Download Stereological estimates of neuronal loss in the primary motor cortex

Stereological estimates of neuronal loss in the primary motor cortex of multiple
sclerosis patients
M.M. Papachatzaki, D. Carassiti, A. McDowell, K. Schmierer
QMUL (London, GB)
Introduction Whilst inflammatory demyelination (ID) is an important feature in the
clinical and pathological diagnosis of MS, evidence suggests mechanisms other than
ID may play an important role for the deterioration of function in people with
progressive MS (pwPMS) (Trapp & Nave. Annu Rev Neurosci 2008; Kolasinski, et al.
Brain 2012). Impaired motor function is one of the most important components of
disability pwPMS accrue over time. Using unbiased sampling techniques applied to
whole central nervous systems of pwPMS we investigate whether a tract specific
pattern of neurodegeneration contributes to the loss of motor function in
pwPMS. Here, we present preliminary data on stereological estimates of neuronal
cell loss in limb specific areas of the MS primary motor cortex (PMC). No limb
specific neuronal cell counts had been reported to date in human brain.
Objective To estimate the absolute number of neurons in the PMC associated with
limb function in pwPMS and a reference case.
Methods The left hemispheres from formalin fixed brains of two women with
primary progressive (PP) MS (age= 67 and 83 years, disease duration= 11 and 14
years) and one reference case (male, age= 82 years) with no known neurological
disease were used. Reference brain was free of any neuropathological findings.
PMCs were delineated with ink on the cortical surface. Hemispheres were then
dissected into 1.1 cm thick coronal slabs and images obtained of each slab following
a standard protocol. Each slab was then embedded in wax, 40µm thick hemispheric
sections were obtained from across the entire motor cortex and stained using a
modified Wolbach's Giemsa method. Sections were chosen in a 1/20 sequence in a
systematic unbiased method and counted using cast-gid® software. Optical
dissectors (50µm x 50µm x 30µm) along the delineated area of interest were
superimposed onto a color monitor at a final magnification of 3000×, using a x60
objective on an Olympus microscope equipped with a motorized stage. The hand
and leg areas were identified anatomically based on a human brain atlas, the
presence of dye in the relevant areas of interest indicating the primary motor cortex
and the presence of Betz cells in the corresponding areas. Neurons were identified
using morphological criteria. Total number of neurons in each area was subsequently
calculated according to NV x VREF stereology method, were VREF was estimated by the
Cavalieri principle.
Results The mean number of neurons in the leg and arm areas of the PMC of the left
hemisphere in pwPPMS was 34 and 32 million in average respectively, whilst the
number of neurons in the reference PMC was 34 and 35 million, respectively. The
coefficient of error (CE) was less than 9% throughout suggesting reliable counting
methodology.
Conclusion Using unbiased sampling methodology our data provide a robust
platform for studies into the relationship between cortical and spinal cord pathology
in MS. Extension of our cohort is key to produce statistically meaningful
results. Subsequently, we will explore the relationship between neuronal-axonal
pathology and other histological features of MS including inflammation and
demyelination.
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