212

International Journal of Radiation Biology, March 2013; 89(3): 212–218

© 2013 Informa UK, Ltd.

ISSN 0955-3002 print / ISSN 1362-3095 online

DOI: 10.3109/09553002.2013.734944

Correspondence: Dr Subash Khushu, NMR Research Centre, INMAS, DRDO Lucknow Road, Timarpur, Delhi, India. Tel: � 91 11 23905313. Fax: � 91 11

23919509. E-mail: skhushu@yahoo.com

(Received 5 October 2011 ; revised 27 August 2012 ; accepted 2 September 2012 )

Altered brain metabolism after whole body irradiation in mice: A preliminary in vivo 1 H MRS study

Poonam Rana 1 , Ahmad Raza Khan 1 , Shilpi Modi 1 , B. S. Hemanth Kumar 1 , Salim Javed 2 , Rajendra Prasad Tripathi 1 & Subash Khushu 1

1 NMR Research Centre, Institute of Nuclear Medicine and Allied Sciences (INMAS), Delhi, and 2 Department of Biochemistry ,

Jamia Hamdard , New Delhi , India

Introduction

Exposure to moderate dose of radiation (1 – 10 Gy) may occur

during the course of radiation therapy or as a result of radia-

tion accident. Th e resulting radiation injuries would be due

to a series of molecular, cellular and systemic processes

(Coleman et al. 2003). Studies from exposed human and

animals indicate that irradiation can aff ect a wide variety of

Abstract

Purpose : In the classical description of acute radiation syndrome,

the role of central nervous system (CNS) is underestimated. It

is now well recognised that ionising radiation-induced oxida-

tive stress may bring about functional changes in the brain. In

this study, we prospectively evaluated metabolic changes in the

brain after whole body irradiation in mice using in vivo proton

( 1 H) nuclear magnetic resonance spectroscopy (MRS).

Material and methods : Young adult mice were exposed to whole

body irradiation of 8 Gy and controls were sham irradiated. In vivo

1 H MRS from cortex-hippocampus and hypothalamic-thalamic

region of brain at diff erent time points, i.e., as early as 6 hours,

day 1, 2, 3, 5 and 10 post irradiation was carried out at 7 Tesla

animal magnetic resonance imaging system. Brain metabolites

were measured and quantitative analysis of detectable metabo-

lites was performed by linear combination of model (LCModel).

Results : Signifi cant reduction in myoinositol ( p � 0.03) and

taurine ( p � 0.02) ratios were observed in cortex-hippocampus

region as early as day 2 post irradiation compared to controls.

These metabolic alterations remained sustained over day 10

post irradiation.

Conclusions : The results of this preliminary study suggest that

the alteration/reduction in the mI and Tau concentration may

be associated with physiological perturbations in astrocytes or

radiation induced neuro-infl ammatory response triggered in

microglial cell.

Keywords: Ionising radiation , oxidative stress , in vivo NMR

spectroscopy

tissue specifi cally those with greater levels of cellular turn-

over and divisions. In the classical pathological concept

of acute radiation syndrome, it is generally assumed that

adult nervous tissue is highly radio resistant and is gener-

ally aff ected only at a dose of 20 – 30 Gy (Prasad 1995). In the

recent years, it has been understood that the exposure to

the whole body radiation also aff ects the functional status

of brain and few studies have considered central nervous

system as radiosensitive in terms of functional criteria (brain

electrical activity and neurochemical metabolism) for doses

as low as 2 Gy (Tofi lon and Fike 2000, Gourmelon et al. 2005).

In fact, it is widely accepted that brain exhibits infl amma-

tion and increased expression of infl ammatory cytokines/

mediators within hours of irradiating the rodent brain (Hong

et al. 1995). Additionally, a recent histological study on dif-

ferent brain regions after whole body radiation of 7.5 Gy in

rats has suggested mild morphological changes as tissue

swelling and mild abnormalities of astrocytes and glial cells

(Tehrani et al. 2010). Another study has reported oxidative

stress induced lipid peroxidation and reduced glutathione

levels in diff erent regions of the brain after whole body radia-

tion exposure (Guney et al. 2005). All these diff erent studies

show signs of radiation-induced infl ammatory response, oxi-

dative stress or mild morphological changes that may bring

about metabolic alterations in diff erent brain regions, which

previously could only be studied in vitro after sacrifi cing

the animals. In the biological sciences, many studies using

animal models are limited by the inability to gather ana-

tomical and physiological information non-invasively and

in a longitudinal manner. Proton ( 1 H) magnetic resonance

spectroscopy (MRS) is a valuable and indispensable tool for

in vivo analysis of brain metabolites in animal models and

human. It is increasingly used in the area of neurodegenera-

tive diseases and other neurological disorders especially due

to the realisation of its potential role in identifying crucial

in vivo biomarkers of these diseases (Kantaraci et al. 2004,

Choi et al. 2007). In the last few years, many 1 H MRS-based

studies have provided metabolite information on late

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In vivo proton MRS of brain in irradiated mice 213

delayed radiation induced injury of the brain (Atwood et al.

2007a, 2007b, Chan et al. 1999) after whole brain irradiation

or targeted fractionated brain irradiation. Radiation-induced

delayed changes observed in the hippocampus and corti-

cal region of the brain in these studies have corroborated

well with impairment in cognitive function. In the recent

years, it has become clear that the biological responses

after single large dose of radiation to brain diff er from those

seen after fractionated brain irradiation (Gaber et al. 2003,

Yuan et al. 2006). Information on the early eff ects of whole

body radiation on brain metabolism is still limiting. Based

on infl ammatory response, oxidative stress-induced and

mild morphological changes as stated in earlier studies, we

hypothesise that whole body irradiation would also prompt

metabolic alterations in diff erent brain regions. In the pres-

ent study, we have made use of in vivo proton MRS for iden-

tifi cation of radiation-induced early metabolic changes after

whole body radiation exposure in mice.

Material and methods

Animal handling and radiation exposure Twenty young adult strain ‘ A ’ male mice of 10 weeks of age

were taken from experimental animal facility located at our

institute and acclimatized for 48 h in polypropylene cages

prior to group allocation and treatment. During the study,

room temperature was maintained within 19 – 23 ° C, daily

relative humidity within 45 – 65%, and fl uorescent lighting

was provided (06:00 to 18:00 h) for 12 h light/12 h dark cycle.

Mice ( n � 10) were exposed to a whole body radiation of a

single dose of 8 Gy through Tele 60 Co unit gamma irradiation

facility (Bhabhatron II, Panacea Medical, Bangalore, India)

with source operating at 2.496 Gy/min in our institute. All

animals were irradiated with a source to surface distance

(SSD) of 80 cm from the centre of the animal with a fi eld

size of 35 � 35 cm. Th e remaining 10 mice served as sham-

irradiated controls. All animal handling and experimental

protocols conformed to the guidelines stipulated by the

institutional animal ethical committee.

Magnetic resonance imaging (MRI) and proton MRS MRI and MRS experiments were carried out pre dose (day 0)

and at diff erent time points during the post irradiation phase

starting as early as 6 h and then followed on day 1, 2, 3, 5 and

10 in control as well as the sham-irradiated group. Anaes-

thesia was induced by i.p. injection of a mixture of xylazine

(10 mg/kg body weight [BW]) and ketamine (80 mg/kg BW).

All animals were placed in a prone position on an animal bed

and then slid into the magnet bore. All MR experiments were

performed on a 7 Tesla Bruker Biospec animal magnetic

resonance (MR) imaging system (Biospec USR70/30, Bruker

Biospin, Switzerland). Radio-frequency (RF) excitation was

accomplished with 72-mm inner diameter (ID) linear bird-

cage coil and 4 channel phase array mouse brain coil was

used for signal reception. A rapid acquisition with relaxation

enhancement (RARE) sequence (echo time [TE], 26 ms; rep-

etition time [TR], 2.5 s) with a fi eld of view � 2.6 � 2.6 cm and

a matrix size of 256 � 256 was used to acquire multislice (11

slices with 0.7 mm slice thickness) T 2 (transverse relaxation

time) weighted images of the mouse brain. Th ese high resolu-

tion images in three orthogonal planes were used as localis-

ers for the position and size of the spectroscopic voxels. Th e

MRS voxels were localised in: (a) Th e hypothalamic-thalamic

region, and (b) in the cortex-hippocampus regions of mouse

brain (1.5 � 3.5 � 3.0 mm 3 ; 15.75 μ l, each). Th e local fi eld

homogeneity was optimised by adjustment of the fi rst and

second order shim coils currents using fast automatic shim-

ming technique by mapping along projections (FASTMAP)

sequence. Th e fi eld homogeneity in a 15.75 μ l voxel typically

resulted in water line widths (FWHM) of 10 – 15 Hz in live

mouse brain. Th e water signal was suppressed by variable

power RF pulses with optimised relaxation delay (VAPOR).

Outer volume suppression combined with a PRESS (Point

Resolved Spectroscopy) sequence with spectral width of

4006.41 Hz, 2048 data points, 512 averages, 2.5 s TR and

20 ms TE with total acquisition time of 21 min was used for

acquiring the MR spectra. Eddy current compensation and

static magnetic fi eld drift correction were applied during the

data acquisitions.

MRS spectral processing and data analysis All spectra were initially processed using Topspin 2.1 software

provided on the scanner (Bruker Biospin, Switzerland). For

quantitative assessment of the brain metabolites, the MRS

raw data was analysed using linear combination of model

(LCModel) (Provencher 1993, 2001). Each spectrum was

fi tted to a set of known spectra from a series of metabolite

solutions with known concentrations. Spectral peaks were

assigned in the reference of water peak (4.82 ppm). To deter-

mine the detectability of each metabolite from measured

spectra, the % standard deviation (SD) of each metabolite

defi ned as the Cramer-Rao lower bound (CRLB) criteria,

was derived as measurable index for quantitation reliability

(Cavassila et al. 2001). Th e % SD was expressed as the per-

centage of the estimated overall concentration; a % SD of

� 20% was considered as an acceptable level of quantita-

tion reliability (Provencher 1993, 2001). Final MRS data in

the irradiated group was interpreted only with n � 6 mice as

4 mice died by day 10.

Statistical analysis Th e Sigmaplot 11.0 (Systat, USA) package was used for statis-

tical analysis of the spectral data. Th e data for each metabo-

lite were tested for homogeneity of variances and Student ’ s

t -test and repeated one way analysis of variance (ANOVA)

was used to compare means.

Results

An in vivo approach, 1 H MRS has been used for identifi cation

of metabolic changes in two important regions of the brain,

i.e., cortex-hippocampus and hypothalamic-thalamic region.

All MR images were reviewed for detectable lesions or

image abnormalities. No noticeable diff erences of image

intensities were observed in the T 2 weighted images in any of

the mouse brain. MR spectral voxels were positioned in the

same brain region of interest for all mice as shown in loca-

liser images (Figure 1).

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214 P. Rana et al.

Figure 1. Representative single voxel in vivo 1 H NMR spectra and localiser images of brain showing voxel positioning in (a) cortex-hippocampus (b) Th alamic-hypothalamic region. Th e metabolites are labelled as mI (myo inositol), Tau (taurine), Cho (choline), Cr � PCr (creatine and phosphocreatine), Glu � Gln (glutamate and glutamine). NAA (N-acetyl aspartate). Th is Figure is reproduced in colour in the online version of International Journal of Radiation Biology.

In the case of the irradiated group, in vivo 1 H MR spectra

of only 6 mice which were alive until the end of all the time

points were analysed using LCModel and 10 metabolites

were identifi ed in the cortex-hippocampus and thalamic-

hypothalamic regions of the mice (Figure 1). Because of

strong cross-correlation between glycerophosphocholine

(GPC) and choline (Cho) arising from the close spectral

similarity, only the sum of GPC and Cho (PCh) was reported.

To accurately extract the dominating metabolic changes

observed during diff erent time points post irradiation, and

to reduce systemic variations among studied animals, a rela-

tive quantifi cation method, using an internal spectral refer-

ence was used. Total creatine (Cr � PCr) spectral intensity

was used as the internal reference for relative quantitation

due to its relatively stable concentration in the brain (Salibi

and Brown 1998). Moreover, earlier MRS studies have also

observed no change in the concentration of creatine dur-

ing acute or delayed response of radiation exposure (Use-

nius et al. 1995, Kaminaga and Shirai 2005). Th e detectable

metabolites and their normalised values with total creatine at

diff erent post dose time points in both control and irradiated

group are given in Table I for both the regions of the brain.

Gamma amino butyric acid (GABA) and lipids observed in

MR spectra could not be included in fi nal comparative rela-

tive levels due to inconsistency in all spectra with SD � 20%.

Th e results explained changes only in cortex-hippocampus

region whereas no signifi cant changes could be observed in Ta

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In vivo proton MRS of brain in irradiated mice 215

Figure 2. Whole body radiation induced signifi cant change in (a) taurine and (b) myo-inositol levels in irradiated group ( � ) compared to controls ( ■ ). Error bars indicate the standard deviation of the mean for n � 6.

Figure 3. Th e relationship between main metabolite ratios (a) mI/Cr � PCr (b) tau/Cr � PCr and time duration post irradiation. Th e solid lines are the best linear fi ts through regression analysis.

any of the metabolites in hypothalamic-thalamic region. Post

irradiation day 2 onwards, statistically signifi cant decreased

levels of myo-inositol (mI) and taurine (tau) were observed

in the cortex-hippocampus region in animals irradiated

with ionising radiation when compared to the control sham-

irradiated group (Figure 2). Repeated measure one-way

ANOVA also observed signifi cant diff erences in both mI

and tau levels between post and pre-irradiation level. No sig-

nifi cant diff erence was observed in other important metabo-

lites, e.g., N-acetyl aspartate (NAA), choline and glutamine/

glutamate (Glx). Time-dependent decrease in mI/Cr � PCr

and tau/Cr � PCr values were detected day 2 onwards and

continued to decrease until day 10 post-irradiation (Figure

3). Signifi cant correlation (0.531, p � 0.01) was observed

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216 P. Rana et al.

between altered metabolite levels. To ensure that there was

no eff ect of spectral quality on relative intensity levels of mI

and tau, data was analysed using ANCOVA by taking FWHM

as a covariate. Results showed insignifi cant correlation

between above mentioned metabolite levels and FWHM at

all time points (Supplementary Table I, available online to

be found online at http://informahealthcare.com/abs/doi/

10.3109/09553002.2013.734944).

Discussion

Th ere are increasing evidences supporting radiation-

induced delayed changes in the brain after fractionated radi-

ation therapy or whole brain irradiation (Usenius et al. 1995,

Kaminaga and Shirai 2005, Atwood et al. 2007a, 2007b). How-

ever, eff ects of whole body irradiation on brain functional

metabolic changes are yet to be elucidated.

With the availability of high fi eld MR systems with

improved homogeneity, in vivo 1 H MRS facilitates highly

specifi c and precise biochemical information (neurochemi-

cal profi le) for particular regions even in the mouse brain.

In the present study, our results showed reduced mI and

taurine levels in cortex-hippocampus region of the mouse

brain, which started appearing day 2 onwards post irradia-

tion and persisted until day 10 (the last time point before the

death of the animals) compared to pre-irradiation level and

sham-irradiated control mice. Although the exact role of the

mI metabolite is unclear, it is an intermediate in the cerebral

inositol poly-phosphate (IPP) cascade and plays an important

role in intracellular signalling pathways (Berridge 1984, Reitz

1991). It is normally present at about 5 mM in human or ani-

mal brain and readily quantifi able by MRS at high magnetic

fi eld strength (Pfeuff er et al. 1999, Govindaraju et al. 2000).

On the contrary, taurine is much more highly concentrated in

rodent brain compared to human brain and present in vary-

ing concentration (Law 1994, Dedeoglu et al. 2004). Some

earlier studies have proposed myo-inositol, taurine and other

organic osmolytes present in the brain together as a part

of the volume regulation process (Kimelberg 1991, Flogel

et al. 1995). An earlier NMR-based in vitro study had shown

decrease in mI in rat brain tissue extracts approximately 14

weeks after total dose of 2 – 20 Gy given as 2 Gy fractions deliv-

ered to the brain (Sokol et al. 2004). Similarly, Atwood et al.

(2007b) observed decreased mI/Cr � PCr ratio in the brain

after administering fractionated radiation dose of 40 – 45 Gy.

Although the cellular, molecular and biochemical mecha-

nism of radiation-induced brain injury are ill-defi ned,

several studies lend support to the hypothesis that radiation-

induced injury is driven in part, via increased oxidative stress

through generation of free radicals (Monje and Palmer 2003,

Robbins and Zhao 2004, Limoli et al. 2006). Th e brain is

specifi cally susceptible to oxidative damage due to its high

content in polyunsaturated fatty acids, which are sensitive

to free radical attack and poor expression of antioxidant

defence mechanisms (Halliwell 1992). Few studies based on

NMR have reported oxidative stress-induced brain damage

(Flogel et al. 1995, Noseworthy and Bray 1998, Brand et al.

1999). An earlier study on cultured astrocytes had shown

oxidative stress-induced changes in the levels of mI, taurine,

hypotaurine using in vitro NMR spectroscopy (Flogel et al.

1995, Brand et al. 1999). Th e study reported dramatic loss

of mI, taurine and hypotaurine that prolonged throughout

the recovery period. Similarly, in our study changes started

appearing on day 2 and continued to decrease until the

time the animals survived. Changes observed in cortex-

hippocampus area in our study could be interpreted as

changes associated with astrocytes or other types of glial cells

and oxidative stress might be involved in dysregulation of the

osmotic control in astrocytes. Th ese changes are well cor-

related with observations attained in the Brand et al. (1999)

study. Astrocytes, like other glial cells, are an important

component of cellular composition of hippocampus which

can exert its infl uence through modulation of the volume,

composition, and the concentration of ions, within extracel-

lular space. Although astrocytes have a high capacity to toler-

ate cellular stress and retain their vitality, metabolic processes

and biosynthetic activities may be altered when exposed to

oxidative stress. Secondly, it has been demonstrated earlier

that membrane-damaging agents such as free radicals might

interfere with the energy metabolism by directly damag-

ing mitochondrial structure and function (Kimelberg 1991).

Consequently, ATP-driven transport systems and ion pumps

may be impaired, including Na � -K � -ATPase, Ca 2 � -ATPase

and the Na � -Ca 2 � exchangers, causing increased infl ux of

Na � and Cl � that results in astrocyte cell swelling (Kimelberg

1991). Dysregulation of the control of ion and volume regula-

tory processes and of the osmotic balance in astrocytes causes

its swelling, which has been observed after traumatic stimuli

and in many pathological conditions, e.g., ischemia, hypoxia

and reoxygenation (Kimelberg and Ransom 1986, Kimelberg

1991). Similarly, swelling of astrocytes after radiation exposure

might have caused altered levels of tau and mI in our study.

Additionally, neuro-infl ammation could be another pos-

sible reason for radiation-induced injury. Th ere are studies

in literature that support radiation-induced neuroimmune

and infl ammatory response (Gourmelon et al. 2005 , Lestaevel

et al 2008). It has already been stated that neuro-infl ammatory

brain response may bring out electrophysiological and bio-

chemical alterations (Lestaevel et al. 2008). In the present

study, change in mI levels post irradiation might be associ-

ated with neuro-infl ammatory response of microglial cells.

However, in our study, lack of change in the NAA/Cr � PCr

ratio supported the hypothesis that radiation did not lead to

a neuronal loss as NAA is considered as a neuronal marker

in 1 H MRS (Tkac et al. 2003). Th e results presented in the

study are in accordance with earlier radiation-related stud-

ies where neuronal loss is apparent only during radiation

induced delayed changes (Chan et al. 1999, Tofi lon and Fike

2000).

Th ere are many published reports that support cognitive

decline, depressive behaviour and aff ective state distur-

bances post radiation exposure (Raber et al. 2004, Atwood

et al. 2007a, Manda and Reiter 2010). Recently, one study

from our group observed memory defi cit, altered anxiety

level and gross behavioural changes based on open fi eld test

and novel object recognition test at day 5 and day 10 follow-

ing whole body radiation exposure of 8 Gy. Th ese changes

corresponded well with decreased fractional anisotropy and

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In vivo proton MRS of brain in irradiated mice 217

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reduced mean diff usivity in diff erent brain regions of mice

post irradiation (Trivedi et al. 2012). In our study, we have

found reduced mI and tau levels in the hippocampus region

in response to whole body radiation exposure. Hippocam-

pus is largely involved in memory and cognitive functions.

Furthermore, previous histological studies also showed that

the fi mbria of hippocampus was more sensitive to radiation

than other myelinated fi bre bundles in animals (Hodges

et al. 1998, Reinhold et al. 1990). Th e present study suggests

that the changes in brain metabolites involved in osmotic

regulation could be associated with cognitive impairment.

In conclusion, 1 H MRS has provided a unique opportu-

nity to acquire brain metabolite information after whole

body irradiation. Th e progressive decrease in taurine and mI

with increasing time duration post-irradiation may refl ect

radiation-induced physiological perturbations in astrocytes

or neuro-infl ammatory response triggered in microglial cell.

Th e absence of NAA loss suggests no change in neuronal cells

during acute radiation sickness. Th e present in vivo MRS

study, though very preliminary in nature, reports very signifi -

cant information about the metabolic changes in the cortico-

hippocampal region after exposure to high dose whole body

irradiation for the fi rst time. Th e present study is limited to

a single dose radiation exposure; however, additional stud-

ies with low and moderate dose radiation exposure would

be benefi cial in understanding the dose response as well as

dose-specifi c changes.

Declaration of interest

Th e authors report no confl icts of interest. Th e authors alone

are responsible for the content and writing of the paper.

Th is work was peformed as part of DRDO sponsored R &

D project INM 308. Th e authors are grateful for the fi nancial

support from Defence Research & Development Organisa-

tion (DRDO), Ministry of Defence, India (grant number:

STP-1-INM-308).

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Supplementary Material available online

Supplementary Table I available online to be found online at

http://informahealthcare.com/abs/doi/10.3109/09553002.

2013.734944.

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