Fractionated manganese injections: Effects on MRI contrast enhancement and physiological measures in C57BL/6 mice

Article (PDF Available)inNMR in Biomedicine 23(8):913-21 · October 2010with57 Reads
DOI: 10.1002/nbm.1508 · Source: PubMed
Manganese-enhanced MRI (MEMRI) is an increasingly used imaging method in animal research, which enables improved T(1)-weighted tissue contrast. Furthermore accumulation of manganese in activated neurons allows visualization of neuronal activity. However, at higher concentrations manganese (Mn2+) exhibits toxic side effects that interfere with the animals' behaviour and well-being. Therefore, when optimizing MEMRI protocols, a compromise has to be found between minimizing side effects and intensifying image contrast. Recently, a low concentrated fractionated Mn2+ application scheme has been proposed as a promising alternative. In this study, we investigated effects of different fractionated Mn2+ dosing schemes on vegetative, behavioural and endocrine markers, and MEMRI signal contrast in C57BL/6N mice. Measurements of the animals' well-being included telemetric monitoring of body temperature and locomotion, control of weight and observation of behavioural parameters during the time course of the injection protocols. Corticosterone levels after Mn2+ application served as endocrine marker of the stress response. We compared three MnCl2  x 4H2O application protocols: 3 times 60 mg/kg with an inter-injection interval of 48 h, six times 30 mg/kg with an inter-injection interval of 48 h, and 8 times 30 mg/kg with an inter-injection interval of 24 h (referred to as 3 x 60/48, 6 x 30/48 and 8 x 30/24, respectively). Both the 6 x 30/48 and the 8 x 30/24 protocols showed attenuated effects on animals' well-being as compared to the 3 x 60/48 scheme. Best MEMRI signal contrast was observed for the 8 x 30/24 protocol. Together, these results argue for a fractionated application scheme such as 30 mg/kg every 24 h for 8 days to provide sufficient MEMRI signal contrast while minimizing toxic side effects and distress.
Received: 16 October 2009, Revised: 23 December 2009, Accepted: 4 January 2010, Published online in Wiley InterScience: 2010
Fractionated manganese injections: effects on
MRI contrast enhancement and physiological
measures in C57BL/6 mice
Barbara Gru¨ necker
, Sebastian F. Kaltwasser
, Yorick Peterse
Philipp G. Sa¨ mann
, Mathias V. Schmidt
, Carsten T. Wotjak
Michael Czisch
Manganese-enhanced MRI (MEMRI) is an increasingly used imaging method in animal research, which enables
improved T
-weighted tissue contrast. Furthermore accumulation of manganese in activated neurons allows
visualization of neuronal activity. However, at higher concentrations manganese (Mn
) exhibits toxic side effects
that interfere with the animals’ behaviour and well-being. Therefore, when optimizing MEMRI protocols, a compro-
mise has to be found between minimizing side effects and intensifying image contrast. Recently, a low concentrated
fractionated Mn
application scheme has been proposed as a promising alternative. In this study, we investigated
effects of different fractionated Mn
dosing schemes on vegetative, behavioural and endocrine markers, and MEMRI
signal contrast in C57BL/6N mice. Measurements of the animals’ well-being included telemetric monitoring of body
temperature and locomotion, control of weight and observation of behavioural parameters during the time course of
the injection protocols. Corticosterone levels after Mn
application served as endocrine marker of the stress
response. We compared three MnCl
O application protocols: 3 times 60 mg/kg with an inter-injection interval of
48 h, six times 30 mg/kg with an inter-injection interval of 48 h, and 8 times 30 mg/kg with an inter-injec tion interval of
24 h (referred to as 3 T60/48, 6 T30/48 and 8 T30/24, respec tively). Both the 6 T30/48 and the 8 T30/24 protocols
showed attenuated effects on animals’ well-being as compared to the 3 T60/48 scheme. Best MEMRI signal contrast
was obser ved for the 8 T30/24 protocol. Together, these results argue for a fractionated application scheme such as
30 mg/kg every 24 h for 8 days to provide sufficient MEMRI signal contrast while minimizing toxic side effec ts and
distress. Copyright ß2010 John Wiley & Sons, Ltd.
Keywords: manganese; neurotoxicity; MEMRI; mouse; contrast agent; stress
Magnetic resonance imaging (MRI) is an important tool in animal
research due to its high resolution and excellent soft-tissue
contrast. Being non-invasive, MRI opens the possibility to pursue
in vivo longitudinal studies. However, anatomical MRI of the
rodent brain is limited by little native contrast between different
cerebral compartments, hampering the delineation of cortical
and subcortical regions. To enhance the regional contrast
specificity, paramagnetic agents are frequently applied (1).
One of the most promising contrast agents in animal studies
is manganese (Mn
). Due to its chemical similarity to calcium
) (2), it may enter neuronal cells through voltage-gated Ca
channels during depolarization (3). Mn
is then taken up into
the endoplasmatic reticulum (4,5). Accumulated in vesicles, it can
be transported anterogradely in axonal tracts (6,7) to the synaptic
cleft, where it is released and taken up by the next neuron
(5,7). Mn
ions reside in the brain for a prolonged period of time
with a half life between 51 and 74 days (8). Its paramagnetic
properties lead to an effective reduction of the spin-lattice
relaxation time constant T
of the surrounding water molecules,
resulting in positive contrast enhancement in T
-weighted MR
images (1).
These basic characteristics of Mn
have led to the develop-
ment of three distinctive applications of MEMRI (9): first, MEMRI is
used to visualise neuroanatomical details after subcutaneous (10)
or intravenous (9,11) administration of Mn
. The regional MEMRI
contrast depends on local neuronal cell density, differences of the
local permeability of the blood brain barrier, and differences in
neuronal activation (9,12). As a second application, neuronal tract
tracing can be performed, exploiting the anterograde Mn
transportation across synapses (4,5,7,13–16). Finally, MEMRI
enables functional imaging in animal models. Since Mn
cells through voltage-gated Ca
channels, it accumulates in
excitable cells. As compared to classical blood oxygenation level
dependent (BOLD) methods used in functional MRI (fMRI), MEMRI
is not primarily dependent on hemodynamic changes as a
correlate of neuronal activity. Rather, it reflects neuronal
( DOI:10.1002/nbm.1508
Research Article
*Correspondence to: M. Czisch, Max Planck Institute of Psychiatry, Neuroima-
ging Group, Kraepelinstr. 2, D-80804 Munich, Germany
aB. Gru¨necker, S. F. Kaltwasser, Y. Peterse, P. G. Sa¨mann, M. V. Schmidt, C. T.
Wotjak, M. Czisch
Max Planck Institute of Psychiatry, Munich, Germany
These authors contributed equally to the study.
Abbreviations used: cþsc, cortical plus subcortical; CV, coefficient of vari-
ation; DG, dentate gyrus; EDTA, ethylenediaminetetraacetic acid; HPA-axis,
hypothalamic-pituitary-adrenal axis; LSD, least significant difference; MEMRI,
manganese enhanced MRI; RI, relative intensity; ROI, region of interest; T1w,
T1-weighted; T2w, T2-weighted; wb, whole brain.
NMR Biomed. (2010) Copyright ß2010 John Wiley & Sons, Ltd.
activation directly at the cellular level. In animal research, fMRI is
difficult to employ due to the need of anaesthesia. The MEMRI
contrast builds up over a longer period of time (17), which allows
exposing awake animals to more complex behavioural para-
digms under less artificial conditions. MEMRI as a functional
imaging tool has already been successfully used in a couple of
murine studies (12,18–21).
A major drawback of Mn
lies in its toxicity when applied in
high concentrations. After systemic application, acute stress to
heart and liver can be observed (22). In humans, overexposure
to Mn
may lead to so-called manganism, a form of
parkinsonism (23), apparently related to the accumulation and
slow clearance of Mn
in the basal ganglia (6,24,25). Therefore,
to ensure minimal toxic side effects in animals while obtaining
optimal MRI contrast enhancement, it is necessary to optimize
the Mn
application scheme. In this respect the long half-life
of Mn
is of advantage as a sufficient cumulative dose can
be achieved by repeatedly administering low dose fractions that
are non-toxic (17). Furthermore, when studying behavioural or
stress-related paradigms e.g. in animal models of psychiatric
disorders, any stress response triggered by the Mn
itself is of particular importance due to interference with the
studied stress paradigm.
These facts make the identification of an optimized application
protocol indispensable. Here we studied the frequently used
C57BL/6N (BL6N) mouse strain to investigate the consequences
of different schemes of fractionated Mn
applications on
vegetative, behavioural and endocrine stress parameters in order
to minimize toxic side effects in MEMRI experiments.
Male C57BL/6NCrl mice were purchased from Charles River
(Sulzfeld, Germany). They entered the experiments at the age of
6–7 weeks. Animals were given at least 1 week to adjust to single-
housing conditions and to an inverse 12:12 day/night cycle (lights
on at 21:00 hs). They had free access to food and water.
The Committees on Animal Health and Care of the local
government (Regierungspra¨sidium Oberbayern) approved all
experimental procedures (AZ 55.2-1-54-2531-133-06 and 55.2-1-
54-2531-41-09), which were carried out in accordance with the
European Communities Council Directive of 24 November 1986
Application of Mn
A solution of 50 mM MnCl
O (Sigma, Germany) was
prepared in 0.9% NaCl. pH was adjusted to 7.0 with HCl and
NaOH. Application protocols were based on a recent publication
by Bock and colleagues (17) who performed fractionated MEMRI
in rats, using 6 30 mg/kg, 3 60 mg/kg, 2 90 mg/kg and
1180 mg/kg injection schemes.
Animals were randomly assigned to one out of four groups
containing six animals per group: The first group received three
injections of 60 mg/kg of the solution with an inter-injection
interval of 48 h (fur ther referred to as 3 60/48), the second
group 8 injections of 30 mg/kg of the solution with an inter-
injection interval of 24 h (further referred to as 8 30/24), and
animals of the third group received a single injection of 99 mg/kg
(1 99). As the first two animals of the third group showed severe
side effects on Mn
shortly after injection and had to be
sacrificed, the latter injection protocol was changed for the
remaining four animals to 6 injections of 30 mg/kg with an inter-
injection interval of 48 h (further referred to as 6 30/48). The
fourth group served as a control group, receiving three injections
of 0.06 ml/10 g of 0.9% NaCl every 48 h (Fig. 1a). All injections
were administered intraperitoneally. To account for the circadian
rhythm of corticosterone secretion, injections were always
performed in the beginning of the dark phase of the mice.
Animals were returned to their home cages after each injection.
Vegetative and behavioural measurements
Behaviour and general health status of the animals was assessed
after each injection and prior to each blood collection (Fig. 1a).
Measures included body weight, body position, tremor, palpebral
closure, coat appearance, whiskers, lacrimation, defecation, gait
and tail elevation. All measures were assessed according to the
European Mouse Phenotyping Resource of Standardized Screens
guidelines (EMPRESS) (
For telemetry, 18 additional animals were randomly assigned
to one out of three groups. Telemetric devices (TA10TA-F20,
PhysioTel Implant, Data Sciences international, St.Paul, USA) were
implanted 14 days before starting of the vegetative and
behavioural recordings. For the implantation procedure, mice
were anesthetized using a combination of ketamine/xylazine
(50 mg/kg ketamine (Essex Pharma GmbH, Germany) þ5 mg/kg
xylazinhydrochloride (Rompun, Bayer Health Care, Germany)).
Transponders were purified and sterilized with benzalchonium
chloride (Sigma, Germany), repeatedly flushed with sterile 0.9%
NaCl and inserted into the abdominal cavity. The wound was
disinfected by Braunol (Braun AG, Melsungen; Gemany). For
analgesia, 0.5 mg/kg Metacam (Vetmedica GmbH, Boehringer
Ingelheim, Germany) was injected subcutaneously right after
surgery. Animals had a recovery period of at least 14 days within
which body weight and health appearance were controlled.
Starting with six animals per group, three animals had to be
excluded due to technical problems and subsequent acquisition
errors which resulted in following group sizes: four animals for
the 6 30/48 group, five for the 8 30/24 and six for the 3 60/
48 group. The monitoring of body temperature and locomotion
was carried out using the Dataquest LabPRO (Data Sciences
International, Version 3.11, USA, Minnesota) acquisition system.
Locomotion is displayed as activity counts for the detected
period. Activity counts depend strictly on the distance the animal
moves horizontally with a velocity of above 1 cm/sec. Following
the recovery period, telemetric devices were turned on for a
three-day baseline recording. As temperature and locomotion
of the animals follow a circadian rhythm, we calculated deviations
of these measures from a mean of the three day baseline. All
animals then received a NaCl injection (0.1 ml/10 g) three days
before the Mn
injection protocols to investigate the influence
of a single vehicle application on temperature and locomotion
(Fig. 1b). These data also served as control for subsequent Mn
injections. Temperature and locomotion measurements were
averaged for bins of 30 min for each animal.
Endocrine measurements
To investigate activation of the hypothalamic-pituitary-adrenal axis
(HPA-axis) in response to the different MnCl
O application
schemes, corticosterone levels were measured in all animals on the Copyright ß2010 John Wiley & Sons, Ltd. NMR Biomed. (2010)
first day, at time points 4 h (d1/4h) and 12 h (d1/12h) after Mn
injection, and on the fifth day after 12 h (d5/12h) (Fig. 1a).
Blood samples were collected in capillary tubes containing
ethylenediaminetetraacetic acid (EDTA, Kabe Labortechnik,
Germany) to prevent clotting. Samples were kept on ice until
centrifuging (8000 RPM, 15 min, 48C), after which plasma was
subtracted and stored at 208C. Plasma corticosterone measure-
ments were performed using an ImmuChem Double Antibody
I-Radioimmunoassay kit (MP Biomedicals,
Eschwege, Germany) with a minimal detectable corticosterone
concentration of 7.7 ng/ml. The coefficients for intra-assay and
inter-assay variation were 7.3% and 6.9%, respectively.
MRI acquisition
MRI experiments were performed on a 7T Avance Biospec 70/30
scanner (Bruker BioSpin, Ettlingen, Germany). Imaging was
performed 24 to 28 h after the last injection.
Mice were anaesthetized with isoflurane (DeltaSelect,
Germany) and fixed in a prone position on a saddle-shaped
receive-only coil, where they were further kept under inhalation
anaesthesia with an isoflurane-oxygen mixture (1.5–1.9 vol% with
an oxygen flow of 1.2–1.4 l/min). Head movements were
prevented by fixing the frontal teeth with a surgical fiber. Body
temperature was monitored with a rectal thermometer (Ther-
malert TH-5, Physitemp Instruments, USA) and kept between
348C and 368C using a heating pad. Pulse rate was continuously
monitored by a plethysmographic pulse oxymeter (Nonin 8600V,
Nonin Medical Inc., USA).
-weighted (T1w) brain images were acquired using a 3D
gradient echo pulse sequence [TR ¼50 ms, TE ¼3.2 ms, matrix
size ¼128 106 106 zero filled to 128 128 128, field of view
(FOV) ¼16 16 18 mm
, number of averages ¼10, resulting in a
spatial resolution of 125 125 140.6 mm
with a total measure-
ment duration of 90 min]. Additionally, 3D T
-weighted (T2w)
images were obtained using a RARE (rapid acquisition relaxation
enhanced) pulse sequence [ TR ¼1000 ms, TE ¼10 ms, matrix
size ¼128 112 112 zero filled to 128128 128, FOV ¼
16 16 18 mm
, N umber of averages ¼2, Rare factor ¼16,
resulting in a resolution of 125 125 140.6 mm
, with a
measuring time of around 30 min]. Total measurement time was
around 2 h. Animals were immediately sacrificed after scanning.
MRI data processing
Images were reconstructed using Paravision software (Bruker
BioSpin, Ettlingen, Germany) and transferred tostandard ANALYZE
format. Further post-processing was performed using SPM2
Figure 1. Experimental schedules: A) Application schemes. Time points of injections, health assessement, corticosterone measurements (tail cut), and
the MR scanning are indicated. Control animals were injected according to the 3 60/48 scheme. B) Telemetric measurements. After implantation of the
telemetric device, animals had two weeks of recovery after which a three day baseline recording was started. A NaCl injection was applied three days prior
to starting point of the Mn
application scheme.
NMR Biomed. (2010) Copyright ß2010 John Wiley & Sons, Ltd.
( T1w- and T2w-images were first co-
registeredusing affine transformations. Imageswere bias corrected
to remove intensity gradients introduced by geometry of the
surface coil. A representative T1w- and T2w-image respectively of
one animal was selected that served as a first template for the
generation of a customized second generator template.
Images of all mice were spatially normalized to the single animal
template. A group template was then produced based on an
averageof all images. Bias correctedimages of all individualanimals
were then normalised a second time to the group template.
For improved normalisation of T1w-images, independent of
extra-brain tissue as well as signal hyperintensity of large vessels in
the T1w-images, normalization steps of T2w-images were carried
out first. Due to the better contrast betweenparenchyma and other
tissue types and no signal hyperintensity of large vessels compared
to T1w-images, a brain extraction step could be performed as
follows: A binary mask defining the intracranial vault without large
vessels (whole brain) was defined (MRIcro,
rorden/mricro.html) on the T2w-group template, and transformed
to native (co-registered) space of each individual animal (by
inverted spatial normalisation). Brain extracted images of the co-
registered and bias corrected T1w-images were then used for the
normalisation steps of T1w-images.
Regions of interest analysis of MEMRI contrast
Regions of interest (ROIs) were defined on the T1w-group
template for selected structures (Fig. 2a), based on the
anatomical atlas of the C57BL/6 mouse by Paxinos and Franklin
(26). As ROIs we defined the bilateral colliculi, CA1þ2 and
CA3þdentate gyrus (DG) regions of the hippocampus, the
caudate putamen, the thalamus and hypothalamus, and a cortical
region as reported in a previous study (17). The extracted whole
brain (wb) also served as a ROI. In addition, an area of the muscle
surrounding the skull was defined to serve as a measure of global
signal intensity. Binary ROI masks were back-transformed into
native space as described for the brain extraction step. Eventually,
intensity measurements of each ROI were performed on the bias-
corrected raw T1w-images of each animal using in-house written
software in (IDL, Mean intensities of the ROIs
within the brain are divided by the mean intensity of the muscle
ROI, and are referred to as ‘relative intensity’ (RI).
To visualize the capacity of the fractionated application schemes
to resolve cerebral fine structure, e.g. cortical layers, the original
image matrix size of 128 128 128 provides insufficient
resolution. Therefore, we reprocessed representative MEMRI
images of each group to an image matrix size of 256 256
256 points with subsequent bias correction. On coronal sections,
truncation artefacts (‘Gibbs ringing’) are visible which hamper
precise evaluation of cortical layer structures, especially parallel to
the skull surface. To avoid contamination by such artefacts, we only
evaluated cortical structures running perpendicular to the skull
surface, e.g. the retrosplenial granular cortex (Fig. 3).
Statistical analyses
Calculated mean intensities within the ROIs of the T1w-images
were divided by the animals mean intensity of the muscle ROI.
Figure 2. MEMRI measurements: A) ROIs as defined on the extracted T1w group average. ROIs are roughly depicted on a representative coronal (left)
and a horizontal slice (right). All ROIs covered multiple slices and were defined bilaterally, based on the anatomical atlas of BL6 mice by Franklin and
Paxinos (26). B) Coronal (top) and horizontal (bottom) slices for control, 6 30/48, 3 60/48 and 8 30/24 application schemes. Mean images of the
different groups are shown. All Mn
images show significantly higher intensities than images of NaCl injected animals. The 8 30/24 group shows
highest intensity and best contrast enhancement, followed by the 3 60/48 and 6 30/48 group. Hippocampus is outlined best in the 8 30/24 group.
Structures of the bulb and the cerebellum are outlined best in the 8 30/24 group (see Figure 3). Copyright ß2010 John Wiley & Sons, Ltd. NMR Biomed. (2010)
Normalised values are referred to as relative intensities (RI). In
additionto the separate ROIs, averageRI over all ROIs combinedwas
calculated, referred to as cortical and subcortical (cþsc) RI. Data
were analyzed by one-way, two-way and repeated measurement
analysis of variance (ANOVA) by means of SPSS 16.0 (Chicago, IL,
USA). Statistica 5.0 (Statsoft, 1995) was used to conduct pair wise
post-hoc comparisons. One-way ANOVA was employed to
investigate the effect of the application scheme on the wb and
cþsc RI values, respectively. To explore regiongroup effects that
would indicate different contrast across regions depending on the
application scheme, two-way ANOVA was applied with factors
group (4-levels) and region (7-levels). To determine if application
schemes produced distinct between-region contrast differences,
two analyses were performed: 1. LSD (Least significant difference)
post-hoc tests of the interregional differences per group were
reviewed and nominally significant pair wise differences counted
per group. 2. On basis of the assumptions that both mean intensity
and interregional differences can be enhanced using Mn
coefficient of variation (CV) across regional RIs was determined for
each animal and correlated with the total dosage of applied
O (0 mg, 180 mg, 240 mg; Spearman rank correlation
Temperature and locomotion were submitted to one-way
ANOVA to examine the effect of the application scheme on the
grand mean of temperature and locomotion drops. LSD test was
used for post-hoc comparison. To explore the development of
temperature and locomotion drops over time we applied
repeated measurements ANOVA to each application scheme
separately. LSD post hoc tests were applied when necessary.
Endocrine measurements were analyzed using one-way ANOVA
to compare corticosterone levels between groups at different
time points. LSD post-hoc tests were used when appropriate.
Body weights were compared using repeated measurements
ANOVA for day 1, day 3 and day 5. LSD post-hoc test was used to
examine detailed differences. Data are presented as mean SEM
using GraphPad Prism 4.0 (GraphPad Software Inc., San Diego, CA,
USA). Statistical significance was accepted if p<0.05.
MRI signal intensity
Figure 2b shows representative coronal and horizontal slices of
T1w-mean images for each group. Visual inspection indicates that
all Mn
groups show higher contrast than the control group,
with the 8 30/24 group exhibiting the highest overall signal and
best contrast enhancement, especially in the hippocampus,
olfactory bulb and cerebellum, followed by the 3 60/48 group
and the 6 30/48 group.
Layers of the olfactory bulb were distinguishable in every
injection protocol involving Mn
with the 8 30/24 protocol
showing best enhancement and contrast (Fig. 3a). Also the
cerebellum showsgood differentiation between laminarstructures
in all Mn
injection protocols (Fig. 3b). Cortical layers of the
retrosplenial granular cortex can be distinguished best in the
830/24 group, followed by the 3 60/48 group. In the 6 30/48
protocol the layers are hardly distinguishable (Fig. 3c).
Both for relative intensity in the whole brain (wb RI) and relative
intensity over all cortical and subcortical regions (c þsc RI), a
significant effect of the application scheme was detected
(p¼0.002 and p <0.0001, respectively), with post-hoc tests
demonstrating a stepwise increased order (control !630/
48 !360/48 !830/24). Within each group, the wb RI was
lower than the c þsc RI value ( p-values <0.038) (Fig. 4a). When all
Figure 3. Visualisation of fine structures: Representative images for each group are shown. A) Coronal sections of the olfactory bulb. Layers are well
distinguishable in every protocol involving Mn
. Different layers are indicated on the Nissl stain (right, http://mouse.brainmap. org/atlas/ARA/Coronal)
(E/OV: ependymal and subendymal layer/ olfactory ventricle, EPL: external plexiform layer, GL: glomerular layer, GrO: granular layer, IPL: internal plexiform
layer, Mi: mitral cell layer, ONL: Olfactory bulb layer). B) Sagittal sections of the cerebellum. Delineation of laminar structure is not present in the control
group, while Mn
application led to improved tissue contrast in ascending order from 6 30/48 to 3 60/48 to 8 30/24. A Nissl stain (right, at approximately the same location is inserted (plf: posterolateral fissure; pcn: precentral fissure; pcuf: preculminate fissure; prf:
primary fissure; psf: posterior superior fissure; ppf: prepyramidal fissure; sf: secondary fissure). C) Coronal sections of the cortex. The location of the
retrosplenial granular cortex (RSG) is indicated by the ellipse in the Nissl stain (right, Layers were
identified according to the Allen mouse brain atlas (, with cortical layer IV not being present in the RSG. Layers indicated on
the Nissl stain are best discriminated on the 8 30/24 MR image.
NMR Biomed. (2010) Copyright ß2010 John Wiley & Sons, Ltd.
ROIs were modelled separately, strong effects of both group
3, 126
¼106.3, p<0.0001) and region (F
6, 126
¼8.5, p<0.0001)
were detect ed, yet the group region interaction was not
significant (F
18, 126
¼1.4, p¼0.154), indicating that the sorting of
regional RIs was similar for all application protocols. The
significant region effect, however, indicated inter-regional
differences of the Mn
accumulation, with the CA3 þDG region
showing highest values for all protocols. Pair wise post-hoc
comparisons between regions were significant for 12 pairs for the
830/24 protocol, as compared with 7, 5, and 3 pairs for the
control, 6 30/48 and 3 60/48 protocol, respectively (data not
shown). A moderate correlation was detected between the CV
(calculated across the 7 ROIs for each animal) and the total dose
of Mn
(Spearman rank test, rho ¼0.66, p¼0.001) (Fig. 4b).
Vegetative and behavioural measurements
Health assessment
The general health status of the animals was assessed after
every injection and blood collection, but failed to reveal any
changes between different application schemes (Table 1).
In contrast, statistical analysis of body weight showed group
and time effect (F
¼5.73, p¼0.006 and F
¼10.52, p¼0.005
respectively). Also the group time interaction showed signifi-
cant differences (F
¼3.77, p¼0.005), indicating different
development of bodyweight over time. As expected, post hoc
comparison revealed no significant differences in body weight
before the first injection (with an average weight of 24.0 0.6 g).
All Mn
groups showed weight loss after the first injection, but
only for the 8 30/24 and the 3 60/48 group those losses were
significant. For all groups body weights recovered after two days
(data not shown).
Grand means of temperature showed significant differences
between groups (F
¼57.25, p<0.0001). Post hoc comparison
revealed significant differences between 3 60/48 and 6 30/48
(p<0.0001) as well as 3 60/48 and 8 30/24 ( p<0.0001).
630/48 and 8 30/24 showed no significant differences
(Fig. 5a, left). All groups differed significantly from the
temperature response to the NaCl injec tion (p <0.002). Tempera-
ture changes returned to baseline levels 7.5 h after Mn
injection (Fig. 5b). Upon repeated injections neither 6 30/48 nor
830/24 showed significant influence of time (F
5, 10
p¼0.58, and F
7, 28
¼1.12, p¼0.38, respectively), if temperature
deviations from baseline were investigated over the time course
of each application protocol separately per group. In contrast, the
Figure 4. MRI signal intensities: A) MRI signal enhancement in the whole brain (wb) and the average relative intensities (RI) over all cortical and
subcortical (cþsc) ROIs. An increase in signal intensities was detectable from control (NaCl) to 8 30/24 in both wb as well as in cþsc (
p<0.001). B) Coefficients of variation across regional RIs calculated for each animal. A Spearman rank test was applied to the CV for the
total Mn
dose delivered to the animals. The correlation coefficient was found to be rho¼0.66, with a p-value of p ¼0.001, indicating that a higher total
amount of Mn
applied provides a better contrast.
Table 1. Health assessment of animals in each group. Measures were assessed according to the EMPRESS guidelines. No detectable
changes in health conditions were observed in animals which were not used for telemetry.
Control 3 60/48 8 30/24 6 30/48
Number of animals 6 6 6 4
Observation period in days 5 5 8 11
Deaths 0 0 0 0
Body position Normal Normal Normal Normal
Tremor Normal Normal Normal Normal
Palpebral closure Normal Normal Normal Normal
Coat appearance Normal Normal Normal Normal
Whiskers Normal Normal Normal Normal
Lacrimation Normal Normal Normal Normal
Defecation Normal Normal Normal Normal
Gait Normal Normal Normal Normal
Tail elevation Normal Normal Normal Normal Copyright ß2010 John Wiley & Sons, Ltd. NMR Biomed. (2010)
Figure 5. Effects of Mn
treatment on vegetative and endocrine measures: A) Body temperature: Grand mean over all days of injection (left) and time
course mean (right) of temperature deviation of the adjacent 7.5 h after injection from the corresponding 0.5h intervals of the three day baseline mean.
B) Temperature drop shown as a mean curve of 0.5h intervals after the 2nd injection (d2 for 8 30/24, d3 for 3 60/48 and 6 30/48). The 3 60/48
application scheme shows a mean temperature drop of around 58C whereas the 8 30/24 group shows temperature drop of maximal 1.58C. The 6 30/
48 group caused a maximal temperature drop of around 28C. C) Locomotion: Grand mean over all days of injection (left) and time course mean (right) of
locomotion deviation of the adjacent 7.5 h after injection from the corresponding 0.5h intervals of the three day baseline mean. Locomotion counts are
strictly dependent on the distance the animal moves with a velocity of above 1cm/s. D) Corticosterone levels at different time points for the different
application schemes. 8 30/24 and 6 30/48 were pooled at d1 as both groups received first injection of 30 mg/kg MnCl
groups started
out with higher corticosterone levels than the control group but reached levels of vehicle treated animals after repeated injection.
NMR Biomed. (2010) Copyright ß2010 John Wiley & Sons, Ltd.
360/48 group became sensitized to Mn
induced hypother-
mia (F
2, 10
¼6.01, p ¼0.019) (Fig. 5a, right).
The locomotion deviation from baseline showed a similar
pattern but with weaker effects. Grand means of
locomotion also showed significant differences between groups
3, 29
¼6.09, p¼0.003) with 3 60/48 being significantly
different to 6 30/48 ( p¼0.023) and 8 30/24 ( p¼0.010) as
well as to the single NaCl injection applied three days before
application of the injec tion scheme ( p<0.0001) (Fig. 5c, left).
Repeated measurements ANOVA for locomotion deviation from
baseline failed to reveal significant differences between different
time points in any of the application schemes (statistics not
shown, Fig. 5c, right).
Endocrine measurements
Figure 5d shows the stress response as measured by corticos-
terone levels to injection of vehicle or Mn
at the different time
points. On the first day (time point d1/4h and d1/12h)
corticosterone measurements for the 6 30/48 and the
830/24 group were pooled (pooled 30), as both groups
received first injection of 30 mg/kg of MnCl
O. All groups
showed the highest corticosterone levels on d1/4h, with mean
levels of 64 49 (mean standard deviation) ng/ml for the
control, 160 59 ng/ml for the pooled 30 and 192 187 ng/ml
for the 3 60/48 group. On d1/12h these levels have declined to
27 19, 80 63 and 122 84 ng/ml for control, pooled 30 and
360/48 group, respectively. All Mn
treated groups showed
increased corticosterone levels on d1/4h and d1/12h compared
to the control group, but not on d5/12h.
Statistical analysis failed to reveal a significant effect of group
¼2.37, p¼0.12) at the first time point (d1/4h). Post hoc
tests showed a trend between control and pooled 30 and 3 60/
48 ( p¼0.1 and p¼0.05, respectively). At the second time point
(d1/12h) a significant effect of group could be detected
¼3.51, p¼0.05) with post hoc tests showing a trend
between control and pooled 30 group (p¼0.1) and significant
differences between control and 3 60/48 ( p¼0.02). At
day 5 (d5/12h) statistical analysis failed to reveal significant
differences (F
¼2.37, p>0.5).
In summary, these results indicate that the first injection with
O causes a considerable amount of stress, but the
animals rapidly tend to habituate to it upon repeated
O injection.
In this study, we compared effects of three different fractionated
application protocols of Mn
on vegetative, behavioural and
endocrine measurements. We found that the protocols with
30 mg/kg doses of injected MnCl
O showed least effects on
health and on animals’ well being.
Of the proposed injection schemes, a daily injection of 30 mg/
kg over 8 days was found optimal in balancing systemic side
effects and satisfying MEMRI contrast. In particular hypothermia
and hypolocomotion were less marked than in the 3 60/48
scheme, while MEMRI contrast features were found to be best.
Application of Mn
led to nominally higher corticosterone levels
4 h and significantly higher corticosterone levels 12 h after
treatment in the 3 60/48 and the pooled 30 compared to
control with no significant differences between 60 mg/kg and
30 mg/kg doses. On day 5 of the experiment, however,
corticosterone levels of all Mn
groups were comparable to
those of the control group. These results imply, that mice
perceiving Mn
treatment as a considerable stressor may readily
habituate upon repeated encounter. In support of this
conclusion, Mn
effects on body weight were only transient,
with recovery after two days.
The 8 30/24 application protocol not only delivered the
highest amount of Mn
to the brain, as measured by RI in T1w
wb and cþsc ROIs, but also provided the best differential contrast
between brain structures.
More specifically, fine structures of the olfactory bulb and
cerebellum could be distinguished with every Mn
protocol. Only at images acquired from mice treated with the
830/24 protocol layers of the retrosplenial granular cortex
became distinguishable. Similar to a study by Lee and colleagues
(27) who showed that MEMRI is able to resolve fine structures of
the mouse cortex using single high doses of Mn
(88 mg/kg or
greater), we could show that visualising fine brain structures is
also possible applying fractionated doses of Mn
in mice.
Sampling at higher resolutions should improve the differentiation
of cortical layers by minimizing artefacts
The contrast differences between brain regions, obtained in
the control group (number of pair wise differences: 7) indicate a
natural variation in T1w-contrast which is flattened by Mn
administration of a low total dosage (number of pair wise
differences for 3 60/48 and 6 30/48: 3 and 5 respectively), but
enhanced by higher total dosages (number of pair wise
differences for 8 30/24: 12). The coefficient of variation,
however, showed that this effect is more dependent on the
total dosage then on the fractionated treatment, with control
group showing lowest variation followed by the groups that
received a total dosage of 180 mg/kg (3 60/48 and 6 30/48)
and the 8 30/24 group which received 240 mg/kg showing the
highest coefficients of variation. However, we can not rule out
that the higher regional and overall contrast of the 8 30/24
group was influenced by the shorter inter-injection interval.
Furthermore, our experimental protocols are limited to 8 days of
injections at maximum. While this provides a reasonable
experimental time frame, we can not rule out that further
extending the low-dosage application protocol would lead to
even better contrast enhancement without impairing animal
well-being. Further studies are necessary to investigate the
timing effect of Mn
application on contrast enhancement and
to clarify, if the application of an extended 8 30/24 protocol
leads to better contrast enhancement.
Regarding well-being of the animals, it should be noted that
despite the highest total dose delivered, the fractionated 8 30/
24 application scheme led to less severe side effects than the
360/48 scheme. This suggests that the toxic effects are not
merely dependent on the total Mn
dose, but also on the
fractionated delivery. While a single application of 99 mg/kg of
O, notably well tolerated in rats (17) and in CD1 mice
(own observations), leads to severe health impairment in BL6N
mice, a total dose of 240 mg/kg delivered over the course of
8 days had only minor consequences on vegetative functions,
behaviour and stress responses. Therefore, taking into consider-
ation the long half life of Mn
, it seems advisable to apply lower
doses of Mn
in a repeated manner, thus avoiding single dose
toxicity and making use of fast recovery after low doses. When
studying sensitive mouse models with potentially compromised
health (e.g. genetically modified animals or mice at early
developmental stages), the fractionated approach may be Copyright ß2010 John Wiley & Sons, Ltd. NMR Biomed. (2010)
particularly useful to avoid unnecessary stress for the animal. Our
data also suggest that pilot experiments on Mn
toxicity have to
include physiological and behavioural measurements rather than
relying solely on general health assessment (cf. Table 1).
MEMRI opens the possibility to study complex paradigms in
freely behaving animals, as only the read-out of the
cerebral Mn
accumulation requires sedation of the animals.
The fact that fractionated application of 30 mg/kg
O did not interfere with the animals’ well-being,
and that corticosterone levels rapidly recovered to baseline levels
after the first injections makes the suggested scheme suitable for
application to different stress paradigms. While fractionated
application protocols lead to good overall contrast enhancement,
studies with repeated readouts are required to determine the
temporal dynamics of the MEMRI contrast. This may help to
optimize the timing of a specific task, if the paradigm cannot be
continuously applied due to experimental reasons or habituation
of the animal.
In conclusion, we provide evidence that higher total doses
of Mn
lead to improved general and between-region MEMRI
contrasts while fractionated application minimises adverse
effects caused by the toxicity of Mn
. More specifically, we
demonstrate that behavioural, vegetative and endocrine markers
of stress are only minimally and transiently affected, when a
fractionated application scheme with low single doses is used.
MEMRI with fractionated Mn
applications is therefore
particularly suitable for paradigms that probe the animals’ stress
system, including complex behavioural paradigms.
We are grateful to Armin Mann for continuous technical support.
We would like to thank Bianca Mayer for performing corticos-
terone measurements.
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NMR Biomed. (2010) Copyright ß2010 John Wiley & Sons, Ltd.
    • "In vivo MEMRI was performed essentially as described before (Grünecker et al., 2010, 2012). Briefly, minimum 2 weeks after the last testing animals received intraperitoneal injections of 30 mg/kg MnCl2 (Sigma, Germany) every 24 h over the course of 8 consecutive days. "
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    Full-text · Article · Dec 2012
    • "The Mn 2+ administration protocol was selected to provide sufficient enhancement of brain tissue signal, i.e., 30%–50%, with minimum effect on animal well-being. This criterion was based on reports on fractionated MEMRI in rats (Bock et al. 2008; Inui-Yamamoto et al. 2010), mice (Grünecker et al. 2010), and on our own pilot studies. A stock solution of 10 mM MnCl 2 (Sigma) in DDW was prepared. "
    [Show abstract] [Hide abstract] ABSTRACT: Memory consolidation is defined temporally based on pharmacological interventions such as inhibitors of mRNA translation (molecular consolidation) or post-acquisition deactivation of specific brain regions (systems level consolidation). However, the relationship between molecular and systems consolidation are poorly understood. Molecular consolidation mechanisms involved in translation initiation and elongation have previously been studied in the cortex using taste-learning paradigms. For example, the levels of phosphorylation of eukaryotic elongation factor 2 (eEF2) were found to be correlated with taste learning in the gustatory cortex (GC), minutes following learning. In order to isolate the role of the eEF2 phosphorylation state at Thr-56 in both molecular and system consolidation, we analyzed cortical-dependent taste learning in eEF2K (the only known kinase for eEF2) ki mice, which exhibit reduced levels of eEF2 phosphorylation but normal levels of eEF2 and eEF2K. These mice exhibit clear attenuation of cortical-dependent associative, but not of incidental, taste learning. In order to gain a better understanding of the underlying mechanisms, we compared brain activity as measured by MEMRI (manganese-enhanced magnetic resonance imaging) between eEF2K ki mice and WT mice during conditioned taste aversion (CTA) learning and observed clear differences between the two but saw no differences under basal conditions. Our results demonstrate that adequate levels of phosphorylation of eEF2 are essential for cortical-dependent associative learning and suggest that malfunction of memory processing at the systems level underlies this associative memory impairment.
    Full-text · Article · Feb 2012
    • "Before scanning, animals were injected intraperitoneally with a 50 mm manganese chloride (MnCl 2 *4H 2 O) (Sigma, Steinheim, Germany) solution in 0.9% NaCl (pH 7). The injection protocol was based on the fractionated application scheme proposed by Grünecker et al. (2010) and was well tolerated by the animals. A total concentration of 180 mg ⁄ kg was injected in three fractionated doses of 60 mg ⁄ kg MnCl 2 with an interinjection interval of 48 h. "
    [Show abstract] [Hide abstract] ABSTRACT: Patients suffering from major depression have repeatedly been reported to have dysregulations in hypothalamus-pituitary-adrenal (HPA) axis activity along with deficits in cognitive processes related to hippocampal and prefrontal cortex (PFC) malfunction. Here, we utilized three mouse lines selectively bred for high (HR), intermediate, or low (LR) stress reactivity, determined by the corticosterone response to a psychological stressor, probing the behavioral and functional consequences of increased vs. decreased HPA axis reactivity on the hippocampus and PFC. We assessed performance in hippocampus- and PFC-dependent tasks and determined the volume, basal activity, and neuronal integrity of the hippocampus and PFC using in vivo manganese-enhanced magnetic resonance imaging and proton magnetic resonance spectroscopy. The hippocampal proteomes of HR and LR mice were also compared using two-dimensional gel electrophoresis and mass spectrometry. HR mice were found to have deficits in the performance of hippocampus- and PFC-dependent tests and showed decreased N-acetylaspartate levels in the right dorsal hippocampus and PFC. In addition, the basal activity of the hippocampus, as assessed by manganese-enhanced magnetic resonance imaging, was reduced in HR mice. The three mouse lines, however, did not differ in hippocampal volume. Proteomic analysis identified several proteins that were differentially expressed in HR and LR mice. In accordance with the notion that N-acetylaspartate levels, in part, reflect dysfunctional mitochondrial metabolism, these proteins were found to be involved in energy metabolism pathways. Thus, our results provide further support for the involvement of a dysregulated HPA axis and mitochondrial dysfunction in the etiology and pathophysiology of affective disorders.
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