skin, skull, and dural placements of the current injection electrode.

Transcranial electrical stimulation (tES) is rapidly becoming an indispensable clinical tool with its different forms. Animal data are crucially needed for better understanding of the underlying mechanisms of tES. For reproducibility of results in animal experiments, the electric fields (E-Fields) inside the brain parenchyma induced by the injected currents need to be predicted accurately. In this study, we measured the electrical fields in the rat brain perpendicular to the brain surface, i.e. vertical electric field (VE-field), when the stimulation electrode was placed over the skin, skull, or dura mater through a craniotomy hole. The E-field attenuation through the skin was a few times larger than that of the skull and the presence of skin substantially reduced the VE-field peak at the cortical surface near the electrode. The VE-field declined much quicker in the gray matter underneath the pial surface than it did in the white matter, and thus the large VE-fields were contained mostly in the gray matter. The transition at the gray/white matter border caused a significant peak in the VE-field, as well as at other local inhomogeneties. A conductivity value of 0.57 S/m is predicted as a global value for the whole brain by matching our VE-field measurements to the field profile given by analytical equations for volume conductors. Finally, insertion of the current return electrode into the shoulder, submandibular, and hind leg muscles had virtually no effects on the measured E-field amplitudes in the cortex underneath the epidural electrodes.

A Sub-Millimeter Lateral Resolution Ultrasonic Beamforming System for Brain Stimulation in Behaving Animals.

In this paper, we present a second-generation wireless ultrasonic beamforming system, aiming for a truly wearable device for brain stimulation in small behaving animals. The fully-integrated, battery-operated system enables a self- contained untethered system. The system is partitioned into two parts for weight distribution: (1) a 1D capacitive micromachined transducer (CMUT) array on a separate head-mountable flexible printed circuit board (PCB), (2) a rigid back-mountable PCB including electronics such as a custom ASIC, a power management unit, a wireless module, and a battery. The newly developed ASIC not only enables a compact electronic system (30.5 mm x 63.5 mm) but also generates 3.4 times higher acoustic pressure (1.89 MPaPP), which corresponds to a spatial-peak pulse-average intensity (ISPPA) of 33.5 W/cm2, at a depth of 5 mm, compared to the first-generation ASIC. The full width at half maximum (FWHM) of the pressure is estimated to be 0.6 mm, achieving a sub-millimeter lateral resolution by using 5-MHz focused waves.

Modulation of Multiunit Spike Activity by Transcranial AC Stimulation (tACS) in the Rat Cerebellar Cortex.

Transcranial electrical stimulation (tES) techniques have garnered significant interest due to their non-invasiveness and potential to offer a treatment option in a wide variety of brain disorders. Among several modulation techniques, transcranial alternating current stimulation (tACS) is favored for its ability to entrain the neural oscillations. The cerebellum is one of the targeted sites because of its involvement in motor and cognitive functions. However, animal studies are lacking in the literature looking into the mechanism of action in cerebellar tACS. In this study, we used a rat model and monitored the activity of the cerebellar cortex, which sculpts the cerebellar output by adjusting the firing rate and timing of the neurons in the deep cerebellar nuclei (DCN). For neural recording, a tungsten electrode was inserted into the cerebellar cortex through a craniotomy hole located over the right paramedian lobule (PML). A helical Ag/AgCl wire electrode was placed atop the skull near the caudal edge to inject a 1 Hz biphasic sinusoidal current. Our results showed that the multiunit activity (MUA) of the cerebellar cortex was strongly modulated by tACS. The negative phase of the electric current enhanced the neural firing rate while the positive phase suppressed the activity. Furthermore, the spike rate showed modulation by the instantaneous strength of the injected current within the sinusoidal cycle. This warrants research to further look into the mechanism of tACS acting on the cerebellar cortex at the cellular level.

Convolutional Networks Outperform Linear Decoders in Predicting EMG From Spinal Cord Signals.

Advanced algorithms are required to reveal the complex relations between neural and behavioral data. In this study, forelimb electromyography (EMG) signals were reconstructed from multi-unit neural signals recorded with multiple electrode arrays (MEAs) from the corticospinal tract (CST) in rats. A six-layer convolutional neural network (CNN) was compared with linear decoders for predicting the EMG signal. The network contained three session-dependent Rectified Linear Unit (ReLU) feature layers and three Gamma function layers were shared between sessions. Coefficient of determination (R 2) values over 0.2 and correlations over 0.5 were achieved for reconstruction within individual sessions in multiple animals, even though the forelimb position was unconstrained for most of the behavior duration. The CNN performed visibily better than the linear decoders and model responses outlasted the activation duration of the rat neuromuscular system. These findings suggest that the CNN model implicitly predicted short-term dynamics of skilled forelimb movements from neural signals. These results are encouraging that similar problems in neural signal processing may be solved using variants of CNNs defined with simple analytical functions. Low powered firmware can be developed to house these CNN solutions in real-time applications.

Carbon Fiber Electrodes for in Vivo Spinal Cord Recordings.

Development of micro electrode arrays for neural recording is an active field that thrives on novel materials and fabrication techniques offered by micro fabrication technology. The material and mechanical properties of microelectrode arrays have a critical role on the quality and longevity of neural signals. In this study, carbon fiber microelectrode (CFME) bundles were developed and implanted in the spinal cord of experimental animals for textbf{\textit{in vivo{recording. Neural data analysis revealed that single spikes could successfully be recorded and sorted. Removal of approximately $75 \mu \mathrm{m}$ of the parylene-C coating at the tips of the fibers increased the signalto-noise ratio. Connecting multiple (three) carbon fiber filaments to the same recording channel did not deteriorate the signal quality compared to that of undesheathed fibers. Immunohistochemistry showed that electrode tips were splayed in tissue after implantation and CF bundles had a small footprint with mild encapsulation around them. These results are very promising for the use of CFME bundles for recordings of spinal cord signals in behaving animals.

Towards an Untethered Ultrasound Beamforming System for Brain Stimulation in Behaving Animals.

In this paper, we present a wireless ultrasound transmit (TX) beamforming system, potentially enabling wearable brain stimulation for small awake/behaving animals. The system is comprised of a 16-element capacitive micromachined transducer (CMUT) array, driven by a custom phased-array integrated circuit (IC), which is capable of generating high-voltage (13.5 V) excitation signals with sixteen phase delays and four amplitude levels. In addition, a Bluetooth low-energy module and a power management unit were integrated into the system, which realizes a battery-operated self-contained unit. We validated the functionality of the system by demonstrating beamforming and steering with a hydrophone measurement setup. We achieved an acoustic pressure output of 554 kPapp at the depth of 5 mm, which corresponds to a spatial-peak pulse-average intensity (ISPPA) of 2.9 W/cm2. The measured 6-dB beamwidth (0.4 mm) is promising in that it can stimulate a specific region of the brain, especially for small animals such as mice. Further smart partitioning of the system will enable a truly wearable device for small animals.

Electric Fields Induced By Transcutaneous And Intracranial Current Injections In The Rat Brain.

As a non-invasive brain stimulation technique, transcranial electrical stimulation (TES) and specifically the transcranial direct current stimulation (tDCS) has gained popularity in recent years for treatment of a wide variety of cognitive and neurological disorders. Recent studies have shown that TES can alter the motor cortex excitability. Animal studies to demonstrate the underlying mechanisms of TES are clearly lacking in literature. Clinical studies have agreed on the critical role of the current intensity and the montage of the electrodes for the treatment to be effective. In this study, we used a rat model for in vivo investigation of the vertical electrical (E) field distribution due to electrodes placed over the skin and through a craniotomy hole. A mono-phasic current pulse was used as a substitute for DC currents by taking advantage of primarily resistive properties of the brain tissue at low frequencies. The electrical potentials induced by the current pulses were recorded with penetrations at 0mm, 2mm, and 4mm away from the stimulation electrode. The results showed that the E-field was maximum immediately under the anodic electrode and decreased both in the vertical and horizontal directions rapidly by distance. The magnitude of the electric field varied from tens of mV/mm to a fraction of mV/mm by distance for a 100 µ A stimulus amplitude. The results also show that the E-field amplitudes and distribution strongly depend on whether the stimulus electrode is placed over the skin or into a craniotomy hole.

Smooth muscle cell differentiation from rabbit amniotic cells.

Amniotic fluid (AF) is the liquid layer that provides mechanical support and allows movement of the fetus during embryogenesis. Mesenchymal stem cells (MSCs), which have differentiation capacity, are also found in AF-derived cells at a low ratio. Smooth muscle cells (SMCs) play an important role in organ function and are frequently used in tissue engineering. We examined the differentiation of AF-derived MSCs (AMSCs) into SMCs. AMSCs were sorted from cultured amniotic cells and differentiated into SMCs using differentiation agents, including platelet-derived growth factor BB (PDGF-BB) and tumor growth factor ß (TGF-ß). Characterization of differentiated SMCs was confirmed morphologically, molecularly (via quantitative polymerase chain reaction [qPCR] and immunocytochemistry [ICC]), and functionally (using a contractile assay and fluo-4 calcium signaling assay). Poly(lactide-co-glycolide) (PLGA) scaffolds were fabricated, and the attachment capacity of AMSCs was assessed via scanning electron microscopy. AMSCs were successfully differentiated into SMCs. Our results indicate that AMSCs change their morphology and exhibit increased expression of ACTA2 and MYH11, which was confirmed via qPCR and ICC. Furthermore, functional experiments revealed that differentiated SMCs had both contraction ability and increased Ca2 concentration in the cytoplasm. Finally, PLGA scaffolds were prepared and AMSCs were successfully planted onto the scaffolds. The AMSCs fully differentiated into functional SMCs, and the PLGA polymer is a suitable scaffold material for AMSCs. With further clinical trials, AF-derived MSC-based SMC engineering may become a highly efficient treatment option.