An Evaluation of Joint Inflammation

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An Evaluation of Joint Inflammation

• A Research Study By
• Vishal Sharma

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A Brief Background

• A Rabbit knee joint is a useful model system
• Joint inflammation is necessary in healing, but
  is a problem when excessive
• Arthritis is a prominent inflammatory joint
  disease
• Synovial tissue responds to injury by permitting
  leukocyte entry
• Hyperplasia and inflammation result
• Previous assessments of inflammation have been
  largely qualitative

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Investigation

• Based on histomorphological features of inflamed
  joints, a measurement of the synovium volume can
  be used as an index of inflammation
• Is estimating the volume of synovium of an
  inflamed rabbit knee joint a useful tool in
  assessing the degree of arthritis ?

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Overview

• Rabbits induced with arthritis in both knee
  joints
• Methotrexate anti-metabolite administered to one
  knee only
• Serial sectioning of the entire joint
• Analysis using Cavalieri estimations of synovium
  volume
• Comparisons to control

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Methods

• A total of 500 slides were prepared for 5 joints
  of interest
• Every 2nd section was mounted, every 4th section
  was stained
• Slides were spaced at 12µm and 15µm for the right
  and left joints, respectively
• Use of Stereo Investigator software and Zeiss
  Axioplan2 microscope
• Blind stereological investigation on joints

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Methods

• Analysis of 50 sections (10 of total)
• 10 sections from each of 5 joints
• Random start, every nth section observed
• Efficient and effective analysis with reasonable
  accuracy and minimal bias
• Cavalieri Estimation
• 250µm grid of points overlaid on section
• Sum of points that hit synovium tissue
• Estimation of average slide thickness

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Methods

• An unbiased sampling of sections
• 200 points counted on 10-15 sections can provide
  an unbiased estimate of the real volume with a
  precision better than 5 (Gundersen et al., 1987)
  
• Normalization with respect to entire joint depth
  is necessary

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Cavalieri Equation

• V(obj) t SA(prof) t S(p a)
• where V(obj) is the volume of any object, t
  is the mean thickness or distance between
  parallel sections, A(prof) is the area of the
  profile of interest, p is the number of Cavalieri
  points on a section, and a is the area
  represented by each Cavalieri point

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Statistical Validation

• For fair comparison, the data were normalized
  with respect to the parasagittal depth through
  the knee joint
• Validation of data significance was done using a
  paired T-test measuring the signal to noise ratio

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Results
Average Joint Depth (µm)
Total number of sections in series
Knee Joint
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Results
Ratio of the experimental knee joint volume to
the control
Knee Joint
Normalized Volume of Synovium Area (µm2)
p 0.029
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Findings

• plt0.05 suggests a statistically significant
  treatment effect
• Methotrexate is effective in reducing joint
  inflammation
• Left joints (no treatment) were 87 more inflamed
  than the control
• Right joints (methotrexate) were 14 more
  inflamed than the control
• Methotrexate-treated joints are 39.5 less
  inflamed than untreated joints

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Synovial tissue characterized by inflammation and
hyperplasia
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Normal healthy synovial tissue with cells of
regular shape and number
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Implications

• The data are statistically meaningful
• Measuring volume of synovium is a worthwhile tool
  for predicting the degree of inflamed versus
  healthy joints
• Measuring synovial volume allows one to determine
  the effectiveness of methotrexate as a treatment
  for arthritis
• Methotrexate has a positive anti-metabolite
  influence on synovium volume

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Challenges

• Time constraints more data is always better
• No expert pathologist made stereological
  observations
• Choosing the synovium contour
• Presence of artifacts
• Scattered lone clusters of synovial tissue
• Measurements were estimations

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Conclusions

• This information has the potential to prove
  useful in the scientific community
• Assessments of synovial tissue volume can relate
  to the degree of inflammation
• Applications in drug effectiveness for
  inflammatory joint diseases (arthritis)
• Unbiased sampling of the entire joint using
  Cavalieri estimations to assess inflammation had
  not been performed
• More data is necessary to strengthen results

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End of Presentation Questions / Comments ?