Allison S. Brown, Ph.D.

1
Ultrasound Biomicroscopy Assessment of
Potential for Bioeffects
Allison S. Brown, Ph.D.
Imaging Research
Sunnybrook and Womens College
Health Sciences Centre University of Toronto
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Applications of UBM Imaging

• 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

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In vivo models
Longitudinal studies with therapeutic
intervention in multiple tumour types

• 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 LHßTAg (spontaneous)

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

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Thermocouple measurement of Temperature Rise

• 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.

Top view
Side view
Couplant
Thermocouple (Proximal)
Thermocouple
6 mm
y direction
Skull
45 mm
Thermocouple (Distal)
x direction
z direction
25 mm
45 mm
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Field parameters in VS40 B mode at 40 MHz
Field parameters in VS40 Doppler mode at 16
cycles, 20 kHz PRF
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Temperature Rise Measurements

• 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)

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Temperature Rise in a Soft Tissue-Bone Phantom
Duckett et al., 2004. UMB 30(5)665-673.
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Mean Temperature Rise in vivo and Postmortem at
Soft Tissue-Bone Interface
Duckett et al., 2004. UMB 30(5)665-673.
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High Resolution Human Ocular UBM Imaging
limbus
scleral spur
corneoscleral junction
Schwalbes line
iris
sclera
lens surface
ciliary body

• UBM has a strong history in ocular imaging
• The lens is an avascular structure

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Assessment of Temperature Rise in Ex vivo Human
Eye
Experimental Setup
Cucevic et al., UMB, in press.
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Mean Temperature Rise Human Eye ex vivo
Cucevic et al., UMB, in press.
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Maximum Temperature Rise Human Eye ex vivo
Cucevic et al., UMB, in press.
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Mean Temperature Rise Rabbit Eye ex vivo
Cucevic et al., UMB, in press.
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Maximum Temperature Rise Rabbit Eye ex vivo
Cucevic et al., UMB, in press.
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• 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).

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

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Brown et al., 2004. UMB 30(9)1223-1232.
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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 (p0.011).

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

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Conclusions

• 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).

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Viviene Cucevic Stuart
Foster Lisa Leamen Angela
Reid
Ontario Consortium for Small Animal Imaging
(ORDCF) Sunnybrook and Womens College Health
Sciences Centre