16771
Effects of Ultrasounds on Human Neuron Connectivity and Microglia Activation: Potential for the Therapeutic Use of Transcranial Ultrasonography in Autism
We and others demonstrated that non-thermal ultrasounds (US) used for diagnostic imaging in transcranial ultrasonography (TUS), improve mental states, reduce chronic pain and anxiety, modulate neurotransmission, and have positive effects on heart frequency, blood pressure and muscle strength (Brain Stimul. 2013 May;6(3):409-15. Neuropsychobiology. 2012;65(3):153-60. The Journal of IiME, vol. 6 (1), p 23-28, 2012). Therefore, it can be hypothesized that TUS might be useful in the treatment of some of the most disturbing symptoms of autism. However, the precise mechanism of action of US on neurons and microglia cells has not been elucidated as yet.
Objectives:
We decided to treat human neurons and microglia cells in culture with US identical to those used in TUS, and to evaluate whether the changes observed in vitro were consistent with the observations reported in vivo.
Methods:
Human neuronal cells SH-SY5Y, and microglia cells were obtained from the Istituto Zooprofilattico Sperimentale della Lombardia e dell’Emilia-Romagna, Brescia, Italy. Cells were routinely maintained at 37°C in a humidified atmosphere of 5% CO2 in Eagle’s minimum essential medium in Earle’s Balanced salt solution (45%), Ham’s F12 medium (45%), foetal bovine serum (FBS) 10%. Cells were treated with US using an Esaote MyLabFive system. A linear probe for TUS was applied onto the Petri dishes for 1 min after having assessed that US were able to cross the plastic well and penetrate the cell monolayer. Immediately thereafter, living cells were observed by phase contrast microscopy without any type of fixation or staining.
Results:
After 1 min US treatment, a significant change in neuronal cell morphology and cell-to-cell connectivity could be observed. US-treated neurons showed a more elongated shape; numerous cytoplasmic bridges between cells could be observed. The increase in neuronal connectivity could be clearly appreciated in those naked areas of the well where thin cytoplasmic elongations connected distant cells. The number of such connections was greatly increased in US-treated neurons. Untreated microglia cells in culture were constantly activated by the presence of serum, and showed the typical round shape that is observed in pathologic, pro-inflammatory microglia activation. After 1 min US treatment, however, microglia cells reverted to the so called ramified state that indicates quiescence and is associated with the lack of production of neurotoxic factors and pro-inflammatory signaling molecules.
Conclusions:
Although TUS is not yet part of autism therapeutic protocols, its effectiveness in areas such as neurological/psychiatric intervention, long-term alertness/wakefulness, behavioural reinforcement, anxiety/stress reduction, cognitive enhancement and pain intervention, suggests that it might be useful in autism treatment as well. Since decreased neuronal connectivity and microglia activation are common neuro-pathological findings in autism (PLoS One. 2013 Jun 18;8(6):e67329), our results demonstrate that the observed effects of TUS can be ascribed to restoration of neuronal connectivity and inhibition of pro-inflammatory microglia activation.