Taking a Closer Look at "Reduction"

Reduction

As we continue to explore each of the 3 R’s as they relate to preclinical research, we move now to the second R as defined by Russell and Burch – Reduction. They state that the goal of Reduction is to minimize the number of laboratory animals used in a study while still obtaining statistically meaningful results.

The latter part of that definition is of upmost importance, researchers must ensure that in Reducing the numbers of animals used does not compromise the validity of the data. Otherwise, they themselves, or others, may feel the need to repeat the study, ultimately utilizing more animals to confirm the outcome of the study.

Another way to look at Reduction is also to think about both the quality and any additional information that may be obtained from the laboratory animal used in a study, which was not initially the goal of the study, but would be beneficial to the researcher or others since the study will already be underway.

When considering Reduction, there are a few approaches that scientists will consider:

  • Improvements to experimental design
    • Standardization of all procedures related to and on the periphery of the study, to help minimize the variability between animals within the study. For example:
      • Surgical techniques
      • Method of acquiring and recording data
      • Time of day that any interactions take place
      • Husbandry practices
      • Etc.
  • Stratification of disease severity, or entry criteria, prior to enrolling the subjects into a study. For example:
      • Tightness of the stenosis in a transverse aortic constriction (TAC) model
      • Confirmed presence and size of lesion in a transgenic tumor model.
      • Selection of appropriate laboratory animal species and strain to best mimic the clinical presentation of the disease being studied.
      • Inbred or immune-compromised mouse strains may help reduce variability but may also remove some of the crucial biological systems and mechanisms which effect clinical outcomes.
      • Some species more closely reflect the clinical disease at a specific stage, for example at onset, or in progression to morbidity.
  • Sample size calculations and statistical analysis techniques
    • When considering the required sample size for a study, it is necessary to examine the expected variability as well as the level of change which is expected, and which would be considered biologically significant not just statistically significant.
    • It is important to include an appropriate number of spare animals in each study group, as inevitably some loss will occur. The number of spare animals will need to be determined for the specific study being considered, and the techniques to be used, along with the skill of those conducting the study.
  • Using advanced technologies 
    • Imaging may be used to non-invasively image the same animal over the course of a longitudinal study. 
      • One animal is used as its own control, and provides data at each imaging timepoint, rather than a different animal being required at baseline, and for each time point. This can dramatically reduce the number of animals required for a study. 
      • Additional animals may be use if post-mortem techniques are desirable at each time-point, for example histological or immunohistochemical staining, but significantly fewer animals would be needed when imaging is used and correlated with the results. 
    • Non-imaging techniques may be needed to help in the stratification of study subjects into groups, or to initiate them into the study, as described previously. 

Taking a deeper look at preclinical imaging, as it relates to Reduction, it is important understand that each modality has it’s own strengths and weaknesses, and researchers will have access to some equipment while others may be harder to access or operate. Ideally, a multi-modal imaging approach is possible, providing anatomical, functional, as well as molecular information at multiple scale levels within the same animal over the various time points within a study.

Below is a quick summary of some of the more commonly used, non-invasive, preclinical imaging modalities.

Modality Resolution Advantages Considerations Main Applications

MRI

~100µm
– Non-ionizing radiation
– Whole body imaging is possible
– Excellent soft tissue contrast
– Clinical translation
– Some systems are expensive and high maintenance costs
**Note M-Series compact MRI does not have these limitations
– No bone contrast
– Cancer Biology
– Neurology
– Organ Imaging
– Cardiac Imaging
– Contrast Agent Imaging

CT

≤50µm
– Whole body imaging is possible
– Excellent bone imaging
– Clinical translation
– Ionizing radiation
– Low soft tissue contrast
– Bone Imaging
– Anatomical Reference for Other Imaging Modalities

PET

~1mm
– Whole body imaging is possible
– High sensitivity to agents used
– Quantifiable data
– Wide variety of applications, based on imaging agents used
– Clinical translation
– Radioactive agents are required
– Specialized infrastructure required
– Proximity to cyclotron, more important for short half-life molecules
– Cancer Biology
– Neurology
– Cardiac Imaging
– Pharmacokinetics and Pharmacodynamic Imaging

Ultrasound

~30-50µm
– High temporal and spatial resolution
– Good soft tissue contrast
– Non-ionizing radiation
– Clinical translation
– Depth of penetration vs resolution
– Operator dependent
– Cancer Biology
– Organ Imaging
– Cardiac Imaging
– Vascular Imaging
– Developmental Biology
– Contrast Agent Imaging

Optical
Imaging

~1mm
– Non-Ionizing radiation
– Whole body imaging is possible
– High throughput
– Wide variety of applications depending on agent used
– Ease of use
– Limited tissue penetration for some photons
– Low spatial resolution
– Semi-quantitative
– Cancer Biology
– Cell Trafficking
– Gene Expression

Photoacoustic
Imaging
(PAI)

~300um
– Whole body imaging is possible
– Penetration depth
– Endogenous and exogenous contrast
– Anatomical & structural information
– Cancer Biology
– Neurology
– Developmental Biology
– Contrast Agent Imaging
– Angiograph

Intravital Microscopy
(IVM)

~1µm
– Multiplex imaging capabilities
– Microscopic resolution
– Dynamic imaging allowing for live cell and molecule tracking
– Wide variety of applications
– Depth of penetration
– Small field of view
– Surgical preparation to access imaging target
– Cancer Biology
– Neurology
– Cardiac Imaging
– Vascular Imaging
– Organ Imaging
– Musculoskeletal Imaging
– Cell Tracking

DEXA/DXA 

~100µm for DEXA
~30µm for highest magnification digital radiography image
– Rapid scan times
– Whole body imaging is possible
– Bone mineral density/content measurements provided
– Lean vs. fat mass measured
– Very low ionizing radiation level per scan, allowing for longitudinal imaging
– Clinical translation
– Images are 2D
– Low soft tissue contrast
– Bone Imaging
– Body Composition Measurements
– Digital Radiography

When taken together, improvements in experimental design, inclusion on advanced techniques such as preclinical imaging, and truly understanding the expected variability and level of change that relates to a biologically significant outcome, there are some truly meaningful changes that researchers can make to aid in the Reduction of the number of laboratory animals used within a study.