UNCORRECTED PROOF

Solid State Nucl. Magn. Reson. ] (]]]]) ]]]–]]]

Investigation of metabolic changes in irradiatedrat brain tissue by means of 1H NMR in vitro

relaxation study

Maria Soko",a,� Waldemar M. Przybyszewki,b andBo’zena Matlasa

aDepartment of Medical Physics, Institute of Oncology, Silesian Technical University, ul. Wybrze’ze Armii

Krajowej, 15 Str., 44101 Gliwice, PolandbDepartment of Radiobiology, Institute of Oncology, Silesian Technical University, ul. Wybrze’ze Armii

Krajowej, 15 Str., 44101 Gliwice, Poland

Received November 7, 2002; revised February 21, 2003

Abstract

The effect of irradiation on concentrations and relaxation behaviour of brain metabolites

was studied by means of high-resolution 1H NMR in vitro. The studies were performed on rat

brains irradiated with the doses of 20Gy applied in fractions of 2Gy. Standard procedures

were used to obtain HClO4 extracts of rat brains. The 1H NMR studies of the extracts

solutions in D2O were performed using a Varian Inova-300 NMR spectrometer. The integral

intensities of the metabolite signals were found to change during the irradiation cycle and after

it. These changes are accompanied by the variations in the T1 relaxation times. N-

acetylaspartate, glycerophosphocholine, phosphocholine, choline, creatine and phosphocrea-

tine, myoinositol and taurine were analysed as potential markers of irradiation injury.

r 2003 Published by Elsevier Inc.

Keywords: Brain; Irradiation; 1H NMR in vitro; PCA extracts; Metabolite Concentrations; Relaxation

1. Introduction

Radiation therapy plays an important role in the treatment of brain tumors,especially in non-surgical cases, post-surgical treatment and in recurrent tumours [1].

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3B2v7:51cGML4:3:1 YSNMR : 157 Prod:Type:FTP

pp:1212ðcol:fig::NILÞED:Rekha

PAGN: Hashmath SCAN: padma

�Corresponding author.

E-mail address: mary@io.gliwice.pl (M. Soko").

0926-2040/$ - see front matter r 2003 Published by Elsevier Inc.

doi:10.1016/j.ssnmr.2003.03.013

UNCORRECTED PROOF

Traditional methods of treatment developed recently provide some tools forradiosurgerical use, such as for example Gamma Knife and a system based on alinear accelerator (known as linac) [2]. Development of radiation oncology has led toprolongation of the patient survival. However, in spite of technical progress, theeffects of radiation exposure induce various pathological changes such asdemyelinization or/and radiation necrosis in extreme [3]. On the cellular level,exposure to ionizing radiation results in immediate and widespread oxidativedamage. Although the lipid membranes destruction may occur, the most importantsub-cellular target is DNA. The cellular responses to DNA damage is apoptosis, cell-cycle arrest or repair [4]. Low-dose radiation stress is known to influence theprocesses involved in cell-cycle control, signal transduction pathways, radiationsensitivity, changes in cell adhesion and growth [5]. There is also increased evidencethat some cells are more sensitive to radiation at low doses, below 2Gy, than theyare at higher doses. As an explanation it is suggested that the resistance at higherdoses is due to the onset of cellular repair processes by the lower conditioning doses[5]. The mechanism described operates also in the case of other stress factors such as,for example, chemical and physical environmental agents. Thus, the irradiatedhuman brain remains under the stress factors leading to multi-factor brain injuriessuch as cytotoxic oedema, ischaemia, and subsequently, vasogenic oedema resultingin an increased extracellular water content.

At present, the delayed radiation injuries are usually monitored by MRI or CT. Asfor MRI, T2-weighted MR imaging of the brain shows that the radiation injuryreveals diffuse high signal areas in white matter [6]. The increase in T2 is alsogenerally observed, which may be attributed to a developing oedema. Although MRimaging allows monitoring of lesion volumes, it cannot provide information on thebrain metabolism—the studies of the latter seem to be very useful in predicting and/or detecting the response to therapy.

Proton in vivo spectroscopy is an ideal tool for this aim, however the literaturedata are sparse and concern mainly late radiation effects [7–14]. In spite of thepotential of the in vivo method, it suffers many limitations. The spectral assignmentsare in case of 1H MR in vivo extremely difficult due to a complex J-pattern and linebroadening induced by inhomogeneity of the static field. Moreover, in case of in vivoproton spectra acquired from the pathological brain areas spectroscopic evaluationsare usually based on the analysis of metabolite ratios thus making it difficult forindividual metabolite concentration changes to be observed. In our previous workwe studied the early response to treatment of the irradiated human brain [15].Though, the explanation of the observed changes requires some model animalstudies. In this study the metabolic changes following radiation were examined bymeans of a high-resolution 1H NMR in vitro using rat brain HClO4 extracts. Alongwith the standard spectra the relaxation measurements were also made.

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2. Material

In the experiment, male WAG rats (aged 6 months) were used (of a weight of 200–250 g). The animals were supplied with standard food and water ad libitum, and theywere housed four per cage, under controlled conditions of temperature (20–22�C),humidity (60–70%) and lighting (12 h light/dark cycle).

The animals were irradiated in Plexiglas boxes with the doses of 20Gy, applied totheir heads in fractions of 10� 2Gy. The external irradiation of animal heads wasperformed using Co-60 gamma-rays (Philips) at a dose rate of 0.8765Gy/min. Aradiation beam was delivered through an anterior field from a distance of 70 cm tothe cranium. In all cases the brain was situated exactly in the field of irradiation andthe rest of the body was shielded with 6 cm thick lead block.

Standard procedures were used to obtain HClO4 (PCA) extracts of rat brains. Thebrains were washed with cooled (4�C) physiological saline, dried with gauze,weighted and froze in liquid nitrogen. The frozen brains (two brains per each sample)were weighed thoroughly and homogenized in a glass-Teflon homogenizer with ice-cold 8% PCA in the 1:2.5 w/v proportion yielding 40% homogenate. Thehomogenate was centrifuged and the supernatant was collected. The precipitatewas re-extracted with 0.3M PCA and centrifuged again. The supernatants werepooled, neutralized with 1.5M KOH and the precipitated KClO4 was removed. Theresulting supernatant was then passed through an ion exchange resin (Chelex),lyophilized and resolved in D2O. The samples, four per each experiment, wereprepared after the doses of 2, 4, 8, 10, 16 and 20Gy as well as after 4, 8, 16, 32 and 96days following the irradiation cycle completion (time=0, total dose=20Gy), inorder to examine the evolution of the changes. The individual NMR measurementswere repeated twice.

The control samples were prepared from non-irradiated rat brains using the sameprocedure. The animals used in the reference study were of the same age and were fedand housed identically as the studied group in order to enable the comparison ofneurochemicals concentrations and relaxation times.

The procedures involving animals were in agreement with the internal rules forhuman and animal treatment accepted in the Institute of Oncology and under thecontrol of the Local Ethical Committee.

3. Methods

High-resolution 1H NMR studies of the extract solutions in D2O were performedusing a Varian Inova-300 multinuclear pulsed NMR spectrometer operating at the1H resonance frequency of 300MHz. Chemical shifts in the in vitro proton spectrawere measured relative to the solution of sodium 3-trimethylsilylpropionate (TSP)serving as a standard. Standard 1H NMR spectra were acquired and the T1relaxation measurements were made (using an inversion-recovery technique and att ¼ 22�C).

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4. Results and discussion

The 1H NMR spectra of the PCA brain extracts were acquired during the wholeirradiation cycle and within 96 days after the cycle completion. The results obtainedfor the treated animals were compared with those for the control, untreated ones.The signals due to the main brain metabolites: N-acetylaspartate (NAA), choline-containing compounds (glycerophosphocholine, GPC; phosphocholine, PC; freecholine (Cho)), myoinositol (mI), creatine and phosphocreatine (Cr)—usuallyanalysed in case of in vivo studies—and taurine (Tau) were under the interest. Thesignals were assigned basing upon the individual metabolite D2O solutions as well ason the literature data [16–19]. The signals intensities were found to differ markedlythan the control values and the most pronounced differences were observed at theonset of irradiation and at the termination of the cycle. The changes in concentrationwere accompanied by the variations in the T1 relaxation times vs. dose and timeafter the irradiation cycle completion. Figs. 1–5a show the dependency of theindividual metabolite concentrations on the dose applied and Figs. 1–5b visualize theT1 changes taking place on irradiation. Figs. 1–5c compare the concentrations of the

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Fig. 1. Integral intensities of the resonances due to N-acetylaspartate (a) and appropriate T1 relaxation

times (b) vs. dose applied as well as NAA integral intensities (c) and appropriate T1 relaxation times (d) vs.

time after irradiation cycle completion. Treated sample: (E) CH3, singlet at 2.02 ppm; (K) CH, doublet of

doublets at 4.4 ppm; (’) half CH2, doublet of doublets at 2.50 ppm; (m) half CH2, doublet of doublets at

2.70 ppm. Control sample: (B) CH3, singlet at 2.02 ppm; (J) CH, doublet of doublets at 4.4 ppm; (&)

half CH2, doublet of doublets at 2.50 ppm; (n) half CH2, doublet of doublets at 2.70 ppm.

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brain metabolites after the irradiation cycle completion, whereas Figs. 1–5d depictthe T1 relaxation behaviour of the metabolite signals in the period of the evolutionof the metabolic changes.

Variations in the macroscopic concentrations of any neurochemicals and in therelaxation behaviour are expected to be dependent on the fluxes of the chemicalsubstances, degradation pathways, cellular density, brain water content and theefficiency of control mechanisms. In case of the relaxation processes also factors likecharge distribution and spatial conformation of the molecule are of importance. Thequestion arises if the model in vitro studies reflect realistically in vivo conditions?The correspondence between animal in vitro studies and in vivo conditions seems tobe confirmed by the consistency of the T1 values for the CH3 groups of the cholines(0.99, 1.1 and 1.53 s for PC, Cho and GPC respectively), creatine/phosphocreatine(1.29 s) and NAA (1.99 s) (Figs. 1–5b), with those from the in vivo report (1.37, 1.34and 1.41 s, respectively) [20].

N-acetylaspartate is a special amino acid predominantly localized to neurons,axons and dendrites within the central nervous system, however, it may also bedetected in non-neuronal cell types, such as mast cells or in oligodendrocytes

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Fig. 2. Integral intensities of the resonances due to choline group N(CH3)3 containing compounds (a) and

appropriate T1 relaxation times (b) vs. dose applied as well as integral intensities of the resonances due to

cholines (c) and appropriate T1 relaxation times (d) vs. time after irradiation cycle completion. Treated

sample: (’) phosphocholine, 3.22 ppm; (~) free choline, 3.20 ppm; (m) glycerophosphocholine, 3.23 ppm.

Control sample: (&) phosphocholine, 3.22 ppm; (B) free choline, 3.20 ppm; (n) glycerophosphocholine,

3.23 ppm.

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[8,13,21–24]. Its functions are still not determined. It has been suggested that NAAmay be an acetyl-group carrier between mitochondria and cytoplasm in neuronalcells [8,24]. A decrease of NAA level is usually interpreted as a reduction in thenumber of neurons [23], but it may also reflect altered function of neuronalmitochondria [8].

As seen from the comparison of the data obtained for the PCA extracts of theirradiated brains and the control results, irradiation leads to a reduction in the NAAconcentration (Fig. 1a) which is accompanied by the increase in the T1 values (Fig.1b). This effect becomes apparent at the doses higher than 8Gy. During the periodof the post-radiation changes evolution both the integral intensities of the NAAsignals and the T1 relaxation times tend to attain the control values (Figs. 1c and d).Thus, the observed NAA depletion seems to be transitory and results presumablyfrom a blood-brain barrier impairment and exposure of the neurons to inflammatorycells and their products. In case of the irreversible process (such as the cell membranebreakdown or cell death), the lost of NAA should correlate with an increase in thecholine containing compounds forming demyelinization products. Interestingly, atthe lower doses (up to 8Gy) the signal intensity due to GPC, a product of membrane

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Fig. 3. Integral intensities of the resonances due to myoinositol (a) and appropriate T1 relaxation times (b)

vs. dose applied as well as integral intensities of myoinositol resonances (c) and appropriate T1 relaxation

times (d) vs. time after irradiation cycle completion. Treated sample: (K) H5, triplet at 3.28 ppm

(overlapped with taurine triplet); (~) H1, H3, doublet of doublets at 3.53 ppm; (’) H4, H6, doublet of

doublets at 3.63 ppm; (m) H2, triplet at 4.06 ppm. Control sample: (J) H5, triplet at 3.28 ppm; (B) H1,

H3, doublet of doublets at 3.53 ppm; (&) H4, H6, doublet of doublets at 3.63 ppm; (n) H2, triplet at

4.06 ppm.

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degradation, increases, whereas, at the higher doses (above 8Gy) all the intensities ofthe cholines signals are found to reduce markedly which is accompanied by anincrease in their T1 times (Figs. 2a and b). In spite of the initial grow of the GPC, PCand Cho signal intensities after the irradiation cycle completion, the final values,measured 96 days after the cycle termination, are still lower than the controls,though, in case of GPC, its short half-life might influence the concentration results[25] (Fig. 2c). The T1 values for the cholines are longer than the control ones withinthe whole evolution period of the metabolic changes (Fig. 2d). Thus, theconcentration changes of the choline-containing compounds seem to reflect theresultant effect of irradiation induced degradation of membranes, oedema andcontrol mechanisms. It may also be supposed that the relaxation behaviour isgoverned not exclusively by the correlation time of motion.

It is appealing to compare the results of the animal model studies with theobservations for irradiated humans. Some authors do not report changes in cholinelevels after irradiation [14,26], other show decrease of choline signal intensity [27] orincrease in the Cho/Cr levels [8,13]. The most comprehensive study was done byChong et al. [28,29] on the patients irradiated due to skull base tumors. In this casethe influence of tumor presence or surgery on brain metabolism is excluded. The

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Fig. 4. Integral intensities of the resonances due to creatine/phosphocreatine (a) and appropriate T1

relaxation times (b) vs. dose applied as well as integral intensities of the resonances due to creatine/

phosphocreatine (c) and appropriate T1 relaxation times (d) vs. time after irradiation cycle completion.

Treated sample: (~) CH3, singlet at 3.02 ppm; (’) CH2, singlet at 3.94 ppm. Control sample: (B) CH3,

singlet at 3.02 ppm; (&) CH2, singlet at 3.94 ppm.

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temporal lobe localized MR spectroscopy following radiation therapy revealed asignificant decrease in the levels of NAA and Cho (for 75% of patients). In ourstudies we observed an increase of the Cho/Cr ratios for the high-dose regions,located in the vicinity of tumor bed—which may be attributed not only to the post-radiation effect but also to the recurrence processes or residual tumor—whereas thevalues corresponding to the low-dose regions were stable [15]. A factor that may beresponsible for the observed changes in the metabolite concentrations and relaxationbehaviour is obviously an increase in total water concentration due to irradiation. Inanimal imaging studies, early histopathological analysis showed macrophageinflammation and vessel thickening reflecting a vascular and glial mechanism ofradiation injury leading to spreading vasogenic oedema [30]. The time sequence ofthe appearance of oedema and metabolic disturbances were not, however,considered. Our results seem to put some light onto this issue. The marked decreasein the GPC integral intensity vs. dose above 8Gy (Fig. 2a) appear to be due tooedema development. It may also be noticed that the intensity of the PC signaldecreases at the same doses as in case of GPC and the relaxation T1 curves for all thecholines change similarly showing the change of their slopes even at the doses lower

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Fig. 5. Integral intensities of the taurine resonance (a) and appropriate T1 relaxation times (b) vs. dose

applied as well as integral intensities of the taurine resonance (c) and appropriate T1 relaxation times (d)

vs. time after irradiation cycle completion. Treated sample: (~) CH2SO3, triplet at 3.41 ppm. Control

sample: (B) CH2SO3, triplet at 3.41 ppm.

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than 8Gy (Fig. 2b). Such variations are not observed for the control data (Figs. 2aand b).

In case of Myoinositol, an organic osmolyte and a glial cells marker, accepted asanother breakdown product of myelin [14,31], the comparison of the integralintensities vs. dose applied with the control ones reveals the most pronounceddifferences at the onset of the irradiation cycle and at its completion (Fig. 3a), thussimilarly as for other metabolites. After the irradiation cycle termination theMyoinositol integral intensities are markedly lower than the control values. Duringthe first 8 days of the evolution period a marked increase in the mI signal intensitiestakes place, however, the further changes are only slight (Fig. 3c). The MyoinositolT1 dependencies on dose and time (Figs. 3b and d) are similar as for the cholines.

Creatine and phosphocreatine are other important metabolites whose totalconcentration and relaxation behaviour seem to undergo variations on irradiationand after the completion of the irradiation cycle (Figs. 4a–d). The dependency of theintegral intensities on dose received is also of two step nature. At lower doses (below8Gy) the variations of the signal intensity are observed, whereas at higher doses thecontinuous decrease of the Cr level takes place followed by a simultaneous increasein the T1 values (Figs. 4a and b). Both the creatine/phosphocreatine integralintensities and T1 times are found to reach the values close to those obtained for thecontrol samples within about a month after the cycle completion (Figs. 4c and d).Although total creatine exhibits a heterogeneous regional distribution, it isfrequently used as an internal standard in in vivo studies. In view of the aboveresults such an approach seems to be a simplification—stress factors, such asirradiation, may change the metabolic status of the brain, thus leading to thedisturbances in the total concentration of these metabolites.

The significant differences between the 1H NMR spectra of the PCA extracts ofthe irradiated brains and the non-irradiated control brains (as well as between therespective T1 relaxation times) are observed for the taurine resonances (Figs. 5a–d).Taurine is another inhibitory amino acid serving osmoregulatory and cell membraneprotective functions [31]. Its role has not been established yet. It is a sulphur-containing amino acid that has been shown to decrease during ageing and is believedto be important for cytoprotection. A decrease in Tau could exacerbate theaccumulation of free radical-induced damage that may lead to cell death.

As seen from Fig. 5a radiation leads to a loss of taurine which in confirmed by theT1 increase (Fig. 5b). After the cycle completion the abrupt increase in the Tauintensity takes place which may indicate the efflux of this osmolyte, however the Taulevel is still lower than the value obtained for the untreated animals (Fig. 5c).Interestingly, the increase in the Tau levels are accompanied by a further increase inthe T1 values (Fig. 5d) which may suggest that the relaxation is not governed by thechanges in the correlation time of motion exclusively.

The correlation between signal intensity and relaxation times is not perfect also incase of the control samples. The most pronounced disturbance of the relaxationdependency on the correlation time of motion is seen for Myoinositol (Figs. 4a–d).The ageing processes and the interindividual differences in the mI levels and otherphysiological parameters are probably of importance here.

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When analysing the T1 behaviour of the N-acetylaspartate, cholines, myoinositol,creatine/phosphocreatine and taurine some similarities may be noticed. Therelaxation times are found to reduce at the beginning of the irradiation cycle, whichis followed by their continuous increase at higher doses. On the other hand, after theirradiation cycle completion the T1’s for all the metabolites are found to attainmaximum values within about 2–3 weeks and then they tend to normalize. It seemsthat the factors responsible for the mentioned variations are developing oedema andthe disturbances in the osmolytes equilibria. It may also be expected that some watersoluble products of the lipid peroxidation formed during the irradiation mayinfluence the relaxation processes. Thus, the overall relaxation is presumablygoverned not only by the changes in the total water content.

5. Conclusions

Creatine/phosphocreatine, taurine and N-acetylaspartate seem to be the mostsensitive markers of irradiation injury.

As revealed by the in vitro measurements the NAA depletion on irradiation isreversible and reflects the mitochondrial dysfunction rather than the loss of neurons.

The integral intensities of the signals due to cholines do not increase onirradiation, thus confirming that in case of in vivo studies the choline related ratiosmay serve mainly as cell proliferation indicators.

The total concentrations of creatine and phosphocreatine change during theirradiation cycle and after its completion, thus excluding using these metabolites asan internal standard of concentration.

The concentration changes of the metabolites seem to reflect the resultant effect ofirradiation induced degradation of membranes, oedema and control mechanisms.

Relaxation measurements provide a valuable tool for the observation of metabolicchanges occurring on irradiation. The overall relaxation is presumably not governedby the changes in the correlation time of motion exclusively.

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