Brain Imaging Center, McLean Hospital, Belmont, and Department of Psychiatry, Harvard Medical School, Boston, Massachusetts, USA
Department of Medical Biophysics, University of Western Ontario, and Diagnostic Radiology and Nuclear Medicine, St Josephs Health Care, London, Ontario
Departments of Medical Biophysics and Psychiatry, University of Western Ontario
Department of Psychology, University of Western Ontario
Department of Medical Biophysics, University of Western Ontario, and Laboratory for Magnetic Resonance Imaging Research, Robarts Research Institute, London, Ontario
Douglas Hospital, McGill University, Montreal, Quebec
Department of Psychiatry, University of Western Ontario
Diagnostic Radiology and Nuclear Medicine, St Josephs Health Care, London, Ontario
Departments of Medical Biophysics and Psychiatry, University of Western Ontario, and Diagnostic Radiology and Nuclear Medicine, St Josephs Health Care, London, Ontario, Canada
Correspondence: Dr J. Eric Jensen, Room 208,Brain Imaging Center, McLean Hospital, 115 Mill Street, Belmont, MA 02478-9106, USA. E-mail: ejensen{at}mclean.harvard.edu
Declaration of interest None. Funding detailed in Acknowledgement.
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ABSTRACT |
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Aims Using improved imaging techniques, previously inaccessible brain regions were examined in patients with first-episode schizophrenia and healthy volunteers with 4.0 T 31PMRS.
Method Brain spectra were collected in vivo from 15 patients with first-episode schizophrenia and 15 healthy volunteers from 15 cm3 effective voxels in the thalamus, cerebellum, hippocampus, anterior/posterior cingulate, prefrontal cortex and parieto-occipital cortex.
Results People with first-episode schizophrenia showed increased levels of glycerophosphocholine in the anterior cingulate. Inorganic phosphate, phosphocreatine and adenosine triphosphate concentrations were also increased in the anterior cingulate in this group.
Conclusions The increased phosphodiester and high-energy phosphate levels in the anterior cingulate of brains of people with first-episode schizophrenia may indicate neural overactivity in this region during the early stages of the illness, resulting in increased excitotoxic neural membrane breakdown.
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INTRODUCTION |
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METHOD |
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Imaging technique
All experiments were performed on a 4.0 Tesla Varian/Siemens/Unity Inova
whole-body magnetic resonance scanner (Varian, Palo Alto, California, USA:
Siemens, Erlangen, Germany) operating at 170.3 MHz. A single-tuned, proton
quadrature hybrid-birdcage volume head coil was used for shimming and imaging.
The individuals to be examined were positioned supine with their heads secured
in a Plexiglas cradle and the global water resonance was manually shimmed. A
two-dimensional fast low-angle shot imaging sequence acquired anatomical,
sagittal and coronal images of the brain for 31P voxel positioning
(repetition time 11 ms, echo time 6 ms, 256 phase-encodes, 0.93 mm x
0.93 mm pixels). Thirty-two T1-weighted transverse images
were acquired on the brain using a magnetisation-prepared (adiabatic inversion
pulse 0.5 s prior to data collection) three-dimensional fast low angle shot
imaging sequence (repetition time 11 ms, echo time 6.2 ms, total acquisition
time <3 min) with a nominal slice thickness of 4.375 mm and 1.09 x
1.09 mm in-plane resolution. These images were used to calculate grey matter,
white matter and cerebrospinal fluid ratios in the 31P chemical
shift imaging volumes.
Image segmentation and partial volume estimation
The transverse image data-set for each brain examined was first separated
into three distinct binary tissue maps grey matter, white matter and
cerebrospinal fluid by thresholding the original high-contrast image
set. Two thresholding values were obtained from the central slice of each
image set, which best displayed the ventricles, corpus callosum and grey
matter of the anterior cingulate. The thresholding coefficients were then
varied until each segmented image type matched the corresponding anatomy in
the original reference image. The thresholding values differed between the
individuals examined since image signal intensity depended on receiver gain
and coil loading. Extraneous tissue with signal intensities similar to neural
tissue or cerebrospinal fluid, such as skin and subcutaneous lipid, was
removed from the segmented images using commercial software (Analyze 4.0 for
PC; Biomedical Imaging Resources, Mayo Clinic) to avoid it being counted as
neuronal tissue in the final volumetric analysis. To obtain the tissue ratio
contribution for each 31P region of interest, the three segmented
image datasets were convolved with the calculated three-dimensional point
spread function of the 31P chemical shift imaging acquisition
(Jensen et al,
2002a). The partial volume contribution for each tissue
type was then expressed as a percentage of total tissue contribution for each
region of interest (see Table
3).
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In vivo 31PMRS
Once the 1H shimming and imaging were completed, the
1H head coil was manually replaced with a single-tuned,
31P quadrature, 14 cm long birdcage head coil, with the person to
be examined still in place in the Plexiglas cradle inside the magnet. Previous
phantom experiments verified that manual coil replacement did not affect the
shim. A 31P external reference standard methylene
diphosphonic acid, 270 mol/l, T1 at 4.0 Tesla about 6 s
was fastened to the Plexiglas head cradle just to the left of the
persons head. The 1.5 cm diameter reference tube was positioned so its
length ran axially, spanning the sensitive region of the 31P radio
frequency coil. Transmit/receive frequency was centred on phosphocreatine, as
measured with a global free induction decay. Tip angle, optimised for the
phosphomonoester/phosphodiester resonances at a 500 ms repetition time, was
32°. In vivo 31PMRS used an optimised
three-dimensional chemical shift imaging sequence
(Jensen et al,
2002a): repetition time 500 ms; sampling bandwidth
±2 kHz; complex points 1600; readout duration 400 ms; pre-pulses 20;
receiver gain 94 dB; pre-acquisition delay 1.905 ms; field of view
(x,y,z) 280 mm; nominal volume 5.4 cm3; maximum
phase-encode matrix dimension (x,y,z) 14 x 14 x 14
(zero-filled out to 16 x 16 x 16 prior to reconstruction). The
three-dimensional chemical shift imaging sequence used a reduced
phase-encoding scheme allowing for the inclusion of spherically bound, reduced
point, weighted k-space acquisition, providing approximately 40% more
signal-to-noise for a given scan time and effective voxel volume over
conventional methods (Jensen et
al, 2002b).
In vivo post-processing and spectral analysis
All in vivo chemical shift imaging/image data were processed and
viewed using Varian Nuclear Magnetic Resonance software (version 6.1B) and
software designed and written on site. Prior to fast Fourier transform
reconstruction to spatially resolve the chemical shift imaging spectra, the
collected k-space data were centred in a 16 x 16 x 16
cubic matrix. Each time-domain free induction decay was then zero-filled out
to 2048 complex points and left-shifted 5 points to remove residual bone and
rigid membrane signal.
Using the 1H images, the three-dimensional chemical shift imaging data grid was shifted in the x, y and z dimensions in order to position each voxel in the appropriate 31P region of interest. For all brains examined, the Brodmann area and Talairach coordinates for voxel centres (x,y,z) were evaluated for every 31P region of interest using statistical parametric mapping (Table 2). Partial overlap with adjacent structures was unavoidable in many of the regions owing to voxel volume and shape (Fig. 1).
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All in vivo spectra were fitted in the time domain using a non-linear, iterative fitting program developed on site (Fig. 2). The fitting routine is based on the MarquardtLevenberg algorithm, using prior spectral knowledge for the relative amplitudes, line widths, line shapes, peak positions and J-coupling constants to model the in vivo 31P brain spectrum (Jensen et al, 2002a). Since it was positioned along the inner face of the coil, the reference standard was subject to different radio frequency power from that used for the brain, and was volume-bound by the x and y tube dimensions (1.5 cm diameter), both requiring correction. Therefore, the methylene diphosphonic acid reference area, modelled with a triplet structure, was corrected for T1 saturation, radio frequency coil sensitivity and volume. Each 31P metabolite area was also T1-corrected and then normalised to the methylene diphosphonic acid signal to obtain absolute millimolar values per unit of wet brain-tissue volume (mol/l) for each 31P metabolite (Jensen et al, 2002a). Human in vivo 31P high-energy phosphate metabolite T1 values at 4.0 Tesla for inorganic phosphate, phosphocreatine and adenosine triphosphate of 3.06 s, 2.98 s and 1.07 s were used for the T1 correction, as well as T1 values of 2.68 s, 1.5 s, 3.92 s, 3.99 s and 2.31 s for phosphoethanolamine, phosphocholine, glycerophosphoethanolamine, glycerophosphocholine and membrane phospholipids, respectively (Jensen et al, 2002a). The fitted metabolite amplitudes are not T2-weighted since the fitting algorithm extrapolates back to time zero. Each 31P metabolite value was finally corrected for cerebrospinal fluid partial volume fraction assuming phosphate concentration in cerebrospinal fluid to be negligible. There was no correction for the ratio of grey to white matter.
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Statistics
Statistical calculations were performed using the Statistical Package for
the Social Sciences, version 10.0 for Windows. A two-tailed t-test
was used to test for significant differences in age, parental education, grey
matter, white matter and cerebrospinal fluid partial volumes between groups
(P < 0.05). In the same fashion as in our previous study
(Jensen et al,
2002b), statistical treatment of all ten 31P
metabolite ratios for each region first involved a one-factor multivariate
analysis of variance (MANOVA) with a between-subjects factor of group.
Pursuant to a significant multivariate test, univariate F-tests were
used to examine group differences, with set at P < 0.05.
In each region where significant group differences in 31P
metabolites were found, Pearson correlations (P < 0.01) were
computed within the patient group between 31P metabolite levels and
each of the following variables: SANS and SAPS scores, age, parental education
and duration of untreated psychosis.
Our application of the region-wise MANOVA represents an optimal compromise between power and type I error protection. Expanding the analysis to include region as a within-subject factor, resulting in the split-plot factorial MANOVA applied to the vector of the metabolites, unnecessarily complicates the analysis when there are a priori reasons to select the targeted regions (Stevens, 1996). The issue of following up a significant MANOVA test, and whether or not to apply Bonferroni correction, has been addressed in the literature (Hummel & Sligo, 1971; Stevens, 1996), justifying the statistical treatment of our data.
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RESULTS |
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DISCUSSION |
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Interpretation of these findings could lead to several conclusions pertaining to how symptoms of schizophrenia develop and evolve over time. One possible explanation might be that the onset of psychosis as an acute, identifiable event is in fact a process that continues over an extended period. The initial excitatory pathophysiological processes involved in the onset of psychosis may continue in the anterior cingulate, while the degenerative process continues in other regions such as the thalamus or hippocampus, which may have already been damaged early in brain development. Eventually, in the chronic stages of illness, the anterior cingulate experiences so much neurodegeneration that its phospholipid profile resembles that of the neurally damaged thalamus, as we observed in our study of patients with chronic schizophrenia (Jensen et al 2002b), and is consistent with early neurodevelopmental lesions suggested in these regions (Lipska & Weinberger, 1993; Jones, 1997; Andreasen, 1999). Also, this suggested neurodegenerative process fits with the finding of increased glutamate-mediated metabolites with 1HMRS in the left anterior cingulate (Theberge et al, 2002) and the reduced tissue volume in this region seen in patients with first-episode disease compared with controls. However, since we are looking at the same metabolites at two discrete time points in different study groups, the question remains whether the hypothetical neurodevelopmental changes in regions such as the thalamus, hippocampus or cerebellum are, in fact, a prerequisite for putative degenerative changes in regions such as the cingulate. More longitudinal human research studies in schizophrenia are needed to support the neurodevelopmental/degenerative model of schizophrenia.
Implications of altered phosphodiester levels
Phospholipase A1 and A2 activity produces the phosphodiester metabolites
glycerophosphocholine and glycerophosphoethanolamine. Phosphodiesterase, a
catalytic enzyme responsible for the breakdown of phospholipid membrane, also
produces glycerophosphocholine and glycerophosphoethanolamine. A decrease in
the activity of this enzyme, and/or an increase in phospholipase A1 and A2
activity, can decrease phosphodiester levels in the brain, and may be involved
in the development of schizophrenia
(Horrobin et al,
1994). However, the increase in glycerophosphocholine seen in the
anterior cingulate may suggest a reversal of this mechanism, leading to
increased membrane breakdown during the acute stages of the illness and
eventual neuronal loss in this region as the illness progresses. This is
supported by our earlier study in patients with chronic illness at 4.0 Tesla
(Jensen et al,
2002b), which found decreased glycerophosphoethanolamine
levels in the anterior cingulate. Interestingly, no difference in any
phospholipid metabolite was detected between groups in either the left or the
right prefrontal cortex between the first-episode schizophrenia group and the
controls. This contrasts with the results of other studies, which report
increased prefrontal phosphodiester levels in first-episode but not chronic
illness (Williamson & Drost,
1999) and decreased phosphodiester levels in drug-naïve (not
first-episode) patients in the left frontal lobe
(Yacubian et al,
2002). However, these earlier studies were conducted at lower
field strengths (<2 Tesla), where (unlike 4.0 Tesla imaging) the broad
phosphodiester resonance is dominant in the human 31P brain
spectrum. This fact, combined with different voxel volumes and analytical
techniques might possibly contribute to our different findings regarding the
prefrontal cortex. Our 31PMRS studies of the brains of
patients with chronic and first-episode schizophrenia at 4.0 Tesla suggest
that the prefrontal cortex may manifest abnormalities in phospholipid
metabolism only later in the chronic stages of illness, suggestive of eventual
neurodegeneration. Indeed, if neurodegeneration is the primary course of
evolution in this illness, then one could speculate that early intervention
with neuroprotective agents could halt or at least slow down
the onset and development of schizophrenic symptoms, lessening the negative
social, physical and economic impact of this disease.
Other metabolites
High-energy phosphate changes seen in this study of first-episode
schizophrenia occurred in the anterior cingulate, with increased
concentrations of inorganic phosphate, phosphocreatine and adenosine
triphosphate. Interpretation of this finding is somewhat difficult as
adenosine triphosphate levels should remain constant, regardless of the
metabolic demands placed on the neural tissue. A decreased metabolic rate
might explain the increased phosphocreatine level in this region, since the
demand for replenishing adenosine triphosphate levels is reduced and
phosphocreatine stores are not being drawn upon, hence the increased levels.
One explanation for the increased adenosine triphosphate level in this region
might be that there is a mitochondrial dysfunction in these neurons, since the
mitochondria are responsible for producing the cells supply of
adenosine triphosphate. Mitochondrial dysfunction in neuropsychiatric
disorders has been alluded to in other illnesses yielding changes in adenosine
triphosphate levels in the brain, such as depression
(Volz et al, 1998).
Since schizophrenia has a strong genetic component, mitochondrial dysfunction
could at least partially explain the energetic anomalies seen in this and
other 31PMRS studies of schizophrenia
(Jensen et al,
2002b). However, this hypothesis has yet to be
tested.
Effect of medication
The effects of neuroleptic and anxiolytic medications on brain phosphorus
metabolites warrant further discussion. Two human studies in vivo
examining the effect of neuroleptic administration on phosphorus metabolites
suggest that phosphodiester levels are decreased in the temporal lobe
(Fukuzako et al, 1999)
and increased in the frontal lobe (Volz
et al, 1999) with neuroleptic medication. In this study,
we observed no differences in phosphodiester levels in either of these regions
between groups. However, it cannot be ruled out that phosphodiester level
differences in these regions do actually exist, but are masked by the effects
of neuroleptics. There are very few studies examining the effects of
benzodiazepine medications on brain phosphorus levels. A study by Miranda
et al (1990) found
that phosphocreatine utilisation is altered in the adult rat brain only after
in utero exposure to benzodiazepine ligands. Extrapolating this
result to the humans investigated in this study, one can only speculate that
phosphocreatine levels might be affected by maternal use of benzodiazepines
during pregnancy. However, it is clear that more human research is needed in
this area to test this hypothesis.
Spectroscopy limitations
The repetition time used for our 31PMRS acquisition (500
ms) was very short compared with the measured T1 values of
the phosphorus metabolites. The resulting 31P spectra are therefore
T1-weighted despite radio frequency pulsing at the Ernst
angle for a T1 of 3 s (the average T1
of phosphomonoester, phosphodiester and phosphocreatine metabolites). It is
possible that our observed changes in metabolite levels between groups might
partly result from different T1 values in the affected
regions, aside from concentration differences. This is unlikely, however,
since T1 values in people with schizophrenia would have to
differ by up to several hundred per cent in order to equalise the metabolite
ratios between the groups, and there is no supporting evidence from other
studies suggesting that brain phosphorus metabolite T1
values are altered in schizophrenia.
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CLINICAL IMPLICATIONS |
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LIMITATIONS |
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ACKNOWLEDGMENTS |
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Received for publication June 27, 2003. Revision received December 12, 2003. Accepted for publication January 6, 2004.
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