Pulsed Transcranial Ultrasound Stimulation and Its Applications in Treatment of Focal Cerebral Ischemia and Depression

The aims of this thesis were to investigate the therapeutic effects of pulsed transcranial ultrasound stimulation (pTUS) on focal cerebral ischemia and depression, respectively, in rodent models. Neurological and psychiatric disorders, such as Parkinson's disease, epilepsy, Alzheimer's disease, stroke (vascular disorder that results in neurological defects), depression, and etc., present an increasing challenge and a substantial social and economic burden for an aging and stressed population. However, conventional treatments, especially pharmacologic interventions, have significant limitations, such as nonspecific effects, insufficient tailoring to the individual, adverse effects such as drowsiness, weight gain and nausea, or inadequate uptake into the brain due to the blood-brain-barrier (BBB). In contrast, neuromodulation techniques have gained more attention, which are able to enhance or inhibit the neural activities in specific cortex, such as motor, somatosensory or other areas related to cognition. Neuromodulation thus could potentially restore the disrupted neural network due to neurological disorders. Capitalizing on its noninvasiveness, high precision (in the scale of mm) and penetration depth (several centimeters), low-intensity (typically <1 W/cm2 spatial-peak-pulse-average intensity-ISPTA) low-frequency (typically <1MHz), pulsed transcranial ultrasound stimulation (pTUS) has been emerging as a promising therapeutic tool for neurological and psychiatric disorders. This thesis provided the first in-vivo demonstrations that pTUS might serve as neuroprotective preconditioning of ischemic brain injury and treatment of depression. Additionally, it also proposed a novel optical imaging-based technique to characterize the neuromodulatory effect of pTUS, which facilitates the parameter optimization of therapeutic pTUS in practice. Both suppressive and excitatory pTUS are applied in this thesis. The corresponding pTUS parameters were: (a) suppressive pTUS (or pTUSS): ISPPA = 8W/cm2, frequency (f) = 0.5 MHz, pulse repetition frequency (PRF) = 100 Hz, and duty cycle (DC) =5%, and (b) excitatory pTUS (or pTUSE): ISPPA = 8W/cm2, f = 0.5MHz, PRF = 1.5 kHz, and DC = 60%, respectively. Before the therapeutic experiments, the neuromodulatory effects of both pTUSS and pTUSE were examined using laser speckle imaging(LSCI) and multispectral reflectance imaging (MSRI) in aspect of the neurovascular responses. Specifically, this thesis consists of: (1) Study on the neurovascular response to pTUS. Compared with other methods, such as pTUS-triggered motor response and visual evoked potentials (VEP), optical imaging allows to measure the neurovascular change at high spatiotemporal resolution (in the scale of μm and ms), including cortical suppression without evoked output. LSCI and MSRI were used to monitor the primary somatosensory response (Chapter 2) to hind limb electrical stimulation before, immediately, and 1 h after 5-min application of pTUSS and pTUSE, respectively. Several indicators, including Response Index, Peak Response, Latency and Response Duration, were derived from optical images to characterize the neuromodulatory effects of pTUS on primary somatosensory cortex. Our results showed that pTUSS could suppress the primary somatosensory cortex across all rats whereas pTUSE only presented excitatory effects in 5 out of 11 rats. The neuromodulatory effects of pTUS were correlated with the baseline cortical excitability. The results showed that: (i) pTUSs could serve in investigating cognitive function by silencing the neurons in the target region; (ii) pTUSE exposure should be treated with caution due to individual differences in neuromodulatory effects, which were associated with the initial brain state of rats; and (iii) optical imaging was useful in evaluating the pTUS neuromodulatory effects. (2) Neuroprotection of preconditioning pTUS. By applying suppressive pTUS, it was investigated whether the severity of stroke could be minimized or alleviated by prior exposure to ultrasound stimulation (Chapter 3). Preconditioning was supposed to increase the tolerance of brain to subsequent ischemic insult. It can potentially be used to prevent the perioperative stroke in patients undergoing cardiovascular surgeries with a series of complications. Considering the noninvasiveness and safety of ultrasound, pTUS may provide a novel preconditioning method. To test the effectiveness of preconditioning pTUS, rats were randomly assigned to control (n=12) and preconditioning pTUS (pTUS-PC) groups (n=14). The pTUS-PC animals received ultrasound stimulation before the induction of photothrombotic stroke, whereas control animals were handled identically except the ultrasound stimulation. The cerebral blood flow was monitored using LSCI in both groups during stroke induction, as well as 24 hours and 48 hours after stroke, respectively. Also, infarct volumes and edema were measured at 48 hours after euthanatizing the rats. Results showed that pTUS-PC rats had smaller ischemic volume during stroke induction, as well as 24 hours and 48 hours after the stroke than the controls. Moreover, the pTUS-PC group showed lower volume of brain edema than the control group. (3) Antidepressant-like effect by pTUS. The potential antidepressant-like effects of pTUS were further investigated in a rat model of depression with excitatory pTUS. Stimulating the left prefrontal cortex (PFC) by TMS has been clinically used for depression treatment, it was thus hypothesize that pTUSE on PFC would act similarly with TMS and result in antidepressant-like effect. To test this hypothesis, pTUS was applied for 2 weeks daily to the left PFC of depressed rats induced by 48-hour restraint. The long-term (3 weeks) efficacy of the depression model as well as the antidepressant-like effects of pTUS were investigated with a group of behavioral tests. In addition, the hippocampal BDNF was measured by western blot to study the mechanisms underlying antidepressant-like effects of pTUS. The safety of long-term (2 weeks) pTUS was assessed by histologic analysis. Results showed that 48-hour-restraint stress could stably lead to at least 3-week reduction of exploratory behavior and protracted anhedonia, whereas pTUSE treatment could successfully reverse the depression-like phenotypes and promote the BDNF expression in the left hippocampus. In addition, H& E staining of brain tissues confirmed the safety of the long-term pTUS treatment. In conclusion, the results in this work suggested that pTUS could serve as preconditioning of perioperative stroke and therapeutics for depression. Additionally, the results also demonstrated that optical neurovascular imaging could measure the neuromodulatory effect of pTUS. This study documented more evidence that pTUS is a promising tool for basic neuroscience and therapeutic applications. KEY WORDS: Neurological and psychiatric disorders, brain stimulation, pulsed ultrasound stimulation, neurovascular imaging, preconditioning, stroke, depression.Ph.D., Biomedical Engineering -- Drexel University, 201

Early brain activity : Translations between bedside and laboratory

Neural activity is both a driver of brain development and a readout of developmental processes. Changes in neuronal activity are therefore both the cause and consequence of neurodevelopmental compromises. Here, we review the assessment of neuronal activities in both preclinical models and clinical situations. We focus on issues that require urgent translational research, the challenges and bottlenecks preventing translation of biomedical research into new clinical diagnostics or treatments, and possibilities to overcome these barriers. The key questions are (i) what can be measured in clinical settings versus animal experiments, (ii) how do measurements relate to particular stages of development, and (iii) how can we balance practical and ethical realities with methodological compromises in measurements and treatments.Peer reviewe

THE EFFECTS OF EARLY LIFE STRESS ON LONG-TERM POTENTIATION IN PATHWAY FROM THE MEDIAL PREFRONTAL CORTEX TO THE BASOLATERAL AMYGDALA

A leading neurocircuitry model of emotional regulation points to the pathway from the medial prefrontal cortex (mPFC) to the basolateral amygdala (BLA). This pathway has been implicated in fear conditioning and extinction studies and its malfunction is hypothesized to underlie affective disorders such as PTSD and anxiety. Interestingly, the mPFC-BLA pathway shows delayed maturation in both humans and rats, rendering it vulnerable to early life stress (ELS). Indeed, several studies have linked ELS to emotional dysregulation as well as changes in the amygdala and PFC. However, no study has ever been done on the effect of ELS on long-term potentiation (LTP) in this pathway. In fact, very few studies on LTP in the mPFC-BLA pathway have been conducted at all which is surprising given LTP’s role in learning and memory and given the mPFC-BLA pathway’s proposed role in fear conditioning/extinction. Therefore, using electrophysiological methods in awake, freely behaving rats, the current study examined whether ELS in the form of neonatal isolation (ISO) affects LTP in the mPFC-BLA pathway. Results indicate that the mPFC-BLA pathway is resistant to LTP in both control and ISO rats following both sustained and theta burst high frequency stimulation (HFS). In fact, rats showed a tendency toward long-term depression (LTD) especially following sustained stimulation at 200 Hz. Small sample sizes prevented a meaningful comparison of LTD across ISO and control groups

Low-Intensity Ultrasonic Neuromodulation of the Rat Hippocampus

Techniques to non-invasively modulate brain activity are important for mapping human brain circuits, and also for the treatment of a host of neurological and psychiatric disorders marked by aberrant brain activity. Though a wide range of techniques for non-invasive neuromodulation have been proposed, the conventional approaches suffer from significant limitations. Most notably, focal stimulation of deep brain regions is presently only possible with invasive optogenetic and chemogenetic approaches that require craniotomies and genetic access to the brain. Transcranial focused ultrasound stimulation (tFUS) possesses many of the characteristics desirable from a neuromodulation approach: non-invasiveness, a spatial resolution in the order of millimeters, the ability to penetrate deep brain regions even through a large cranium, and compatibility with MRI. Although it is known that ultrasonic pressure waves may be focused through the skull and mechanically stimulate neurons in targeted brain areas, there is considerable uncertainty about the mechanism of action, and importantly, how to tailor tFUS to produce a desired electrophysiological change (e.g. excite or inhibit activity in a targeted brain region). In particular, the influence of baseline brain state on the sensitivity to tFUS is unknown. Another important gap in knowledge is the effect of the stimulation parameters, for example the acoustic intensity and waveform shape, on stimulation outcome. Addressing these gaps is critical to advancing tFUS as a non-invasive neuromodulation technique. The aims of this dissertation are two-fold: (1) to identify the influence of pre-stimulation brain state on the neuronal response to tFUS, and (2) to identify the effect of waveform and intensity (collectively termed “the dose”) on the neuronal response to tFUS. Our working hypothesis in Aim 1 is that the level of synaptic input into the neuron leading up to stimulation, as captured by the power spectrum of the local field potential (LFP), predicts sensitivity to tFUS. Our working hypothesis in Aim 2 is that amplitude-modulated stimulation will produce qualitatively different outcomes compared to the conventionally tested pulsed and continuous wave stimulation. To test these hypotheses, we conducted electrophysiological recordings from the hippocampus during tFUS in n\u3e100 anesthetized rats. Our main findings are that: (1) high levels of gamma band (30-200 Hz) and theta band (3-10Hz) and low levels of delta band (1-3 Hz) LFP power leading up to tFUS promote successful neuromodulation (Chapter 3), (2) novel low-intensity amplitude modulated tFUS is capable of bimodal modulation of theta band powers (chapter 4), and (3) mechanical displacement of the probe caused by tFUS leads to an unexpected dominant electrophysiological artifact in the LFP: consisting of a slow time-locked response to sonication onset that is largely preserved across amplitude modulated, continuous wave, and pulsed wave stimulation (Chapter 5). These findings emphasize the importance of considering ongoing brain state when performing tFUS, while shedding light on the neurophysiological substrate of low-intensity ultrasonic neuromodulation

Somatosensory stimuli trigger coordinated oxytocin neurons activity during social interaction

The hypothalamic neuropeptide oxytocin (OT) promotes social communication via its central release in the mammalian brain. However, how social interaction affects electrical activity of OT neurons is still unclear. To address this question, I used cell-type specific viral vectors in combination with optoelectrode-based techniques. I performed the in vivo single-unit recording of optically identified OT neurons in the paraventricular nucleus (PVN) of hypothalamus in adult female rats during their social interactions with unfamiliar female conspecifics. Simultaneously, we monitored behavior and recorded ultrasonic vocalizations. The results showed that active social interactions events induce an increase of PVN OT neurons spiking activity as well as a re-organization of the firing pattern from regular to bursting. The action potentials of simultaneously recorded OT neurons were synchronized and phase-locked with the PVN theta oscillations precisely at the time of social interactions, but not during non-social exploratory behavior. To decipher which sensory stimuli trigger OT neuron activity, I performed experiments with partial deprivation of specific sensory modalities. Direct physical contact between rats, or even gentle skin stimulation, led to a profound increase in OT firing rates. In contrast, presentation of visual, auditory and olfactory social-relevant stimuli alone did not significantly alter OT neuron activity. This led to the conclusion somatosensory component of social interaction drives OT neurons synchronous activity. To further explore the effects of tactile stimuli on the OT system, I examined the expression of the marker of neuronal activity c-Fos after repetitive somatosensory stimulation; it appeared to be significantly increased in a particular subpopulation of OT neurons named parvocellular OT neurons. Employing in-vivo calcium recording via fiber photometry, I investigated the role of parvocellular OT neurons in regulating the activity of the general population of PVN OT neurons, finding that parvocellular OT neurons mediate the activation of the OT system in response to somatosensory stimuli. Next, I selectively modulated the activity of parvocellular OT neurons in awake freely moving rats via pharmacogenetics: activation of this population of neurons resulted in increased social interaction, while inhibition leaded to decrease of social interaction. Finally, I studied the effect of intracerebral infusion of an OT receptor antagonist which induced a substantial reduction of social interaction time, even when parvocellular OT neurons were activated. Altogether, these results indicate that somatosensory stimulation is essential to activate OT neuron ensembles and, hence, can induce central neuropeptide release in socially interacting female rats. This opens perspectives for studying functional and anatomical connectivity between the somatosensory and OT systems in normal and psychopathological conditions

Tissue acidosis associated with ischemic stroke to guide nimodipine delivery

Background: Ischemic stroke is a leading cause of death and disability worldwide. Yet, the effective therapy of focal cerebral ischemia has been an unresolved challenge. We propose here that ischemic tissue acidosis, a sensitive metabolic indicator of injury progression in cerebral ischemia, can be harnessed for the targeted delivery of neuroprotective agents. Ischemic tissue acidosis, which represents the accumulation of lactic acid in malperfused brain tissue is significantly exacerbated by the recurrence of SD events. Deepening acidosis itself activates specific ion channels to cause neurotoxic cellular Ca2+ accumulation and cytotoxic edema. These processes are thought to contribute to the loss of the ischemic penumbra. Importantly, acidosis in the ischemic penumbra may also be used to guide therapeutic intervention. Nimodipine, an L-type VGCC antagonist dilates cerebral arterioles, but its systemic administration may cause potential side effects (mainly hypotension). We have constructed chitosan nanoparticles as drug carriers, which release nimodipine in response to decreasing pH typical of cerebral ischemia. Here we have set out to evaluate this nanomedical approach to deliver nimodipine selectively to acidic ischemic brain tissue. Methods: Two sets of experiments are presented in this thesis. In Experimental Project I, nimodipine was applied in solution (100 μM), then global forebrain ischemia was induced in half of the animals by bilateral common carotid artery occlusion under isoflurane anesthesia. Functional hyperemia in the somatosensory cortex was created by mechanical stimulation of the contralateral whisker pad under α‐chloralose anesthesia. SD events were elicited subsequently by 1 M KCl. LFP and CBF in the parietal somatosensory cortex were monitored by electrophysiology and LDF. In Experimental Project II, nimodipine was associated with pH-sensitive nanoparticles in suspension. After washing the nanoparticle suspension with or without nimodipine (100 μM) on the exposed brain surface of anesthetized rats, both common carotid arteries were occluded to create forebrain ischemia. SDs were elicited by 1 M KCl to deepen the ischemic insult. LFP, CBF and tissue pH were recorded from the cerebral cortex. Microglia activation and neuronal survival were evaluated in brain sections by immunocytochemistry. Results: Nimodipine in solution attenuated evoked potentials and SD. In addition to the elevation of baseline CBF, nimodipine augmented hyperemia in response to both somatosensory stimulation and SD, the drug effect was particularly discernable under ischemia. Ischemia-induced tissue acidosis initiated nimodipine release from nanoparticles, confirmed by the significant elevation of baseline CBF. Nimodipine shortened the duration of both SD itself, and the associated tissue acidosis, moreover it enhanced the SD-related hyperemia. Chitosan nanoparticles did not activate microglia. Conclusions: The administered nanoparticles release nimodipine in acidic tissue environment, which reliably delineates sites at risk of injury. The data support the concept that tissue acidosis linked to cerebral ischemia can be employed as a trigger for targeted drug delivery. Nimodipine-mediated vasodilation and SD inhibition can be achieved by pH-responsive chitosan nanoparticles applied directly to the brain surface. Ultimately, this approach may offer a new way to treat stroke patients with the hope of more effective therapy, and better stroke outcome

High precision optoacoustic neural modulation

Manipulation of brain circuits is a critical to understanding how brain controls behaviors under normal physiological conditions and how its dysfunction causes diseases. Ultrasound stimulation is an emerging neuromodulation modality that allows activation of neurons with acoustic waves. However, the piezo based transcranial ultrasound stimulation offers poor spatial resolution, which hinders the understanding of its mechanism as well as application in region specific activation in small animals. To address this limitation, we developed a series of neuromodulation techniques utilizing the photon to sound conversion capability offered by the optoacoustic effect. In chapter 2, we developed a fiber based optoacoustic converter th-at allows neural stimulation at submillimeter spatial precision both in vitro and in vivo. In chapter 3, the spatial resolution was further improved by tapered fiber optoacoustic emitter to achieve stimulation of single neurons and even subcellular structures in culture. In chapter 4, we developed photoacoustic nanoparticle based neural stimulation that allows direct activation of neurons through optoacoustic waves generated by nanoparticles bonded to the neuronal membrane. Finally, in chapter 5, in an effort to improve penetration depth, a split ring resonator based microwave neuromodulation was developed that allows wireless stimulation and inhibition of neurons with subwavelength spatial resolution. Together, these methods offer an enabling platform with opportunities to understand the mechanism of acoustic neural stimulation as well as potential for treatment of neurological diseases with high precision neuromodulation

Induction of a torpor-like hypothermic and hypometabolic state in rodents by ultrasound

Torpor is an energy-conserving state in which animals dramatically decrease their metabolic rate and body temperature to survive harsh environmental conditions. Here, we report the noninvasive, precise and safe induction of a torpor-like hypothermic and hypometabolic state in rodents by remote transcranial ultrasound stimulation at the hypothalamus preoptic area (POA). We achieve a long-lasting (\u3e24 h) torpor-like state in mice via closed-loop feedback control of ultrasound stimulation with automated detection of body temperature. Ultrasound-induced hypothermia and hypometabolism (UIH) is triggered by activation of POA neurons, involves the dorsomedial hypothalamus as a downstream brain region and subsequent inhibition of thermogenic brown adipose tissue. Single-nucleus RNA-sequencing of POA neurons reveals TRPM2 as an ultrasound-sensitive ion channel, the knockdown of which suppresses UIH. We also demonstrate that UIH is feasible in a non-torpid animal, the rat. Our findings establish UIH as a promising technology for the noninvasive and safe induction of a torpor-like state