Neurosurgery typically requires craniectomy and meticulous dissection to achieve sufficient exposure for subsequent surgical intervention. This highly invasive process requires hours of operating time, long recovery periods and leaves patients with visible surgical scars. Non-invasive high-intensity focused ultrasound (HIFU) has shown some promise yet remains challenged by the attenuation of ultrasonic waves while passing through the skull. Consequently, the clinical impact of this technology remains limited, particularly in the treatment of neuro-oncology. In order to compensate for acoustic attenuation, excessive use of power for HIFU devices has been investigated, although it is undesirable from a regulatory and patient safety standpoint. Here, we report the design and development of a novel HIFU device prototype for neurologic lesion ablation. This device concept is envisioned to access the ventricular space via a minimally invasive ventriculostomy, allowing ultrasound to reach targets deep in the brain, while eliminating the need for high power to penetrate the skull.
Angiogenesis is a key factor for the growth and expansion of malignant tumors. Recently, non-invasive imaging techniques have been largely employed to observe the functional neovascular status of tumor progression. In this study, we present an integrated hybrid-resolution photoacoustic microscopy (PAM) capable of both optical-resolution (OR: a tightly focused beam for finer lateral resolution at shallower region) and acoustic-resolution (AR: a deeper imaging depth based on its ultrasound-dominated detection with relatively large illumination area) imaging for monitoring the progression of angiogenesis. The hybrid-resolution design is achieved by using a liquid lens to adjust the beam size for OR/AR mode selection. A multimode fiber with small core diameter is used to maintain the fine lateral resolution and deliver the laser light with higher energy for OR and AR illumination, respectively. The imaging resolutions of the proposed PAM are demonstrated by phantom experiments: the lateral resolution of OR mode is ~20 μm at a depth of 1 mm, while the resolution of AR mode is ~80 μm at depths of 2 to 3 mm. Additionally, in vivo experiments are conducted to show the capability of this PAM. Angiogenesis imaging of a subcutaneous tumor model in mice is presented using its intrinsic optical contrast (i.e., hemoglobin). Besides, information of oxygen saturation is also acquired using two wavelengths to indicate the hypoxic region of the tumor. In summary, the developed hybrid-resolution PAM is able to monitor the angiogenesis and provide hemodynamic information of tumor covering a broader depth range with high resolutions.
Tumor blood vessels have been known as being heterogeneous because of their chaotic and abundant distribution. Thus, imaging techniques which reveal hemodynamic information of tumor vasculature play significant roles in tumor studies. Photoacoustic (PA) imaging could acquire hemodynamic information based on the intrinsic characteristics of hemoglobin, while ultrasound (US) imaging provides information of structure and blood flow. Therefore, an integrated system was developed for both US microvascular imaging and PA imaging of the tumor region. To further improve the imaging performance, a liquid filled dual-modality microdroplets was designed for both ultrasound flow and PA imaging. The microdroplets were manufactured using the microfluidics technique to produce consistent microbubble with diameters between 23 µm to 25 µm, determining the vascular size for imaging. Additionally, the microdroplets were filled with saline diluted organic nanoparticles as contrast agents for PA imaging, while commercial microbubbles are filled with inert gas. Both in vitro and in vivo studies have been conducted for evaluating the designed contrast agent and system. Results of in vitro experiments, which performed with microtubes submerged in a scattering medium, demonstrated different flow speeds and directions of the designed phantom. Subcutaneous tumor was next tested during in vivo studies. Based on the organic nanoparticle-doped droplet, we were able to obtain the information of total hemoglobin concentration, oxygen saturation and blood flow speed of the tumor angiogenesis region with a higher sensitivity. In the future, our microdroplets could be applied to more applications, such as slow drug release based on its specific structure.
Traditional optical imaging systems are designed for imaging with a single contrast mechanism, and therefore can interrogate only a single neurophysiologic variable. However, the biological complexity underlying neurophysiological function and its alteration in neurodegenerative diseases, requires the simultaneous interrogation of multiple neurophysiologic variables to arrive at a better understanding. Today’s multicontrast optical imaging systems satisfy this need, but suffer from some inherent limitations. Owing to the need to integrate multiple contrast mechanisms, these imaging systems tend to be benchtop-based and unportable, often requiring animals to be anesthetized, custom built and expensive. This limits their widespread adoption. Miniaturization, although technically challenging, remains a potential solution to these limitations. To address this unmet need, here we present the design considerations and practical guidelines for building inexpensive, miniaturized, and portable multicontrast optical neuroimaging systems that allow comprehensive interrogation of brain function in freely behaving rodents. We then showcase an example tri-contrast miniature neuroimaging system and demonstrate the implementation of our guidelines. We conclude by demonstrating the utility of such a miniature multicontrast neuroimaging system by interrogating in an awake rodent the tumor extent, angiogenic vascular sprouting, flow establishment in the newly formed vessels, as well as anomalies in resting-state microvascular fluctuations in a preclinical model of brain tumor progression.
Cathodal-transcranial direct current stimulation induces therapeutic effects in animal ischemia models by preventing the expansion of ischemic injury during the hyperacute phase of ischemia. However, its efficacy is limited by an accompanying decrease in cerebral blood flow. On the other hand, peripheral sensory stimulation can increase blood flow to specific brain areas resulting in rescue of neurovascular functions from ischemic damage. Therefore, the two modalities appear to complement each other to form an integrated treatment modality. Our results showed that hemodynamics was improved in a photothrombotic ischemia model, as cerebral blood volume and hemoglobin oxygen saturation (SO2) recovered to 71% and 76% of the baseline values, respectively. Furthermore, neural activities, including somatosensory-evoked potentials (110% increase), the alpha-to-delta ratio (27% increase), and the (delta+theta)/(alpha+beta) ratio (27% decrease), were also restored. Infarct volume was reduced by 50% with a 2-fold preservation in the number of neurons and a 6-fold reduction in the number of active microglia in the infarct region compared with the untreated group. Grip strength was also better preserved (28% higher) compared with the untreated group. Overall, this nonpharmacological, nonintrusive approach could be prospectively developed into a clinical treatment modality.
Different brain regions exhibit complex information processing even at rest. Therefore, assessing temporal correlations between regions permits task-free visualization of their ‘resting state connectivity’. Although functional MRI (fMRI) is widely used for mapping resting state connectivity in the human brain, it is not well suited for ‘microvascular scale’ imaging in rodents because of its limited spatial resolution. Moreover, co-registered cerebral blood flow (CBF) and total hemoglobin (HbT) data are often unavailable in conventional fMRI experiments. Therefore, we built a customized system that combines laser speckle contrast imaging (LSCI), intrinsic optical signal (IOS) imaging and fluorescence imaging (FI) to generate multi-contrast functional connectivity maps at a spatial resolution of 10 μm. This system comprised of three illumination sources: a 632 nm HeNe laser (for LSCI), a 570 nm ± 5 nm filtered white light source (for IOS), and a 473 nm blue laser (for FI), as well as a sensitive CCD camera operating at 10 frames per second for image acquisition. The acquired data enabled visualization of changes in resting state neurophysiology at microvascular spatial scales. Moreover, concurrent mapping of CBF and HbT-based temporal correlations enabled in vivo mapping of how resting brain regions were linked in terms of their hemodynamics. Additionally, we complemented this approach by exploiting the transit times of a fluorescent tracer (Dextran-FITC) to distinguish arterial from venous perfusion. Overall, we demonstrated the feasibility of wide area mapping of resting state connectivity at microvascular resolution and created a new toolbox for interrogating neurovascular function.
KEYWORDS: Imaging systems, Ischemia, Real time imaging, Photoacoustic spectroscopy, Brain, Hemodynamics, In vivo imaging, Point spread functions, Lead, Blood vessels
Stroke is the second leading cause of death worldwide. Rapid and precise diagnosis is essential to expedite clinical
decision and improve functional outcomes in stroke patients; therefore, real-time imaging plays an important role to
provide crucial information for post-stroke recovery analysis. In this study, based on the multi-wavelength laser and 18.5
MHz array-based ultrasound platform, a real-time handheld photoacoustic (PA) system was developed to evaluate
cerebrovascular functions pre- and post-stroke in rats. Using this system, hemodynamic information such as cerebral
blood volume (CBV) can be acquired for assessment. One rat stroke model (i.e., photothrombotic ischemia (PTI)) was
employed for evaluating the effect of local ischemia. For achieving better intrinsic PA contrast, Vantage and COMSOL
simulations were applied to optimize the light delivery (e.g., interval between two arms) from customized fiber bundle,
while phantom experiment was conducted to evaluate the imaging performance of this system. Results of phantom
experiment showed that hairs (~150 μm diameter) and pencil lead (500 μm diameter) can be imaged clearly. On the
other hand, results of in vivo experiments also demonstrated that stroke symptoms can be observed in PTI model poststroke.
In the near future, with the help of PA specific contrast agent, the system would be able to achieve blood-brain
barrier leakage imaging post-stroke. Overall, the real-time handheld PA system holds great potential in disease models
involving impairments in cerebrovascular functions.
Monitoring brain activities in awake and freely moving status is very important in physiological and pathological studies
of brain functions. In this study, we developed a new standalone micro-device combining electrophysiology and optical
imaging for monitoring the cerebral blood flow and neural activities with more feasibility for freely moving animals.
Myelination is governed by axon-glia interaction which is modulated by neural activity. Currently, the effects of subcellular activation of neurons which induce neural activity upon myelination are not well understood. To identify if subcellular neuronal stimulation can enhance myelination, we developed a novel system for focal stimulation of neural activity with optogenetic in a compartmentalized microfluidic platform. In our systems, stimulation for neurons in restricted subcellular parts, such as cell bodies and axons promoted oligodendrocyte differentiation and the myelination of axons the just as much as whole cell activation of neurons did. The number of premature O4 positive oligodendrocytes was reduced and the numbers of mature and myelin basic protein-positive oligodendrocytes was increased both by subcellular optogenetic stimulation.
The cranial window preparation provides optical access to the rodent brain for high-resolution in vivo optical imaging. Two types of cranial windows are commonly employed, namely the open-skull window and thinned-skull window. Chronic in vivo laser speckle contrast imaging (LSCI) through the cranial window permits characterization of neurovascular morphology and blood flow changes over days or weeks. However, the effects of window type and their long-term stability for in vivo LSCI have not been studied. Here we systematically characterize the effect of each cranial window type on in vivo neurovascular monitoring with LSCI over two weeks. Imaging outcomes for each window were assessed in terms of contrast-to-noise ratio (CNR), microvessel density (MVD) and total vessel length (TVL). We found that the thinned-skull window required a shorter recovery period (~ 4 days), provided a larger field of view and was a good choice for short-term (i.e. < 10 days) in vivo imaging, but not for the long term because of the confounding effects of skull regrowth after ten days. The open-skull window required a longer recovery period, as made evident by the decrease in window quality within the 10-day period. In spite of this, the open-skull window would be preferable for chronic (i.e. < 10 days) in vivo imaging applications. Overall, this study informs about the pros and cons of each cranial window type for LSCI-based neurovascular imaging.
Restoring perfusion to the penumbra during the hyperacute phase of ischemic stroke is a key goal of neuroprotection. Thrombolysis is currently the only approved treatment for ischemic stroke. However, its use is limited by the narrow therapeutic window and side effect of bleeding. Therefore, other interventions are desired that could potentially increase the perfusion of the penumbra. Here, we hypothesized that bilateral peripheral electrical stimulation will improve cerebral perfusion and restore cortical neurovascular response. We assess the outcomes of bilateral forepaw electrical stimulation at intensities of 2 and 4 mA, administered either unilaterally or bilaterally. We developed a combined electrocorticogram (ECoG)-functional photoacoustic microscopy (fPAM) system to evaluate the relative changes in cerebral hemodynamic function and electrophysiologic response to acute, focal stroke. The fPAM system is used for cerebral blood volume (CBV) and hemoglobin oxygen saturation (SO2) and the ECoG for neural activity, namely somatosensory-evoked potential (SSEP), interhemispheric coherence, and alpha-delta ratio (ADR) in response to forepaw stimulation. Our results confirmed the neuroprotective effect of bilateral forepaw stimulation at 2 mA as indicated by the 82% recovery of ADR and 95% improvement in perfusion into the region of penumbra. This experimental model can be used to study other potential interventions such as therapeutic hypertension and hypercarbia.
As a critical basis of functional brain imaging, neurovascular coupling describes the link between neuronal and
hemodynamic changes. The majority of in vivo neurovascular coupling studies was performed by inducing sensory
stimulation via afferent inputs. Unfortunately such an approach results in recruiting of multiple types of cells, which
confounds the explanation of neuronal roles in stimulus evoked hemodynamic changes. Recently optogenetics has
emerged to provide immediate control of neurons by exciting or inhibiting genetically engineered neurons expressing
light sensitive proteins. However, there is a need for optical methods capable of imaging the concurrent hemodynamic
changes. We utilize laser speckle contrast imaging (LSCI) to obtain high resolution display of cerebral blood flow (CBF)
in the vicinity of the targeted neural population. LSCI is a minimally invasive method for imaging CBF in microvessels
through thinned skull, and produces images with high spatiotemporal resolution, wide field of view. In the integrated
system light sources with different wavelengths and band-passing/blocking filters were used to allow simultaneous
optical manipulation of neuronal activities and optical imaging of corresponding CBF. Experimental studies were carried
out in a rodent model expressing channalrhodopsin (ChR2) in excitatory neurons in the somatosensory cortex (S1). The
results demonstrated significant increases of CBF in response to ChR2 stimulation (exciting neuronal firing) comparable
to the CBF response to contralateral forepaw stimulation. The approach promises to be an exciting minimally invasive
method to study neurovascular coupling. The complete system provides a novel approach for broad neuroscience
applications.
Laser speckle contrast imaging (LSCI) is a contrast agent free imaging technique suited for longitudinal assessment of vascular remodeling that accompanies brain tumor growth. We report the use of LSCI to monitor vascular changes in a rodent glioma model. Ten rats are inoculated with 9L gliosarcoma cells, and the angiogenic response is monitored five times over two weeks through a thinned skull imaging window. We are able to visualize neovascularization and measure the number of vessels per unit area to assess quantitatively the microvessel density (MVD). Spatial spread of MVD reveals regions of high MVD that may correspond to tumor location. Whole-field average MVD values increase with time in the tumor group but are fairly stable in the control groups. Statistical analysis shows significant differences in MVD values between the tumor group and both saline-receiving and unperturbed control groups over the two-week period (p<0.05). In conclusion, LSCI is suitable for investigation of tumor angiogenesis in rodent models. In addition, the statistical difference (p<0.02) between MVD values of the tumor (24.40±1.41) and control groups (15.40±1.60) on the 14th day after inoculation suggests a potential use of LSCI in the clinic in distinguishing tumor environments from normal vasculature.
We report the novel use of laser speckle contrast imaging (LSCI) at multiple exposure times (meLSCI) for enhanced in vivo imaging of the microvascular changes that accompany angiogenesis. LSCI is an optical imaging technique that can monitor blood vessels and the flow therein at a high spatial resolution without requiring the administration of an exogenous contrast agent. LSCI images are obtained under red (632 nm) laser illumination at seven exposure times (1-7 ms) and combined using a curve-fitting approach to obtain high-resolution meLSCI images of the rat brain vasculature. To evaluate enhancement in in vivo imaging performance, meLSCI images are statistically compared to individual LSCI images obtained at a single exposure time. We find that meLSCI reduced the observed variability in the LSCI-based blood-flow estimates by 30% and improved the contrast-to-noise ratio in regions with high microvessel density by 41%. The ability to better distinguish microvessels, makes meLSCI uniquely suited to longitudinal imaging of changes in the vascular microenvironment induced by pathological angiogenesis. We demonstrate this utility of meLSCI by sequentially monitoring, over days, the microvascular changes that accompany wound healing in a mouse ear model.
Information processing and propagation in the central nervous system is mostly electrical in nature. At synapses, electrical signals cause the release of neurotransmitters like dopamine, glutamate etc., that are sensed by post-synaptic neurons resulting in signal propagation or inhibition. It can be very informative to monitor electrical and neurochemical signals simultaneously to understand the mechanisms underlying normal or abnormal brain function. We present an integrated system for the simultaneous wireless acquisition of neurophysiological and neurochemical activity. Applications of the system to neuroscience include monitoring EEG and glutamate in rat somatosensory cortex following global ischemia.
Simultaneous mapping of multiple electrical or chemical properties of
neural activity facilitates understanding neurological phenomena and
their underlying mechanisms. We present a track-and-hold potentiostat
performing simultaneous acquisition of 16 independent channels of
current ranging five orders of magnitude in dynamic range over four
scales down to hundreds of picoamperes. Sampling rate ranges from DC
to 200KHz. The system features programmable current gain control,
configurable anti-aliasing log-domain filter, triggered current
integration and provides differential output ready for asynchronous
external analog-to-digital conversion over a compressed dynamic range.
We present system description, circuit implementation and experimental
results of real-time neurotransmitter concentration measurements from
the 16-channel prototype fabricated in a 1.2 micron CMOS process.
Stroke has been shown to cause exitotoxic injury, two of its manifestations being cellular and mitochondrial swelling. In vitro models of stroke attempt to reproduce the effects of stroke by treating brain tissue with excitotoxins or hypotonic solutions. To further resolve the mechanism of stroke injury, we have designed a dual-angle scatter imaging (DASI) system sensitive to particle size. The DASI system has been used with a hippocampal slice preparation to contrast cellular swelling, induced by hypotonicity, and combined cellular and mitochondrial swelling caused by excitotoxicity. We found that both hypotonic end excitotoxic treatments caused changes in light scatter. However, only excitotoxic treatment caused a significant change in DASI.
Currently, methods for the detection of brain edema in patients or laboratory experiments are invasive or inconvenient for continuous monitoring. We have performed experiments on two models of brain edema to determine the validity of differential near infrared spectroscopy (NIR) as a real-time, low cost and noninvasive method of monitoring brain edema. As a chemical in-vitro model, we prepared serial dilutions of Liposyn III, a fat emulsion, to simulate varying degrees of brain water content. NIR light at two wavelengths (703 nm and 957 nm) was used to assess water content of Liposyn solutions. We demonstrated a strong relation between wavelength specific light interactance and water content, for (n equals 4) serial dilutions from 97.6% to 80.0% water, R2 equals 0.985 plus or minus 0.017. Secondly an in vitro brain tissue model was developed to test the NIR method against wet-to-dry water content measurements. A total brain water content range of from 83.5 to 67.6 water was investigated (n equals 4). Using 695 nm and 957 nm NIR light, a correlation between NIR interactance and brain water content was again obtained, R2 equals 0.957 plus or minus 0.027. Our preliminary results suggest differential NIR spectroscopy may serve as an accurate and useful technique for monitoring surface brain edema in clinical and laboratory settings.
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