Changes of relaxation times T1 and T2 in rat tissues after biopsy and fixation

Magnetic Resonance Imaging, Vol. 3. pp. 245-250. Printed in &heUSA. All rights reserved.

1985

073~725X/85 13.w + .I0 Copyright 0 1985 Pergamon Press Ltd.

l Original Contribution CHANGES OF RELAXATION TIMES Tl AND T2IN RAT TISSUES AFTER BIOPSY AND FIXATION R. Departments

L.

KAMMAN,*

K.

G.

Go,?

G.

H.

J. C.

BERENDSEN.*

P. STOMP,*

C.

E.

HULSTAERT,$

of *Physical Chemistry and tNeurosurgery, $Centre for Medical Electron Microscopy, of Groningen, Nijenborgh 16, 9747 AG Groningen, The Netherlands

University

NMR spectroscopical measurements of relaxation times were conducted on muscle, intestine, fatty tissue and cerebral cortex and white matter of the rat at various time intervals following removal of the tissue. It appeared that most tissues can be stored at 4°C up to 24 hours without noticeable effects on NMR relaxation parameters. Exceptions are the T2 of muscle and the T, and T2 of intestine, which tended to change in the first hour after biopsy. Relaxation parameters change considerably after fixation of the tissues. Therefore the effects of fixation have to be taken into account when carrying out NMR measurements on fixed tissues. Keywords: NMR, Proton spin relaxation, Rat tissue, Biopsy, Sample handling, Fixation.

INTRODUCTION

extent NMR parameters of biopsy tissues change with time. It has been reported that T,values for rat tissues as spleen, muscle and kidney stored at 4OC, do not change significantly within one day after removal.” The goal of our study was to give special attention to the shortest time intervals (min). during which any changes of the parameters T,and T2 might develop. In the case of pathological tissues, samples are mostly available for measurements after fixation. Therefore we also investigated the effects of fixation on the relaxation characteristics of healthy tissues.

Recent interest in the application of nuclear magnetic resonance (NMR) imaging for medical purposes has led to a widespread use of this technique in clinical medicine. However, many NMR measurements are carried out on biopsied tissues and generally the interpretation of these measurements is considered to apply also to the in vivo situation without taking into account the effect of storage time after dissection on the stability of the relaxation parameters. Only a few publications deal with this problem,‘~3~8~9~‘2 and in a recent article Bottomley et al. published a review of all the studies that were done so far on the subject.2 Therefore we investigated the stability of relaxation parameters of various rat tissues as a function of time after dissection, fixation and environmental conditions as humidity and temperature. Interpretation of NMR images requires insight in the relaxation parameters T,, Tzand proton density of the imaged tissues. However, in in vivo NMR imaging experiments it is very difficult to determine absolute T, and T2 values. Usually relative values are presented. Absolute values can be obtained by immediate measurements on tissues removed during operation. Before a meaningful comparison between in vivo and in vitro data can be made, it is imperative to know to what

RECEIVED

l/1

185; ACCEPTED

Address correspondence Kamman.

MATERIALS

3/20/85.

and reprints

AND METHODS

Biopsies were taken from Wistar rats of 200 g average weight anesthetized with 1.2 ml of urethane (25 g/100 ml). The animals were pretreated with antibiotics to minimize bacterial action. The preparation of fatty tissue, intestine, muscle, cerebral cortex and cerebral white matter samples were carried out in a glovebox in a controlled humidity environment. immediately after biopsy, the samples, which had an average volume of about 0.1 ml, were placed in a N MR tube with an inner diameter of 4 mm. This tube was appropriately sealed to preserve the water content of the tissues. T, and T2 relaxation time measurements were car-

requests to Dr. R. L. 245

246

Magnetic Resonance Imaging 0 Volume 3, Number 3, I985 Table I. T, and T, relaxation

time values for normal rat tissues obtained T, (ms) f SD. mean initial value

Muscle (n = 4)* Fatty tissue (n = 4) Intestine (n = 5) Cerebral cortex (n = 4) Cerebral white matter (n = 2)

460 187 377 523 364

f t + + +

in vitro at 10.6 MHz and 35OC. Tz (ms) 2 S.D. mean initial value

T, (ms) ranget

22 39 59 31 24

420-500 160-260 220-450 465-560 305-435

41 111 63 86 72

T2 (ms) ranget

+ 6 + 13 + 6 + 3 A4

35-54 98-132 52-75 73-90 59-76

*n represents number of rats. tRange over a period of 24 hours

Meiboom-Gill pulse sequence experiments (90(TE180-TE)n). The 90”-pulse length was 2.0 WS,the echo time TE was 4 ms and the number of ethos (n) varied from 100 to 250. Tl and T2 values were calculated using a non-linear least squares fitting roytine. A double-exponential fitting routine’ was used in those cases where a single exponential could not properly fit the data. T, and T2 measurements were carried out as a function of time. The elapse between removal of the tissue and measurement in the spectrometer varied from 30 s to 24 h. In the first hour after taking the biopsy, T, and T2 measurements were carried out as often as possible. Long term changes in relaxation

ried out at 35°C which is the rat’s body temperature, and at a resonance frequency of 10.6 MHz. The probehead of a Bruker SXP 4-100 high power pulse spectrometer (Bruker G.M.B.H., Karlsruhe, F.R.G.) as well as the sample tubes were stabilized at the same temperature. Spin-lattice relaxation time T, values were obtained from an inversion recovery pulse sequence (180-TD90), in which the amplitude of the free induction decay (FID) was measured as a function of the interpulse distance TD. TD varied from 1 ms to at least five times T,. Spin-spin relaxation times T2 were calculated from the decay of the echo amplitudes of Carr-Purcell200

T2 (ms)

o Fatty

Tissue Intestine l White Matter 8 Cortex A Muscle

0

11

-4+ P

I

I

I

I

I

I

I

OO

100

200

300

LOO

500

600

Tims)

Fig. 1. changes

T,/T, correlation diagram for normal rat tissues measured at 10.6 MHz and 35“C. The arrows in T, and T2 values measured over a period of 24 h after biopsy of the tissue.

indicate

the maximal

Relaxation times T, and T,0 R. L.KAMMAN ETAL.

parameters were measured by repeating the experiments at respectively 2,4,6,8 and 24 h after taking the biopsy. During the intervals between the measurements the tissue samples were kept at 4OC. Shortly before each measurement the tissue samples were warmed up to 35°C in a water bath. The effects of immersion fixation on the relaxation parameters of the tissues were studied by fixing the tissues in 10% formaldehyde. T, and T, changes were followed for total fixation periods up to five days. The effect on tissue of perfusion fixation with formaldehyde were studied as a function of the perfusion time. Details of these techniques are described elsewhere.5~”

600

(a)

(‘1,;’

TI

300 RESULTS Proton spin-lattice (T,) and spin-spin (TJ relaxation times were measured for rat muscle, intestine, fatty tissue and cerebral cortex and white matter. The results of these measurements are shown in Table 1. In general it appeared that the changes in T, and T2 over a period of 24 h after biopsy do not exceed the variations in the relaxation parameters of a given tissue in different animals. Characterisation of tissue can be achieved by depicting the relaxation times in a T,/T2 correlation diagram. Such a diagram is presented in Fig. 1 by using the data of Table 1. It appeared that in our measuring period of 24 h most tissues are clearly distinguishable according to their relaxation parameters. For white matter and intestine the values overlap, but in practical imaging this is not likely to cause any problems. When we focus our attention on the changes of T, and T, of different tissues (Figs. 2-5) specific information about time effects are visible. Because of the variations within the same tissues of different rats, all curves are shifted to a certain mean value in order to show the time changes more clearly. This mean value has been calculated from all the T, and T2 values for one particular tissue, measured at 2, 4 and 6 h after dissection. It is remarkable that most tissues do have stable relaxation parameters over a period of 24 h after taking the biopsy. When considerable changes were observed, they appeared during the first hour. The relaxation times of cerebral cortex and white matter are almost constant over the whole measuring period (Fig. 2). The T2 values of brain tissue show a tendency to decrease. The most prominent changes in relaxation times pertain to the T, and T2 values of intestine and in the T2 values of muscle (Fig. 3). In the first 30 min an increase in T2 was measured for muscle (up to 20% of the initial value) as well as for intestine (up to 15%). After this period a constant level was reached. The intestine showed a remarkable decrease (up to 20%) in

I

-L-w--Hhours

(b)

90

T2

(msl

+

80 b 70 k=a White

Matter

50 -I-

‘i

I

1

I

2

#---A-

hours

Fig. 2. Rat cortex and white matter T,(a) and T,(b) values as a function of storage time after biopsy. Curves are shifted to a mean value calculated from the T, and T, values measured at 2,4 and 6 h after biopsy (see text). The different symbols refer to different samples. T, during the first two h. Such an effect was not noticed in any other tissue. T, and T, of fatty tissue did not show any change over the period of 24 h as is illustrated in Fig. 4. Fatty tissue T, data were analyzed using a double exponential as well as a single exponential fitting routine. The former, however, fitted the data more properly. Two components could be discerned: T2 slow ( T2s, about 210 ms) and T2 fast (T,f,

Magnetic Resonance Imaging0

248

Volume 3, Number 3, 1985

lms)

Futty

Tissue

180 Tl 160

.

Intestine

120

I==Q

L.

100

I

1

_L

T2 ---+k--,

I

2 hours

a---k-

-I--

I

I

I

1

2

;I

l----k

hours

70

r (b)

Intestme

Fig. 4. T, and T2 values of fatty tissue measured over a period of 24 h after biopsy. Data are calculated using a single exponential fitting routine. All curves are shifted to a mean value (see text). The different symbols refer to different samples. on tissue structure, water content and molecular mobility. We investigated the effect of formaldehyde, a fixative which is widely used in routine histology, on T, and T2. Considerable changes in both parameters were noticed after immersion fixation (Table 2) and after fixation by perfusion (Table 3).

60 T2 Imsl 50

DISCUSSION

I

1

I

2

:I

l--A-

hours

Fig. 3. Rat muscle and intestine 7’,(a) and T,(b) values as a function of storage time after biopsy. All curves are shifted in the same manner as indicated in Fig. 2. The different symbols refer to different samples.

about 60 ms), which were both stable over a period of 24 h (Fig. 5). In order to compare in vivo and in vitro relaxation time studies it may be necessary to preserve tissue for a long period, up to several days and sometimes weeks, without inducing changes in their relaxation characteristics. As T, and T2 reflect tissue structure and mobility of molecules, the employment of fixatives has also to be taken into account because of their influence

Changes of T, and T2 have been reported for various tissues after biopsy.‘,*%‘* In most of these studies time effects on relaxation parameters were observed starting about one h after dissection of the tissues. We were particularly interested in the early effects within the first few minutes after removal of the tissue. Dissection interrupts the supply of nutrients and oxygen to the tissue and causes an acute energy deficit. This interferes with the energy dependent functions, among which the derangement of the cellular ionic pumps soon induces osmotic and ionic shifts within the tissue. Depending on the kind of tissue, degradation of structural elements becomes manifest after some time by the action of degrading enzymes (autolysis) or by the action of bacterial contamination. In our study most tissues showed little or no change in relaxation times over a period of 24 h. This was expected for fatty tissue because this predominantly consists of triglycerides6 and to a lesser extent of proteins. Brain tissue such as cortex and white matter exhibited no changes in T, and T2 values during 24 h. Only a very small decrease in T, was noted in the same period which can probably be ascribed to a slight loss of water

Relaxation

2LO (rnSl

times T, and T2 0 R. L. KAMMANET AL.

1

200-k T%

Fatty

1604

01l-r 1

Tissue

2

L

2L

hours Fig. 5. Using a double exponential fitting routine, two components could be calculated from the data of the T, measurements. A fast one ( T2 fast, about 60 ms) and a slow one (7’* slow, about 2 IO ms) which both can be assigned to different protons. The different symbols refer to different samples.

Table

2.

249

from the tissue, even in a sealed tube, on account of the very small size of the tissue samples used. A loss of water means stronger effects of relaxing components, yielding a shortening of the T, relaxation time value. Loss of tissue water cannot explain the increase of T, in muscle and intestine nor the decrease of T, in intestine during the first hour after biopsy. Firstly, because the tubes were sealed to preserve the water content and secondly, because a loss of water would result in a decrease of T, and T,. Anoxia can be a possible explanation for the prolongation of the relaxation times shortly after biopsy. Oxygen is a paramagnetic compound and is present in large quantities in tissues such as muscle and in tissues with a large content of blood.13 A loss of oxygen after biopsy can decrease the paramagnetic effect and the result will be a prolongation of both the T, and T2 values. Because T, for muscle did not increase, anoxia can not be the only explanation for the increase in T,. To account for the considerable changes in intestine, the influence of bacterial flora should not be neglected. These changes are probably due to enzymatic breakdown and bacterial activity. Some special remarks have to be made with respect to fatty tissue. This tissue showed, especially in the T2 relaxation curve, a multi-exponential decay, which was not noticed in the other tissues. Performing a doubleexponential fitting analysis, Tz values for two components could be calculated from the data. From previous studies6 the fast component could be assigned to the mobile lipid proton whereas the slow component was partly assigned to water protons and partly to other

T,values for rat tissue before and after immersion Measurements

were performed

fixation with 10% formaldehyde. at 10.6 MHz and 3Y’C. Formaldehyde

T, (ms) + SD

Muscle

Fatty Tissue

Intestine

White Matter

Cortex

( 10%)

Native tissue 24 h fixation 5 days fixation

410 t 30 l56k 5 -

250 & IO 185 + 2 I46 2 2

420 + 30 175 f 4

320 + 20 222 t 5 195 -r 5

500 i 30 208 -t 6 132 5 2

2650 + 50

Table 3. T, and Tz values of rat muscle and brain as a function of perfusion Measured at 10.6 MHz and 35%.

_

time with formaldehyde.

Muscle

T, i S.D.

Tz i S.D.

Brain Tissue

T, r S.D.

T2 f S.D.

15m Ih 24 h

614 k 21 47.5 * 5 463 + 6

36 -r 2 41 k2 45 + 2

45 m 90 m 24 h

556 + 12 523 i I5 451 + 4

54 + I 61 + 1 65 ? 2

Magnetic Resonance Imaging 0 Volume 3, Number 3, 1985

250

lipid protons. Both components showed a constant behavior over a period of 24 hours (Fig. 5). There are cases in which in vitro studies on biopsy materials cannot be carried out within several hours. In these cases precautions have to be taken to preserve the relaxation characteristics over long periods. Most pathological specimen have been preserved with fixatives. From our study on tissues fixed by perfusion or immersion we must conclude that these samples cannot be regarded as representatives for the native tissues with respect to their relaxation times. Therefore these fixed tissues cannot be used for interpretation studies of in vivo imaging experiments. In all our experiments it appeared that T, was considerably shortened with respect to the T, of unfixed tissue. This can be explained by the fact that fixatives such as formaldehyde cause structural changes of the tissue macromolecules such as denaturation which promote the dissipation of energy from the proton spins. Our main conclusion from these studies is that relaxation parameters measured in vitro are quite stable from the time of biopsy, and hence are expected

to correlate very well with in vivo measurements when tissues are stored at low temperatures (4°C) in sealed tubes. According to our studies there are changes present in both T, and T2 after the tissues were removed from the body by biopsy. But in all cases these changes were not exceeding the individual changes between the same tissues of different rats. Brain tissue and fatty tissue characteristics do not change during the first 24 hours. For muscle, there is a change in T2 that is not noticed in T1. For intestine changes are seen in both T, and Tz especially in the first hour. These changes must be due to enzymatic breakdown and the influences of bacteria in the tissue. Early studies reported” that the relaxation values of tissues change considerably after fixation. This was confirmed by our experiments. The explanation for these changes are to be found in changed tissue structure and restricted mobility of molecules in the tissue after fixation. Our results support our contention that in vitro determinations of tissue characteristics correlate very well with the in vivo situation and therefore they can be used as a basis for the understanding of in vivo imaging and spectroscopy experiments.

REFERENCES methods for biological sample 1. Beall, P.T. Practical handling. Magnetic Resonance Imaging 1:165-l 8 1; 1982.

8.

2. Bottomly, P.A.; Foster, T.H.; Argersinger,

3.

4.

R.E.; Pfeifer, L.M. A review of normal tissue hydrogen NMR relaxation times and relaxation mechanisms from l-100 MHz: Dependence on tissue type, NMR frequency, temperaand age. Medical Physics ture, species, excision, 11:425-448; 1984. Escayne, J.M.; Cane& D.; Robert, J. Frequency dependence of water proton longitudinal nuclear magnetic relaxation times in mouse tissue at 20%. Biochim. Biophys. Acta 721:305-313; 1982. Fullerton, G.D.; Potter, J.L.; Dornbluth, N.C. NMR relaxation of protons in tissues and other macromolecular water solutions. Magnetic Resonance Imaging 1:209-228; 1982. D. Fixation and fixatives. In Theory and Practice of Histological Techniques, J.D. Bancroct, A. Stevens, eds. pp. 20-41. New York: Livingstone; 1982.

5. Hopwood,

9.

10.

11. 12.

6. Kamman,

7.

R.L.; Go, K.G.; Stomp, G.P.; Muskiet, F.A.J.; Van Dijk, P.: Berendsen, H.J.C. Proton spin relaxation studies on fatty tissue and cerebral white matter. Magnetic Resonance Imaging 3:2l I-220; 1984. Marquardt, D.W. An algorithm for least-squares esti-

13.

mation of non-linear parameters. J. Sot. Indust. Appl. Math. 11:431-441; 1963. Moseley, M.E.; Nishimura, B.A.; Pitts, L.H.; Bartkowski, H.M.; James, T.L. Proton nuclear magnetic resonance spectroscopy of normal and edematous brain tissue in vitro: Changes in relaxation during tissue storage. Magnetic Resonance Imaging 2~205-209; 1984. Peemoeller, H.; Shenoy, R.K.; Pintar, M.M.; Kydon, D.W.; Inch, W.R. Improved characterisation of healthy and malignant tissue by NMR line-shape relaxation correlations. Biophysical Journal 38:27 l-276; 1982. Raaphorst, G.P.; Kruuv, J. Nuclear magnetic resonance spin-lattice times of normal and transformed cultured mammalian cells and of normal and neoplastic animal tissues. Physiol. Chem. Physics 13:251-258; 1981. Riemersma, J.C. Biological Techniques in Electron Microscopy. New York: Academic Press: 1970. Thickman, D.I.; Kundel, H.L.; Wolf, G. Nuclear magnetic resonance characteristics of fresh and fixed tissue: The effect of elapsed time. Radiology 148:183-185; 1983. Thulborn, K.R.; Waterto, J.C.; Mattheus, P.M.; Radda, G.K. Oxygenation dependence of the transverse relaxation time of water protons in whole blood at high field. Biochim. Biophys. Acta 714:265-270; 1982.