A MINIATURE LASER SPECKLE CONTRAST IMAGER FOR MONITORING OF THE NEURO-MODULATORY EFFECT OF TRANSCRANIAL FOCUSED ULTRASOUND STIMULATION

Abstract

Transcranial focused ultrasound stimulation is a neuromodulation technique that is capable of exciting or suppressing the neural network. Such neuro-modulatory effects enable the treatment of brain diseases non-invasively, such as treating stroke. The neuro-modulatory effect on cerebral hemodynamics has been monitored using laser speckle contrast imaging in animal studies. However, the bulky size and stationary nature of the imaging system constrains the application of this imaging technique on research that requires the animal to have different body positions or to be awake. We present the design of a system that combines a miniature microscope for laser speckle contrast imaging and transcranial focused ultrasound stimulation, as well as, test its capability to monitor cerebral hemodynamics during stimulation and compare the result with a benchtop imaging system.

1. INTRODUCTION

Transcranial focused ultrasound stimulation (tFUS) is a neuromodulation technique that delivers ultrasound pulses to a specific brain region which in turn, excites or suppresses neural activity. In comparison to other neuromodulation techniques, tFUS provides a deeper penetration depth and higher spatial resolution via non-invasive methods. It has been reported that tFUS can be used to treat many diseases. For example, Min et al. showed that tFUS can suppress chemically induced acute epilepsy [1]. Zhang et al. showed that prefrontal ultrasonic stimulation improved the depression-like behaviors of depressed rats [2]. Other studies have shown that tFUS can mitigate ischemic brain injury either as preconditioning or immediately after the injury was induced [3, 4].

Many techniques have been used to monitor the neuro-modulatory effects of tFUS, such as local field potential to monitor neural activity, electromyography (EMG) to monitor muscle movement response, and functional magnetic resonance imaging (fMRI) to monitor cerebral hemodynamic response [5]. Laser speckle contrast imaging (LSCI) is a dye-free, wide area, continuous blood flow imaging technique that has been successfully applied with tFUS in imaging blood flow in the cerebral cortex [3, 6]. As mentioned previously though, the bulky size and stationary nature of the imaging system constrains the application of this imaging technique on research that requires the animal to be awake or to have different body positions. With the goal of performing ultrasound stimulation and using optical imaging to record the stimulation effect, a small-size, integrated imaging system is needed.

In our group we are interested in studying various applications of ultrasound in the clinic [8–13] and in this work we aim to study the effects of ultrasound neuromodulation using real-time optical monitoring technique. We have made a miniature LSCI imager that is capable of monitoring cerebral blood flow (CBF) in a freely moving animal [7]. In this study, we aim to combine this miniature LSCI imager and a tFUS system to stimulate the rat’s brain and monitor the hemodynamic changes, and compare the results with a benchtop LSCI imaging system.

2. METHODS

2.1. Animal preparation

All experimental procedures were approved by the Johns Hopkins Medical Institute Animal Care and Use Committee,and conducted in accordance with the National Institutes of Health guidelines for the care and use of laboratory animals.

One male adult Wistar rat (375g, 10 weeks old; Charles River, Wilmington, MA) was used to test the setup. Anesthesia was induced with 2% isoflurane (Baxter, IL, USA) carried by 50% oxygen and 50% nitrogen gas at 4 L/min via nose cone. The rat was placed on a stereotaxic apparatus and the head was fixed using ear bars. A median axial incision was made in the scalp, and a 5 by 5mm window over the left parietal bone of the skull was removed to expose the brain using a high-speed dental drill (Vogue Professional 6000, USA). After the surgery, the rat along with the stereotaxic apparatus was moved to the imaging bench. The miniature LSCI microscope was mounted on the cranial window when obtaining images of cerebral blood flow (Fig.1).

FIGURE 1:

A: Schematic of the experiment setup. Left: benchtop LSCI system setup; Right: miniature LSCI imager setup. (1) CCD camera and the objective lens; (2) laser source; (3) ultrasound transducer with collimator; (4) miniature LSCI imager; (5) optic fiber. B: A real picture of the experiment setup which shows the miniature LSCI imager and the benchtop LSCI camera.

2.2. The tFUS System

We used a custom-built tFUS system, which generated a pulsed signal and amplified it to 180 Vpp to drive a single element immersion type transducer (25.4 mm in diameter, V301, Olympus, USA). A custom-designed acoustic collimator (7 mm diameter output aperture) filled with ultrasound gel was used to transmit the ultrasound waves to the rat’s brain. The transducer was tilted 45 degrees vertically to avoid interference with the imaging system. The pulsed signals used for stimulation were: (1) fundamental frequency 500kHz; (2) tone burst duration 500us; (3) number of tone bursts 30; (4) duty cycle 75%, 50% and 25%. The stimulation was repeated every one second, that is pulse repetition frequency 1Hz (fig. 2 D).

FIGURE 2:

A: example view of the laser speckle contrast imaging which shows the 5 by 5 mm cranial window and the stimulation site. B & C: enlarged view of the cranial window before and after ultrasound stimulation, the brighter blood vessels in the after stimulation indicates an increase of blood flow. D: an example of the ultrasound pulse sequences.

2.3. Laser Speckle Contrast Imaging

In this study, we used a miniaturized LSCI imager suitable for head mounting and CBF monitoring. Coherent 780 nm laser light generated by a laser light source illuminated the cortex through a multimode optical fiber bundle fixed onto the supporting frame of the imager (Fig.1). The reflected light was collected by a macro lens system and imaged by a high-resolution CMOS camera at a frame rate of 15 frames/s [7]. The benchtop LSCI system consists of a CCD camera, an objective lens, and a 780 nm laser light source. A lifting platform placed under the stereotaxic apparatus was used to adjust the distance between the objective lens and the mouse’s head, for image focusing. The laser speckle images were acquired at 15 frames/s and the exposure time was 5 ms.

2.4. Test Protocol and Data Processing

After the surgery and setup of all the instruments, the test to compare the miniature LSCI imager and benchtop system was conducted. The test consisted of two phases, in the first phase the benchtop system was used and in the second phase the miniature imager was used. There were different strengths of the stimulation (different duty cycles) in each phase, and we performed 5 trials of tFUS for each duty cycle. Each trial consisted of a baseline period of 10 seconds and a stimulation period of 20 seconds. The tFUS started at the beginning of the stimulation period and continued until the end of the stimulation period. The timing and synchronization of the imaging and tFUS were controlled by the computer.

After performing the two phases of tFUS, the raw image data was processed to compare these two systems. We used custom-coded MATLAB scripts to process all of the raw image data. An example view of the LSCI image is shown in Fig. 2 A–C. The blood flow within the cranial window was computed for each trial. The blood flow data was normalized to the average value in the baseline period for each trial, and was averaged among all trials in the same phase for each time point to show the change of cerebral hemodynamic following tFUS. The average value of blood flow in the stimulation period of each trial and each duty cycles was calculated and plotted as a boxplot to show the difference between different duty cycles.

3. RESULTS AND DISCUSSION

We have done a preliminary experiment and the result is shown in Fig. 3. This is one of a series of experiments with a goal to demonstrate the feasibility of the experimental setup and compare the two systems to monitor the effect of tFUS. This preliminary result will help us to improve our experimental procedure and generate a final result. The sample size is still small, so we are planning more experiments. More data will be presented after finishing the following experiments.

FIGURE 3:

Preliminary results. A: The blood flow change recorded by the benchtop LSCl system. B: The blood flow change recorded by the miniature LSCI imager. The blood flow data was normalized to the average value during the baseline period and was averaged among all trials for each time point. C: Boxplot of the average of CBF during stimulation. The boxplots are separated by different duty cycles, and the label “B” is for benchtop LSCI system and the label “M” is for miniature LSCI imager.

The result shows that the normalized CBF in the stimulation period was increased as compared to the baseline period, which indicates that the tFUS induced a change in cerebral hemodynamics (Fig. 3 A & B). The boxplot shows that the highest increase is observed when the duty cycle is 75%, and the increase of CBF is similar when duty cycle is 50% or 25%. Generally, the change of CBF detected by the miniature imager is similar to the benchtop system. Unfortunately, we didn’t observe the return of the CBF to baseline because the imaging was stopped at 30 seconds. The boxplots show the increase of CBF more clearly. The mean and variance of the change of CBF is similar between the miniature imager group and benthop system group, except for 25% duty cycle, where the benchtop system detected a higher variance of change of CBF.

This result demonstrates that our experimental setup is able to detect the cerebral hemodynamic change induced by tFUS, and there is a graded change of blood flow with different duty cycles. The change of CBF detected by the miniature imager is similar to the benchtop system.

4. CONCLUSION

This study seeks to demonstrate the feasibility of using a miniature LSCI imager to record the cerebral hemodynamic changes induced by tFUS. The preliminary result demonstrated the experimental setup is feasible. Following experiments will be conducted to compare the two systems using the protocol derived from the preliminary experiment.

Nomenclature

tFUS

Transcranial focused ultrasound stimulation

LSCI

Laser speckle contrast imaging

CBF

Cerebral blood flow