Andre Obenaus1,2, Eli Kinney-Lang1,3, Duke Shereen4, Ana Solodkin3,4, and Tallie Z Baram2,3
1Pediatrics, Loma Linda University, Loma Linda, CA, United States, 2Pediatrics, University of California, Irvine, Irvine, CA, United States, 3Anatomy/Neurobiology, University of California, Irvine, Irvine, CA, United States, 4Neurology, University of California, Irvine, Irvine, CA, United States
Synopsis
The
amygdaloid complex, including the basolateral nucleus (BLA) are critically
important in mediating emotional and adaptive responses to stress. However, lack of contrast between the BLA and the
surrounding gray matter (GM) has hampered routine imaging. We report a novel
DTI paradigm to identify the BLA in the rodent brain. We derived BLA volumes and
confirmed these with histological measures. Our approach can be used to study
the morphological and functional consequences of emerging neuropsychiatric
diseases, both in experimental and clinical studies.Purpose
The amygdaloid complex and its associated nuclei play
an essential integrative role of sensory, visceral, emotional and behavioral
functions. The basolateral nucleus (BLA) is critically important in mediating
emotional and adaptive responses to stressors. Virtually no MRI studies report the anatomical
characteristics of the BLA, due to the lack of sufficient contrast
between the BLA and the surrounding gray matter (GM). Given its relevance we
undertook a high-resolution study to obtain BLA volumes in conjunction with
imaging approaches to improve contrast to accurately demarcate the BLA within
the rodent brain.
Methods
Eight ex vivo
adult rat brains (60d of age) underwent MR imaging using an 11.7 T Bruker
Avance. Standard anatomical imaging sequences were obtained, including: 1) T2-weighted
imaging (T2WI) sequence with the following parameters: TR/TE=2900/10.2ms (10
echo), 2X2cm FOV, 256
2 matrix, 4 averages, 0.5mm slice thickness with 25 slices spanning the
entire cerebrum; 2) 3D Rapid Acquisition with Relaxation Enhancement (3D RARE) with
a 256
3 matrix, 2X2 cm FOV, 78 µm slice thickness, 3)
An additional DTI scan was acquired on a 9.4 T Bruker with 30 directionally encoded diffusion gradients using a 4-shot EPI sequence with a matrix 128
2 and then zero-filled to 256
2 with a TE/TE=12500/36ms, 50 slices at 0.5mm, a 1.92X1.92cm FOV, 4 averages, b value of 0 (5 images) and 3000 s/mm
2 (30 images, non-collinear directions). This DTI sequence was run prior to the gradient echo DTI for volumetric determinations, and 4) a gradient echo diffusion tensor imaging (DTI) sequence with TR/TE=3000/40ms, 2cm
FOV, 2 averages, b=3000 s/mm
2 and 0.6mm slice thickness with 20 slices covering the brain. . DTI images were analysed using DSI Studio and fractional
anisotropy (FA) and primary, secondary and tertiary diffusion eigenvector maps
were calculated.
Gadolinium (Gd) enhancement was
undertaken in an attempt to improve neuroanatomical demarcation, with 3
approaches: 1) 4% PFA fixation only, 2) 4% PFA fixation+1% Gd, and 3) 4% PFA
fixation followed by 48hr incubation in 1% Gd with phosphate buffer.
Signal (SNR) and contrast to noise ratios (CNR)
focusing on the BLA region of the amygdala were assessed. Volumetric BLA and
whole brain analysis was performed manually on each coronal slice that
contained the BLA using Cheshire image software (Hayden Image/Processing Group)
using anatomically defined landmarks.
After
MR imaging, brains were processed for histological analysis, using cresyl
violet staining at 30um sections, using every 10th section.
Results
Standard
T2WI and high resolution 3D RARE sequences were unable to unequivocally define
the anatomical boundaries of the BLA. Similarly, Gadolinium enhancement strategies
did not improve identification of the BLA. DTI parametric maps found increased
CNR within the BLA region, particularly on the FA and λ1 maps (Fig. 1). Further
inspection of each of the individual 30 directionally encoded eigenvector
images (0.6mm thickness) and subsequent CNR analysis revealed that the BLA
could be optimally observed in 2 specific directions (2, 11) (Fig. 2A). In these
orientations, both the right and left BLA had the highest CNR and were visible
in multiple slices. For volumetric
analysis of the BLA we rescanned the brains using only a single DTI vector
profile (11) at 0.6mm/slice, but shifted the slice packet by 0.2mm (3X’s) to
obtain an effective slice thickness of 0.2mm/slice. This strategy allowed a
3-fold increase in number of slices for volumetric analysis while still
providing sufficient SNR. Using this approach we obtained BLA volumes for the
right 1.35±0.12 mm
3 and left BLA of 1.39±0.16 mm
3 (Fig
2B). MRI derived volumes were validated by comparison to histologically measured
BLA volumes which were smaller by 5.7% compared to MRI (Fig. 2C).
Discussion
MR related studies have demonstrated volumetric alterations in the brain that
correlate with neuropsychiatric conditions (i.e. schizophrenia). The amygdala
is important in emotional processing, fear, reward, anxiety and cognitive
functions with the BLA having critical reciprocal connections to the
hippocampus, prefrontal cortex, and other cortical regions. However, there are
virtually no volumetric imaging studies evaluating the amygdala and in particular the BLA.
We report on a novel strategy where selected directionally encoded DTI
images can provide excellent anatomical demarcation from the surrounding GM. We
further found that a slice-shifting paradigm with an effective slice thickness
of 0.2mm provided accurate volumetric assessments of the BLA in the rodent.
Conclusion
The
poor contrast between the BLA and surrounding gray matter has been a barrier in
neuroimaging studies. DTI combined with a slice-shifting approach found that we
can reliably differentiate the BLA within GM for volumetric studies. Future,
clinical and experimental studies could use a similar approach to evaluate the
role of the BLA in neuropsychiatric diseases.
Acknowledgements
Supported by National Institutes of Health Grant P50MH096889 (to TZB).References
No reference found.