PhD Research Work

1. 

   Photoacoustic Imaging of alcohol-induced changes and identification of affected neural circuits

   
   

   A 2D multispectral photoacoustic imaging system shown in Figure 1, was used to analyze the changes in cerebral vessel diameter and hemoglobin oxygen saturation in High and Low Alcohol Preferring (LAP) mice. Evaluation of these two major parameters helped to understand the alcohol-induced changes in the mouse brain as well as in identifying the neural circuits for targeted modulation.

   
   

   Fig 1: Photoacoustic imaging system for non-invasive in vivo imaging of the cerebral vasculature in alcohol preferring mice. TM-Thermoelectric thermometer with water heater, TLS-Translational linear stage.


2. 

   PAT Imaging of Alcohol-Induced Changes in Cerebral Vessel Diameter

   
   

   A significant difference in optical absorption between hemoglobin, deoxyhemoglobin, and surrounding tissues was observed in HAP and LAP mice, resulting in good contrast and enabling the clear identification of cerebral blood vessels in the PAT images. First, a normalized Gaussian curve was fitted and then its full width half maximum (FWHM) was calculated to extract and calculate the blood vessel diameters.

   
   

   Fig 2: Estimation and comparison of selected blood vessels between HAP and LAP. A, Details of a Normalized Gaussian fit for one of the vessels of interest from which full width at half maximum (FWHM) is estimated (ICV- Inferior cerebral vein, MCA- Middle cerebral artery, Acer: -Anterior Cerebral Artery). B, Results of statistical analysis and comparison between selected vessels for 5 HAP and 5 LAP (3 x 5 vessels for each animal model). C(i) and C(ii), Typical PAT image of HAP and LAP mouse cerebral cortex respectively at 800nm wavelength.

   
   

   The results demonstrates that vessel diameter is statistically and significantly larger in low alcohol preferring mice, compared to their high alcohol preferring counterparts.


3. 

   PAT Imaging of Alcohol-Induced Changes in Hemoglobin Oxygen Saturation

   
   

   To evaluate the changes in hemoglobin oxygen saturation, photoacoustic signals were collected at 720nm and 840nm wavelengths, considering the molar extinction coefficient spectra of oxyhemoglobin (HbO) and deoxyhemoglobin (HbR). Figure 3 (a-c) shows the oxygen saturation maps over time as well as the comparison of percentage change in vessel diameter and hemoglobin oxygen saturation.

   
   

   Fig 3: Estimation and comparison of percentage change in vessel diameter and hemoglobin oxygen saturation with time for a selected vessel between HAP and LAP. A: Demonstration of drop in vessel diameter for the selected vessels. B: Comparison of the change in vessel diameter for HAP and LAP, C: Corresponding change in hemoglobin oxygen saturation for the same vessel.


4. 

   Finite Element Modeling (FEM) for optimizing LIFU parameters and its guiding process

   
   

   An FEM-based simulation model featuring a focused transducer and a detailed mouse head, including skull and brain tissue, was developed to investigate acoustic signal distribution within an animal brain. This simulation assessed several key parameters:
   
   

   1. the efficiency of LIFU frequencies from 0.5 to 1.3 MHz
   2. the effects of sound velocity and attenuation in mouse brain tissue on LIFU propagation, and
   3. how these parameters influence the power density and dimensions of the focal zone.

   
   

   Fig 4: Distribution of acoustic pressure, intensity, and heat around the focal zone. A. Distribution of axial acoustic pressure and intensity. B. Distribution and Analysis of intensity distribution at focal zone. The lateral beam width at focus is estimated to be 0.6 mm while the axial beam width is 2 mm. C/E Distribution of temperature at focus after 2 s of sonication. Tissue temperature rises by 0.08° C, less than 0.1° C.


5. 

   Low Intensity Focused Ultrasound (LIFU) Stimulation for the Treatment of Alcohol Use Disorder

   
   

   Using a mouse brain atlas, the stereotaxic coordinates of the medial and lateral NAc were marked on the scalp using an indelible marker. A coronal line was then marked across these points to prepare the animal for PAT imaging. A coronal slice of the mouse brain was taken along a plane passing through the marked line to reveal the NAc. Then the PAT image was analyzed to determine the depth of the NAc. This depth was used to adjust the focal length of the LIFU transducer for accurately focusing the ultrasound to the NAc.

   
   

   Fig 5: Animal preparation for LIFU stimulation. A: Illustration of mouse in stereotaxic frame with B: Mouse scalp showing the bregma and lambda lines. C: Stimulation point for the lateral and medial NAc. D: LIFU stimulation setup. The mouse is wrapped with a custom wrap which restraints and keeps it warm and placed beneath the degassed water tank. A thin plastic membrane is used to cover mouse head as it protrudes beneath the water tank and ultrasound gel used for coupling.


6. 

   Activation of the NAc Following LIFU Stimulation

   
   

   PAT images were acquired for 1s each before, during and after LIFU stimulation, for a total of 3s. and averaged for each second. The signal amplitude across the lateral and medial NAc (for left -L and right-R NAc) shell/core and the change in PAT signal during across each NAc were measured. Through this stimulation process, LIFU affects neuronal excitability of NAc in two ways: by applying mechanical stress that changes the flexibility of cell membranes, and by influencing proteins in the membrane that are sensitive to mechanical forces, which in turn alters the opening and closing of receptor channels. Furthermore, LIFU also activates voltage-gated sodium and calcium channels, leading to the generation of action potentials and the transmission of signals between neurons located at NAc.

   
   

   Fig 6: Change in PAT signal intensity before, during and after LIFU stimulation. A: PAT functional Images B: Signal Intensity at each NAc. C: Change in PAT signal intensity across each NAC.


7. Validation of the UT imaging platforms with phantom experiments
   
   
   Several phantom experiments were conducted to validate the quantitative capabilities of our UT system. For the phantom, glycerin was used to emulate the sound speed of brain tissue, adjustable from 1500 to 1750 m/s by varying the glycerin concentration, and graphite to mimic tissue sound attenuation, adjustable from 0.3 to 1 dB/cm with varying graphite concentration. The acoustic parameters of these phantoms were cross validated by measuring their physical properties with a 1 MHz unfocused transducer and a needle hydrophone. This involved comparing the literature values and physical acoustic values of the phantoms with the average values from our UT system’s quantitative images, achieving an error margin of less than 6%.

   
   

   Fig 7A: (a) Schematic of phantom with an internal target. (b) Table depicting the parameters of four phantoms with varying sizes and acoustic properties. (c) Photographs of the four phantoms.

   
   

   
   

   Fig 7B: Reconstructed UT images of acoustic velocity and attenuation.