a brown 4 february2005

Allison S. Brown, Ph.D.Imaging Research

Sunnybrook and Womens College Health Sciences Centre University of Toronto

Ultrasound Biomicroscopy: Assessment of Potential for Bioeffects

• Characterization of mouse models of human disease and therapeutics• 3D analysis of tumour volume growth and progression • Assessment of antiangiogenic and antivascular agents in xenografts, orthografts and spontaneous tumour models.• Multi-modality comparisons using MRI, microCT, OPT and associated contrast agents/stains.• Image guided injections and probe positioning• Image guided Doppler studies of flow hemodynamics• Targeted molecular imaging with high frequency US

Applications of UBM Imaging

In vivo models

– Melanomas MeWo, WM1341 (xenograft models)

– Mammary tumours Polyoma Middle T (spontaneous) MDA-MB-435, MDA-MB-231 (xenograft

models)– Prostate tumours

TRAMP, LadyTRAMP (spontaneous)PC3, LNCaP (xenograft models)

– Retinoblastoma SV40 LHβTAg (spontaneous)

Longitudinal studies with therapeutic intervention in multiple tumour types:

Mouse models of embryonic and postnatal development, vascular development and regression, and models of human disease (e.g., glaucoma, cirrhosis).

• Ultrasound biomicroscopy (UBM) utilizes high ultrasound frequencies, generating localized high intensity levels.

• Absorption is higher at high frequencies, therefore more heat is generated.

• The small dimensions of the focal zone promote rapid dissipation of heat because of the steep thermal gradients that arise within the beam.

• The use of high frequency ultrasound in avascular structures which lack potentially cooling perfusion, or at strongly attenuating bone-tissue interfaces, are a concern with respect to thermal bioeffects.

• K-type (Ni-Cr, Ni-Al), 50 m diameter thermocouple embedded in 15% porcine gelatin at the proximal or distal surface of an isolated adult mouse skull.

• OMEGA OMK-TDA4 Parallel Port Data Acquisition System used to collect data to PC.

Thermocouple

Top view

x direction

40 MHz transducer

UBM

PC

gelatin

Side view

y direction

45 mm

25 mm

45 mm

6 mm Thermocouple (Proximal)

Thermocouple (Distal)

Skull

z direction

Couplant

Thermocouple measurement of Temperature Rise

Field parameters in VS40 Doppler mode at 16 cycles, 20 kHz PRF

Transducer Frequency

(MHz)

MI p- (Mpa)

Image Rate (Hz)

Ispta (mW/cm2

)

40 0.83 5.218 4 2.6

Field parameters in VS40 B mode at 40 MHz

Transducer Frequency

(MHz)

MI p- (Mpa)

Ispta (W/cm2)

40 1.05 7.32 11.9

Temperature recording taken every 200ms– 30s baseline; 3 min during insonation; 2 min after

turning UBM off– 40 MHz, 16 cycles, 20 kHz PRF, 0 dB attenuation;

maximum acoustic output – worst case scenario)

Temperature Rise Measurements

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-0.5

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Time (s)

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rees

Cel

siu

s)

Proximal Distal

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Focus 1 mm x 2 mm x 1 mm y 2 mm y 1 mm z

Measurement Position

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per

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re R

ise

(deg

rees

Cel

siu

s) Proximal Distal

Temperature Rise in a Soft Tissue-Bone Phantom

Duckett et al., 2004. UMB 30(5):665-673.

Mean Temperature Rise in vivo and Postmortem at Soft Tissue-Bone Interface

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Measurement Position

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In vivo Postmortem

Duckett et al., 2004. UMB 30(5):665-673.

lens surface

ciliary body

sclera

scleral spur

limbus

corneoscleral junction

Schwalbe’s line

iris

High Resolution Human Ocular UBM Imaging

• UBM has a strong history in ocular imaging

• The lens is an avascular structure

Assessment of Temperature Rise in Ex vivo Human Eye

Experimental Setup

Cucevic et al., UMB, in press.

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RF cycles - PRF (kHz) - Attenuation (dB)

Mea

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Lens Ciliary Body

Mean Temperature Rise – Human Eye ex vivo


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Lens Ciliary Body

Maximum Temperature Rise – Human Eye ex vivo


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)

Rabbit lens

Mean Temperature Rise – Rabbit Eye ex vivo


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Rabbit lens

Maximum Temperature Rise – Rabbit Eye ex vivo


• No significant differences in mean temperature rise from insonation of ex vivo human lens and ciliary body.

• No significant difference between mean temperature rises obtained from insonation of human versus rabbit lens at any setting examined.

• Ex vivo rabbit eye may be a representative model system for ex vivo human eye for thermal bioeffects studies.

•Attenuation parameters show a significant age effect in the lens, and sclera expresses a significant age effect for ultrasound velocity and attenuation characteristics.

some differences may be expected in ocular tissue younger than the donor ages employed in this study (66-90 years of age).

Embryonic high frequency ultrasound exposures

•Pregnant CD-1 nulliparous mice were exposed to inhalant anesthesia and restraint, 40 MHz B mode, or 40 MHz Doppler (16 cycles, 20 kHz PRF, 0 dB attenuation, 3 min per embryo) for 1 hour at embryonic day (E) 8.5 or E10.5.

•Pups were measured and weighed every week from the first day of postnatal development until sacrifice and postmortem examination at 6 weeks.•Hearts and kidneys were excised and fixed in formalin at postmortem, and later weighed and measured.

Total Body Length

Pup Length - E8.5 Exposure (all litters combined)

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P1 P8 P15 P22 Postmortem

Time

Le

ng

th (

mm

)

Cage Control Anesthetic Control B mode Exposed Doppler Exposed

Brown et al., 2004. UMB 30(9):1223-1232.

Pup Weight - E8.5 Exposure (all litters combined)

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Brown et al., 2004. UMB 30(9):1223-1232.

• Exposure of pregnant CD-1 mice to 40 MHz ultrasound at E8.5 does not appear to affect gestation length, litter size, or gender ratio of pups.

• Total body length and crown-rump length were not statistically significantly different between any animal group; body weight of Doppler exposed pups deviated in the second week and was significantly different versus controls or B mode exposed litters at postmortem (1.5 g at 6 weeks).

• Morphological abnormalities were rare; the sole abnormality was renal agenesis (1 pup; B mode exposure) of 246 pups assayed. No other visceral, skeletal or other morphological abnormalities were observed.

Pup Length - E10.5 Exposure (all litters combined)

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th (

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Brown et al., 2004. UMB 30(9):1223-1232.

Pup Weight - E10.5 Exposure (all litters combined)

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•At postmortem, mean body weight of the E10.5 Doppler exposed group was significantly different only from the cage control group (p=0.011).

Brown et al., 2004. UMB 30(9):1223-1232.

• Exposure of pregnant CD-1 mice to 40 MHz ultrasound at E10.5 does not appear to affect gestation length, litter size, or gender ratio of pups.

• Body length and crown-rump length were not statistically significantly different between any group at postmortem.

• Body weight of Doppler exposed pups transiently deviated in the 3rd and 4th weeks but was not significantly different versus controls or B mode exposed litters at 6 weeks.

•Morphological abnormalities were extremely rare and included kinky tail (1 pup- Doppler), slightly abnormal/patchy fur (1 pup- Doppler) of 249 pups assayed. No other visceral, skeletal or other morphological abnormalities were observed.

•Soft tissue-bone interfaces insonated with high frequency ultrasound do not exhibit significant perfusion-induced cooling, and ultrasound-induced temperature rises decrease with distance from the beam focus.

• Ocular insonation should utilize maximum settings of 9 cycles, 10 kHz PRF, 0 dB attenuation to limit temperature rise to >1ºC.

• Other, subcellular effects may occur during exposure to high frequency ultrasound which were not detected in this study.

•We have also shown high frequency ultrasound (40 MHz) does not appear to generate gross biological effects from B mode or Doppler exposure of mice in utero (429 pups from 35 dams; Brown et al., 2004; UMB 30(9):1223-1232).

Conclusions

Ontario Consortium for Small Animal Imaging (ORDCF)

Sunnybrook and Women’s College Health Sciences Centre

Viviene CucevicStuart Foster

Lisa LeamenAngela Reid

a brown 4 february2005

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