Optison

Spermatogonial stem cell transplants were performed 9–15 weeks after busulfan treatment (autologous – unilateral; allogeneic - bilateral). In biopsied animals, autologous transplants were performed into the contralateral testis. Cryopreserved donor cells were recovered for transplant from storage in liquid nitrogen, as described (Hermann et al., 2009 (link); Hermann et al., 2007 (link)). In some cases, donor cells were enriched for spermatogonia, including SSCs, on a 24% Percoll cushion (GE Healthcare Life Sciences, Piscataway, NJ) prior to transplant (see Figure S1 and Table S2). Cells were then suspended at approximately 100 × 10⁶ cells/ml in MEMalpha medium (Invitrogen) containing 10% FBS, 20% trypan blue, 20% Optison (ultrasound contrast agent; GE Healthcare, Waukesha, WI) and 0.7mg/ml DNase I in a total volume of ≤ 1ml, depending on recipient testis size and available cells. SSC transplants were performed using ultrasound-guided rete testis injections (Figure 1 and Movie S1). For this purpose, a 13MHz linear superficial probe was used to visualize the rete testis space on a MicroMaxx ultrasound machine (Sonosite, Bothell, WA) and guide a 25Ga 2′ spinal needle into the rete testis. Cells were injected under slow constant pressure and chased with saline.

In order to establish the contrast agent best suited for in vivo SHAPE for lower frequency applications (< 7 MHz), such as cardiac and portal vein pressures, five contrast agents were compared: Definity (Lantheus Medical Imaging, N Billerica, MA, USA), Sonazoid (GE Healthcare, Oslo, Norway), Levovist (Schering AG, Berlin, Germany), Optison (GE Healthcare, Princeton, NJ, USA), and ZFX (Zhifuxian,Xinqiao Hospital, the Third Military Medical University, Chongqing, China). Properties of each agent can be found in Table 1.
An experimental setup, similar to the one previously employed by our group [6 (link)], was constructed to quantify the best imaging parameters for SHAPE (Figure 1). A programmable function generator (Model 8116A; Hewlett Packard, Santa Clara, CA) produced pulses for transmission. The transmit signals were first amplified in a broadband 50 dB RF power amplifier (Model 3100L; ENI, Rochester, NY) and then supplied to an acoustic transmit transducer. Scattered signal from microbubbles were sensed by a receive transducer and amplified with a low-noise RF amplifier (Model 5052 PR, Panametrics, Waltham, MA). The amplified signals were then acquired using a digital oscilloscope equipped with mathematical functions (Model 9350AM, LeCroy, Chestnut Ridge, NY). The data transfer from the digital oscilloscope was controlled by a PC via LabView (National Instruments, Austin, TX). The digitized signals were further processed using fast Fourier transform (FFT) spectrum analysis in the oscilloscope. The amplitude of the fundamental, harmonic and subharmonic signal components were obtained from spectra averaged over 64 sequences to minimize noise and produce a well defined peak.
The contrast signals at different hydrostatic pressures were measured using a 2.25 L water tank (cube with inside dimensions of 13.1 cm) that can sustain pressure changes in excess of 200 mmHg. The water tank is also equipped with an acoustic window made out of thin plastic (thickness: 1.5 mm). The pressure inside could be varied by injecting air through an special inlet on the tank and was monitored by a pressure gauge (OMEGA Engineering Inc., Stamford, CT). A single-element focused transducer with a bandwidth of 86 % and a center frequency of 3.6 MHz (Etalon, Lebanon, IN) was used as the transmitter and another transducer with a bandwidth of 38 % and a center frequency of 2.2 MHz (Staveley, East Hartford, CT) was used as the receiver. Both transducers had a diameter of 2.5 cm and a focal length of 5.0 cm. The two transducers were positioned confocally at an angle of 60° to each other (Figure 1b) and 64 cycle acoustic pulses were transmitted at a pulse repetition frequency (PRF) of 5 Hz. Acoustic pressures were determined with a 0.2 mm broadband acoustic hydrophone (Precision Acoustics Ltd, Dorchester, England)
All measurements were carried out in triplicate at room temperature (25 ± 2^o C) with direct injection of contrast agents (dosage: 0.1 ml/L) into saline (Isoton II; Coulter, Miami, FL) immediately after activation of the agent. The mixture was kept homogenous by a magnetic stirrer and a waiting period of 30 s was allowed prior to data acquisition to ensure complete mixing. Data acquisition took on average 4 minutes (including the mixing period).
At a transmit frequency 4.4 MHz and acoustic pressure 0.42 MPa changes in the first, second, and subharmonic amplitudes of the five ultrasound contrast agents were measured over a pressure range from 0 to 186 mmHg which is similar to the range of blood pressures encountered in the human body (excluding extreme hypertension). Linear regression analysis was conducted to establish the relationship between changes in hydrostatic pressure and in contrast signal amplitude (calculated separately for first, second, and subharmonic signal components). The statistical software package Stata 9.0 (Stata Corporation, College Station, TX) was used.
Further studies were then conducted for the three agents that showed the most promise at 4.4 MHz and 0.42 MPa. For those three agents the transmit frequency was varied from 2.5 to 6.6 MHz and the acoustic pressure from 0.35 to 0.6 MPa (corresponding to the growth phase of the agents) to establish the optimal parameters and agent needed for SHAPE.

The testing microbubble data set was obtained from a female New Zealand white rabbit. The experiment conformed to the policy of the Institutional Animal Care and Use Committee (IACUC) at Mayo Clinic. The rabbit was anesthetized with ketamine (35.0 mg/kg) and xylazine (6.0 mg/kg). The right kidney of the rabbit was imaged. For ultrasound data acquisition, a Verasonics Vantage system (Verasonics Inc., Kirkland, WA) and a linear array transducer L11-4v (Verasonics Inc., Kirkland, WA) were used. A 5-angle (−4° to 4° with a step size of 2°) plane wave compounding with effective pulse-repetition-frequency (PRF) of 500 Hz was used (center frequency = 8 MHz). The mechanical index (MI) of the transmit pulse was 0.4 [1 (link)]. The spatial resolution of the ultrasound data was 0.172 mm. A total of 1000 ensembles were acquired (corresponding to 2 s time duration), with 200 ensembles (0.4 s time duration) acquired in each second (total data acquisition time = 5 s). The rabbit was allowed to have free breathing and freehand scanning was conducted. The imaging field-of-view (FOV) was carefully chosen so that only in-plane kidney motion caused by breathing was observed (i.e., no out-of-plane tissue motion). Before data acquisition, a 0.1 ml (5.0–8.0 × 10⁸ microspheres per mL) bolus injection of Optison (GE Healthcare, Milwaukee, WI) suspension was administered through the marginal ear vein followed by a 1 ml flush of saline. Ultrasound data acquisition started after the kidney was fully perfused by microbubbles.