C (?- \

IN VIVO NUCLEAR MAGNETIC RESONANCE IMAGING

CONTRACT NAS9-16442, P3

FINAL REPORT - OCTOBER 1984

DEPARTMENT OF MEDICINE

BAYLOR COLLEGE OF MEDICINE

(NASA-CR-171928) IN VIVO NUCLZAB MAGNETIC N86-211C5

RESONANCE IMAGING Final Beport, Oct. 1984

(Baylor Coll. of Hedicine) 25 p

HC A02/MF A01 CSCL 06P Unclas

G3/52 08475

Principal Investigator:

Co-Investigators:

Adrian LeBlanc, Ph.D.

Harlan Evans, Ph.D.

R. Nick Bryan, M.D.

Phillip Johnson, M.D.

Ernie Schonfeld, M.S.

Satish G. Jhingran, M.D.

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 ll*^f, • ,-,

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SUMMARY

A number of physiological changes have been demonstrated in bone, muscle and

blood after exposure of humans and animals to microgravity. Determining mechanisms and the development of effective countermeasures for long duration

space missions is an important NASA goal. Historically, NASA has had to rely

on measuring tapes, x-ray and metabolic balance studies requiring collection

if excreta and blood specimens to study this problem. The advent of tomoJjraphic nuclear magnetic resonance imaging (NMR or MRI) gives NASA a way to

greatly extend early studies of this phenomena in ways not previously possible; NMR is also noninvasive and safe. NMR provides both superb anatomical

images for volume assessments of individual organs and quantification of chemical/physical changes induced in the examined tissues. The purpose of this

work is to determine and demonstrate the feasibility of NMR as a tool for

human physiological research as it is affected by microgravity.

The animal studies employed the rear-limb suspended rat as a model of muscle

atrophy that results from microgravity. This technique unloads the rear limbs

without immobilization and causes a caudal shift in body fluid similar to

spaceflight. NMR measurements were made using a number of resonant frequencies (100, 20, 10 MHz) and after various periods of limb suspension. Despite

significant muscle atrophy (soleus and gastrocnemius) and blood flow changes,

none of the NMR measurements (T]_, T?, T? diffusion) demonstrated a significant

change. This data suggests that atrophy itself wil l present a different NMR

spectrum than pathological induced atrophy. Also, volume measurements of

atrophied muscle wil l not be affected by concomittant changes due to muscle

atrophy.

Bedrest of normal male subjects was used to simulate the effects of microgravity on bone and muscle. A technique was developed to image the lower limbs of

subjects before and after bedrest which includes an improved method for producing images calculated from T?, a computer scheme for classifying tissue

according to different T2 relaxation times and quantifying tissue volumes. To

date, six bedrest subjects have been imaged before and after bedrest. Based

on cross-sectional images through the lower leg, the gastrocnemius and soleus

muscle areas decreased 9% while total muscle area in this same section decreased 6%, demonstrating differential muscle loss. Bone marrow T2 was

decreased 14% (P < 0.001) after bedrest indicating an increase in marrow fat,

a change which may be related to red cell mass and bone changes observed after

exposure to weightlessness or bedrest. Muscle T2 did not change, suggesting

that tissue hydration is not changed by atrophy.

With the acquisition of the 2.3 Tesla system by Baylor in early 1985, these

studies will be extended to high energy phosphate measurements. A technique

to display fat and H20 proton quantitative images simultaneously wil l be

investigated and techniques to measure vessel and/or limb muscle blood flow

will be developed. Limb investigations will be extended to muscles in the

back and bone marrow in the spinal vertebrae.
BACKGROUND

a. Development of Nuclear Magnetic Resonance Imaging

In 1946, Bloch, Hanson, Purcell and Pound showed that certain elements give

rise to observable nuclear magnetic resonances (1-4). Magnetic moments,

oriented in a random fashion in the absence of a magnetic field, align

i themselves within an external field in a limited number of orientations.

* The energy resulting from changes between states lie within conventional

1 radio frequency (rf) bands. Hence, magnetically aligned or polarized nuclei may be detected by energizing them with incident rf waves. The resultant NMR signals characterize many aspects of the chemical and physical

state of a sample. Significantly, NMR is a noninvasive technique in the

sense that the chemical nature of the investigated material is not altered.

High-resolution NMR spectrometry has been applied in biochemistry with

increasing regularity. Its ability to investigate biologically important

molecules using probes for hydrogen, phosphorus, carbon and nitrogen has

resulted in substantial progress in the analysis of complex biological compounds. Recently, high-resolution NMR methods have been adapted to in vivo

studies of 31p. These experiments rest on the ability to distinguish in

the intact animal several phosphorus compounds - inorganic (iP), carbohydrate phosphorus, phosphocreatinine (PCr), ATP, and perhaps ADP . For

these experiments, excitation and detection are done by an rf coil attached

to the sample, surface coils. In general, it detects material close to the

coil and it has been used to study 31p Of skeletal and heart muscles, kidney, liver and brain (5). Ackerman et al have shown PCr to ATP ratios of

1.93 which is higher than the best values attained by in vitro rapid freezing techniques, suggesting a nearly instantaneous start to the degradation

process. In addition, they found that mobile ADP and iP concentrations

determined by NMR were much lower than in other analytical techniques.

They and others have reported that localized ischemia of rat muscle and

brain produce significant differences in the 31p spectra. Ischemic tissue

has lower PCr and ATP values and a markedly higher iP. They observed also

a chemical shift in iP indicating a lower tissue pH (6-10). NMR techniques

offer great potential in the investigation of acute ischemic tissue metabolism.

Typical high-resolution proton NMR experiments assume that NMR signals

indicate the total number of nuclei observed, or the spin density (SO) of

the sample. While this may be true under precisely defined conditions, the

observed SD, in complex compounds and tissues is altered by two other NMR

parameters, the transverse, or spin-spin (T£) and longitudinal, or spinlattice (Ti), relaxation times. Differences in T£ and TI reveal molecular

level organizational differences. It is convenient to think of relaxation

times as memories. The phase memory, or T2, is "short-term", i.e., 10-200

msec while the polarization memory, or Tj is "long-term", i.e., 150-1500

msec in biological tissues (11-12). Measurement of relaxation times has

been used to evaluate water in the complex forms it exists in biological

tissues. This technique can characterize some of the physical properties

of hydrogen nuclei (mainly H20) in biological samples and allow differentiation of normal organ tissue from pathologic tissues. While it is an

empirical fact that different tissues have distinctive proton
parameters, a proven theoretical basis for these variations in biologic

samples is not available (13-16).

In the 1960's and early 1970's several investigators, including Damadian

and Lauterbur in the United States and Mansfield and Hinshaw in England,

modified high resolution NMR for the partial chemical analysis of relatively small volumes with coincident identification of the spatial coordinates

of the analyzed volume (17-23). If these data are obtained from a tissue

sample, images based on tissue chemistry can be formed. This important

step has led to a recent flurry of interest in NMR in vivo imaging called

zeugmatography or NMR tomography. NMR imaging is now undergoing initial

clinical trials and has been shown to have excellent anatomic resolution.

Early results also indicate that the technique provides unique pathologic

information (24-27).

b. Physiological Changes from Exposure to Microgravity and from Disuse

In this section we do not attempt to exhaustively review the literature but

will summarize major animal or human findings from spaceflight, bedrest,

and animal modeling of spaceflight as they relate to the proposal, i.e.,

application of NMR techniques.

Limb volume change associated with exposure to microgravity probably

involves two factors: 1) Loss of fluid and changes in venous pooling followed subsequently by; 2) Muscle atrophy. Both processes involve fluid

shifts, one mostly extracellular, the second intracellular. NMR techniques

are ideal for measuring changes in water content of tissues and is both

noninvasive and quantitative.

Urinary nitrogen excretion has been shown to be increased in bedrest and

during weightlessness indicating muscle wasting or deconditioning (28-31).

Muscle wasting would be expected to produce muscles less able to perform

work. Muscular deconditioning was documented during Apollo and Skylab

missions and has been shown in bedrest subjects (32). Pre and post flight

muscle strength testing in Skylab demonstrated that an adequate exercise

program may have been devised which protected the arm muscles against wasting. Locomotion in weightlessness uses the arms more than the legs. Not

surprisingly, exercise regimes designed to protect the leg muscles were

only partially successful. Leg volume changes in Skylab 2 and 3 were about

-4.5% while in Skylab 4 they were approximately -2%. Presumably, the

smaller loss of leg volume by the Skylab 4 crew was due to an increase in

programmed and personal preference daily exercise which occurred. This is

supported by the leg strength measurements which showed a similar pattern,

i.e., -25% loss after Skylab 2 vs. -8% loss after Skylab 4. These results

suggest that a properly designed exercise program might preserve muscle

volume and strength even during long duration missions.

The muscle volume loss and the strength loss produced by weightless exposure probably affects some muscles more than it does others. In Skylab data

this was suggested by the observed differences between the leg flexors and

extensors. Prior to NMR there would be no way in which this could be studied in the intact human except by the Skylab methods of estimating differential strength which are subject to error because of psychological factors. It is also not possible to differentiate soleus from gastrocnemius
which are both extensors. Estimating back muscle strength may be even more

difficult.

Animal studies have shown that differential muscle atrophy (soleus vs. gastrocnemius) occurs with weightlessness and in animal models of weightlessness but not with immobilization. Cast immobilization of the rear limbs of

rats for 21 days produces a 52% and 45% atrophy by weight of the soleus and

gastrocnemius muscles, respectively (40,41). Similarly, immobilization of

the knee and ankle joints produces a 58% loss of the soleus mass within two

weeks (42). Tenotomy produces a 25% decrease in the gastrocnemius in three

weeks, and a decrease of 50% by 4 - 9 weeks (43). Nerve resection causes a

50% weight loss in the gastrocnemius by 4 - 5 weeks (44). Young rats suspended for two to three weeks showed a 40 - 45 % and a 15 - 20 % loss in

the soleus and gastrocnemius, respectively (45). Rear limb suspension of

older rats for up to 90 days showed similar muscle loss which maximized by

the 30th day (46). Rats flown aboard the Cosmos spacecraft for approximately 2 weeks demonstrated similar differential muscle loss in the soleus

and gastrocnemius (47).

We have shown that leg area measurements using NMR are possible with a precision of about 0.2%; fat and muscle cross-sectional area may be accurately

measured in a given anatomical section; differential muscle atrophy can be

investigated (soleus vs. gastrocnemius) along with tissue hydration

changes. It may also be possible to distinguish intracellular from extracellular water (33).

Limb immobilization reportedly alters the percentage of slow twitch muscle

fibers in the soleus to the extent that twitch characteristics become similar to that of a fast twitch muscle (41). This is supported by our measurements of relative blood flow in suspended rats, i.e., the ratio of soleus to gastrocnemius blood flow of 2.3 in controls is reduced to 1.4 in

suspended animals (46). Reis (48) concluded that there is a high correlation between blood flow and contraction time in normal skeletal muscle. It

is reasonable then that the blood flow of the two muscles would become similar as the fiber twitch characteristics become similar. The ATP and Phosphocreatinine (Per) concentration of fast twitch muscle is about double

that of slow twitch while the inorganic phosphate (iP) concentration in

fast twitch is half that of slow twitch (49). Using NMR surface coils

placed over these respective muscles the P-31 spectra of ATP, Per, iP can

be used to monitor these high energy phosphate changes (50-52) and therefore noninvasively monitor changes in fiber type.

In Skylab a crude estimate of limb blood flow, through the belly of the

gastrocnemius, was obtained by using an occlusion cuff and a leg volume

measuring band (55). Measurements taken during flight suggest that limb

flow was elevated compared to pre and post flight measurements. This is

interesting because in contrast we find that muscle blood flow in suspended

rats, an analogue of weightlessness, was probably decreased (46). NMR can

be used for monitoring changes in blood flow (34-39).

Fluid shifts were documented in Skylab by visual observations of blood vessel distention, rapid changes in limb volume, center of mass measurements

and subjective description such as puffy faces and head fullness (56).

The spin density NMR image is essentially equal to hydrogen density which
is closely related to water content. A total or partial body spin density

survey of bedrest subjects could give quantitative data regarding fluid

shift not possible by any other technique.

It has been reported that the muscle water content of biopsied soleus muscles decreased following 8 hours of head down tilt bedrest (53). Tissue,

TI and T£ values are very sensitive to tissue water. For example a 2%

change in tissue water may produce a 16% change in the relaxation time of

tissue (54).

It is generally accepted that osteoporosis as well as disuse bone atrophy

from bedrest and presumably spaceflight results from a relative increase of

bone resorption over formation (57-62). Ultimately this manifests itself

as a lower bone mineral content characterized by a larger medullary canal

width and a smaller cortical width and area. For longitudinal studies a

morphometric measurement of bone along with densitometry would be most useful. Because of the inherently large contrast between the bones and soft

tissue NMR signals, NMR imaging is a noninvasive way for performing longitudinal bone morphometry studies.

It is known from bone biopsies that marrow fat increases 100% during the

first 20 weeks of paralysis (63). Rats flown aboard Cosmos (19-21 days)

have shown increases in marrow fat of 150 - 200 % in the tibia and humerus

while rats undergoing • rear limb suspension have shown even larger increases, +685 and +500% in marrow fat of the tibia and humerus respectively

as well as decreases in bone formation (64).

The exact function of marrow adipose cells and their relation to bone metabolism and hemopoiesis is unknown. The hematological changes and the theoretical basis for these changes resulting from weightlessness are documented elsewhere and will not be discussed (65). It is known that-bone

marrow adipose cells have a reciprocal relationship to hemopoietic tissue.

As the hemopoietic tissue expands the lipid fraction of the adipose cells

decreases while the opposite occurs during decreases in hemopoiesis. Thus

the metabolic activity of the marrow adipose cells are coupled to the hemopoietic activity of the marrow. In response to acute and total starvation

lipolysis occurs in the extramedullary cells but not in the marrow adipose

cell which continue to produce fat at an unaltered rate (66). In contrast

to its remarkable resistance to starvation, the marrow adipose cell is

highly sensitive to hemopoietic stimuli. The primary function therefore of

marrow adipose cells appears to be related to the intensity of hemopoiesis. Therefore the study of marrow fat as it relates to hematological

changes in weightlessness or bedrest could provide new information concerning the loss of red cell mass during space flight. With NMR the possibility exists to monitor these changes in vivo. This is especially important

since fat containing cells of long term marrow cultures are not considered

equivalent to the in vivo marrow adipose cells. These studies may also

have relevance to bone atrophy that occurs under these same conditions,

i.e., increased fat with decreased hemopoiesis. As it is speculated that

adipose cells may serve as a store of energy required for hemopoiesis, it

may serve a similar purpose for control of osteocytic or osteoblastic

activity.
ANIMAL EXPERIMENTS

During the first year the rat muscle measurements were completed. The enclosed tables summarize this work which includes NMR parameter measurements

(

Tl» T2» T2 diffusion) using resonant frequencies of 200 MHz, 20 MHz, and 10

MHz. Female Sprague-Dawley rats (250-300g) were suspended by the tail for

periods of 90 days. Suspended animals were compared with age matched controls

i/hich were kept in individual rat cages. In both groups, water was ad libitum

l/hile the amount of food fed to the experimental group was increased slightly

to maintain body weight similar to controls.

Just prior to sacrifice approximately 800K carbonized microspheres were injected into the left ventricle (LV) of the anesthetized rats via a percutaneous puncture of the LV. After sacrificing, the left soleus and the gastrocnemius were removed for NMR analysis while the right leg muscles were removed

for determination of wet weight, dry weight, percent H20, and percent uptake

of labeled microspheres. The percent uptake of labeled microspheres was normalized by muscle weight and are labeled in the tables as BF%/gm. The latter

is an indicator of relative blood flow when compared with the microsphere

values of the control animals (non-suspended).

The soleus atrophies about 55% in 20 days which does not increase with an

additional 60 days of suspension. Similarly the gastrocnemius shows about a

25% loss in muscle mass. These changes are similar to those seen in rats

flown in space but are less than that seen with other forms of immobilization. Muscle water is decreased in the soleus at 90 days while no change was

seen in the gastrocnemius. Blood flow, which is 223% higher in the soleus

compared to the gastrocnemius in control animals is decreased after suspension

in both muscles but to a greater degree in the soleus; the soleus is only 120%

higher than the gastrocnemius after suspension. This change in blood flow can

be compared to the reported change in the composition of the soleus muscle

with immobilization. It has been reported that the soleus, which is normally

slow twitch changes to fast twitch,* the predominant fiber type in the gastrocnemius.

In none of the NMR measurements performed to date was there a significant

change in the NMR parameters measured. In view of the rather significant

physiological changes occurring in the suspended rat muscle, this is surprising. Further work is needed before speculating as to why the NMR parameters

are not altered. It does suggest that atrophy itself will present a different

NMR spectrum than pathological changes in muscle. Volume measurements of

atrophied muscle wil l not be affected by concomitant changes due to muscle

atrophy.
200 MHZ MEASUREMENTS

T2 cliff. Net Weight (g) BF%/gm

f

Soleus

IT

SD

Soleus

x (n)

± SD

Soleus

"x

± SD

CONTROLS

1.336

1.338

1.553

1.517

1.461

1.441

0.100

1.420

1.420

1.380

1.429

1.515

1.441

0.068

25.2

30.2

25.6

27.3

27.9

2.7

28.2

28.2

31.3

28.0

29.6

2.3

33.5

37.2

31.1

36.5

37.5

35.2

2.8

30 DAY

35.5

35.5

90 DAY

32.7

35.5

32.6

0.1

0.1199

0.1433 (9)

0.015

SUSPENDED RATS

0.0567

0.0641 (9)

0.0130

SUSPENDED RATS

0.0578

0.0753

0.0548

0.0626

±0.011

74.0

74.0 (9) 0.371 (9)

0.75 0.165

71.41 0.106

71.84 (9) 0.083 (6)

1.32 0.036

65.06

67.71 0.133

63.75 0.128

65.51 0.130

2.02 0.004
200 MHZ MEASUREMENTS

T2 diff. Net Weight (g) BF%/gm

f

!

Gastroc.

IT

SO

Gastroc.

"x

SD

Gastroc.

X

± SD

1.293

1.312

1.450

1.496

1.

1.

0.

1.

1.

1.

0.

1.

1.

1.

0.

702

451

165

514

320

417

137

619

518

568

071

31.

30.

31.

28.

30.

1.

26.

31.

29.

3.

32.

34.

33.

1.

9

5

5

8

7

4

9

3

1

1

2

8

5

8

39.

34.

39.

36.

37.

2.

30 DAY

34.

33.

33.

0.

90 DAY

37.

37.

CONTROLS

5

0

2 1.

4

3 2.

6 0.

SUSPENDED

0 1.

7

8 1.

2 0.

SUSPENDED

1.

9 1.

1.

9 1.

0.

7405

0415 (9)

246

RATS

3678

4923 (9)

144

RATS

3756

7386

5342

5495

182

73.

74.

2.

72.

73.

0.

74.

75.

73.

74.

1.

22

07 (9)

10

47

18 (9)

60

06

77

60

48

14

0.166 (9)

0.039

0.085

0.069 (6)

0.015

0.099

0.081

0.090

0.013
200 MHZ MEASUREMENTS

diff. Net Weight (g) BF%/gm

i

Soleus

x" 1.441 27.9

SD 0.100 2.7

Gastroc.

x" 1.451 30.7

SD 0.165 1.4

CONTROLS

35.2

2.8

37.3

2.6

0.1433 (9) 74.0 (9) 0.371 (9)

0.015 0.75 0.165

2.0415 (9) 74.07 (9) 0.166 (9)

0.246 2.10 0.039

SUSPENDED/CONTROL

90 DAY SUSPENSION

Soleus 1.00 1.06

Gastroc. 1.08 1.09

Soleus 0.99 1.01

Gastroc. 0.98 0.95

0.93

1.02

30 DAY

1.00

0.91

0.44 0.88 0.35

0.76 1.00 0.54

SUSPENSION

0.45 0.97 0.22

0.73 0.99 0.42
200 MHZ MEASUREMENTS (Old T Meter)

Net Weight BF%/gm

j

Soleus

X

SD

13.6

13.2

13.6

12.3

12.7

12.3

13.0

0.6

15.7

16.3

16.2

22.3

21.3

21.6

22.7

0.5

CONTROLS

0.1527

0.1349

0.1328

0.1548

0.1395

0.1746

0.1482

0.0158

74.74

74.49

74.73

73.64

74.20

72.60

74.07

0.83

0.429

0.334

0.318

0.539

0.158

0.356

0.141

30 DAY SUSPENDED RATS

Soleus

X

SD

13.3

13.5

13.0

13.1

13.2

0.2

15.4

15.7

20.8

19.3

20.0

1.1

0.0793

0.0842

0.0652

0.0650

0.0734

0.0098

72.71

71.97

69.40

71.73

71.45

1.43

0.045

0.028

0.082

0.052

0.027
20 MHZ MEASUREMENTS (Old T Meter)

TI Net Weight BF%/gm

f

Gastroc.

X

SD

Gastroc.

X

SD

Soleus

Gastroc.

12.8

13.0

13.3

12.6

12.8

12.8

12.9

0.2

12.8

12.6

12.8

12.6

12.7

0.1

1.02

0.98

16.0

16.3

15.4

22.1

20.4

19.9

20.8

1.2

15.0

15.6

20.9

20.8

20.8

0.1

0.95

1.03

CONTROLS

2.0301

2.5738

1.9056

2.0072

1.9960

2.2651

2.1363

0.2470

1

30 DAY SUSPENDED RATS

1.7423

1.7081

1.5406

1.4318

1.6057

0.1456

SUSPENDED/CONTROL

0.49

0.75

73.60

79.65

73.29

73.20

73.30

74.08

74.52

2.53

73.49

73.59

73.67

73.76

73.63

0.12

0.96

0.99

0.116

0.191

0.161

0.161

0.147

0.155

0.027

0.049

0.066

0.062

0.059

0.009

0.15

0.38
20 MHZ MEASUREMENTS (NEW)

Rat

Soleus

Gastroc,

10F

11F

10F

11F.

CONTROLS

315

329

308

297

29

26

26

23

Soleus

Gastroc,

10 MHZ MEASUREMENTS (IBM)

0.337 28.7

CONTROL

Soleus

Gastroc,

0.330

0.345

35.8

23.4
The above measurements of muscle in rats rear-limb suspended for 14 - 90 have

shown that despite severe muscle atrophy and changes in muscle blood flow, Tj

and ?2 NMR parameters are within normal values. Possible explanations for

this are that changes in blood flow, or sacrificing and removing the muscle

specimen alters the NMR signal to mask the effect from atrophy. The alternative is that atrophy produced in this manner does not alter relaxation times

unlike muscle dystrophy. A set of experiments were conducted to determine 1)

whether NMR parameters are altered significantly with blood flow and 2) to

determine if NMR signals are similar in muscles from live versus dead animals. Two rabbits, weighing 2.5 kg. were anesthetized and imaged with the 6

MHz whole body imager (head probe in). The pulse sequence was a 90° pulse

followed by a train of 16, 180° pulses spaced at 3 msec. The rabbits were

imaged with the cross-sectional slice just superior to the kidneys. Using a

technique developed at Baylor (see attached), a calculated T2 image was produced - 1) with the animal alive, 2) with the animal alive but with the

descending aorta occluded, and 3) 15 minutes after sacrifice, using a region

of interest cursor, the value of the supraspinal muscles was obtained for each

image. The calculated ~\2

 was approximately 19 msec and was not altered by

arterial occlusion or death.
HUMAN STUDIES

An initial series of images were taken of the lower limbs of one of the investigators using the spin echo technique and the 6 mHz instrument. Three slices

were obtained corresponding to regions through the belly of the soleus, the

belly of the gastrocnemius and the distal femur. These pictures demonstrated

the potential to differentiate individual muscle groups and clearly image various structures. The possibility of monitoring differential muscle atrophy is

apparent. As discussed previously we found that in the suspended rat model

the soleus (slow twitch) atrophies 50% in two to four weeks while the gastrocnemius (fast twitch) atrophies 25%. Since the fluoride bedrest studies are in

progress the opportunity to make measurements under controlled conditions

where atrophy should occur is possible. In order to quantitate the NMR images

in units of cm? or cm35 the pixel size and slice thickness would have to be

known each time an image is obtained. Since the pre and post bedrest images

might be separated by weeks or months, a direct calibration is necessary. A

jig was designed to rigidly hold the right limb immobile during NMR acquisition. A typical T2 image requires about 4 minutes. A schematic of the device

is given in Figure One. In addition to rigidly holding the leg, the jig unambiguously determines slice location, pixel size and thickness. This is accomplished by a series of machined grooves in the holder which are filled with an

NMR sensitive material. After experimenting with a number of vegetable fat

substances, Crisco was chosen to fill the grooves. Four grooves (A-D) are

used for calibrating the pixel size and hence the x-y plane dimensions. The

image length produced from the E grooves after simple geometric calculation

gives slice thickness. The image of the series of grooves labeled F is used

to calibrate the precise location on the jig where the NMR slice is acquired.

This permits exact anatomical repositioning by carefully positioning the leg

in the jig in the same manner each time. Calibration is accomplished by noting the distance between grooves A, E, and F. A digital histogram through the

two spots of the F grooves gives a crude estimate of image resolution. The

large tube labeled G is used to obtain calculated relaxation times which are

used to monitor system stability for comparing tissue relaxation times.

All imaging to date "has been accomplished using the 6 mHz Bruker magnet and

head probe to enhance image resolution. Calf area images of two normal male

volunteers were obtained. In one subject (swimmer) a series of 5 slices (A-E)

3 cm apart, starting from the heel were obtained. The sixth slice (F) was obtained at the level of the proximal tibia. The first volunteer subject was

then removed from the magnet and a second volunteer subject (jogger) positioned within the magnet. Two slices were obtained 3 cm apart, starting at 35

cm from the heel. The second volunteer got back in and a slice (I) was obtained which corresponded to slice C to test reproducibility. The NMR technique was spin echo using a 180° slice select pulse followed 20 msecs later by

a 90" read pulse and 16, 180° spin echo pulses separated by 9 msec. A delay

of one second is used between the end of the spin echos and the next slice

select pulse. Reconstruction was accomplished from 120 projections at 1.5°

intervals in a collection matrix of 256 x 256. For each projection a series

of 16 echoes were collected and reformatted into four echoes by summing successive echoes in groups of 4. After reconstruction, each of these are

displayed as separate images. From these four images, T? and spin density

images are computer calculated. The methodology and documentation for producing accurate calculated T? NMR images has been published by investigators at

Baylor and a copy is included in the appendix.
Attached are copies of 4 images from slice C, figure 2, from the first volunteer. The upper left images of each are reconstructed from the first four

echoes (CP1); the upper right image from the third set of four echoes (CP3);

the lower left is the T£ and the lower right the spin density image. Muscle

contrast appears to be best in the images obtained during the latter portion

of the echo train as evidenced by comparing CP3 vs CP1. There are four marker

spots surrounding each image produced from NMR sensitive material in the jig.

iSince the distance between the points are known exactly, the pixel size can be

Icalculated, which for this image sequence was 1.1 mm. The markings below the

'image are used to calculate slice thickness, 1.5 - 2.0 cm in this determination. Two spots which appear below and to the left of each image serve to

unambiguously determine the distance from the subject's heel at which the NMR

slice was taken. A pixel intensity vs distance histogram through these markers give an estimate of image resolution, <2 mm. Grossly, the two slices

appear very similar, indicating"adequate reproducibility for a scan through a

given anatomical location. Bone width, cross-sectional areas and shape are

readily obtained from CP1 or spin density images. Marrow, fat, vessels, and

muscle are best distinguished in the T£ calculated image.

NASA has considerable experience in image processing resulting from the remote

sensing program (Earth Observations). Typically NMR imaging requires considerable image processing and therefore benefits from NASA technology. A number

of techniques for combining multiple NMR images (spin density, Tj, T2» early

vs late spin echo) are being investigated such as a 3 color combination, parallelepiped, principal component, etc. In addition an effort is ongoing to

improve NMR images, i.e., contrast, edge detection, resolution, signal to

noise ratio. Figure 3 is a black and white image of CP1 slice C from subject

number one using the shadow technique to enhance contrast. Figure 4 is a

reconstructed image of this same slice using 3 color combination. The NMR

picture was constructed by coding the CP1 picture red, CP3 green and the ratio

of CP1/CP3 as blue. These three images are then combined to form the NMR picture of. Figure 4. It is not clear at the present time what advantage these

various display methods wil l have, but work is progressing to determine the

optimum combination of resolution and contrast. For example, the highest S/N

in the T2 images is observed in CP1 while the greatest contrast is seen in

CP3-4. Another series of images from TI experiments will result which will

have different relative tissue intensities compared to T£ images. Earth

resource techniques allow the combination of several of the NMR images in various ways to produce enhanced information in a compact/accessible form. A

more detailed description of these techniques is contained in Appendix B. One

major objective is to be able to distinguish individual muscle groups. Figure

4 shows a considerable amount of anatomical detail. The soleus is clearly

demonstrated as well as the lateral and medial lobes of gastrocnemius. Other

muscles that appear to be observable are the anterior and posterior tibialis,

peroneus longus and brevis, and the extensor hallucis and digitorum. The

anterior and posterior tibial and preoneal vessels are clearly seen as are the

compact bone and marrow of the tibia and fibula. It is expected that images

such as Figure 4 will best demonstrate muscle size changes.
We are also attempting to characterize and quantitate tissue by differences in

T£. A preliminary classification is given below.

Tissue

Muscle

Fascia

Fat

Marrow

Blood (vessels)

Bone

T? (msec)

40 - 60

60 - 80

85 - 126

122 - 138

150 - 240

n il

Color Code

Red

Magenta

Yellow

Blue-White

White Gray Scale

Black

By assigning a color to pixels having these ranges of T£ an image can be

formed. Figure 5 is a T? colored image based on the above scheme. It is

important to realize that the color scheme is not uniform but corresponds to

the above ranges of ?2- Since we can internally calibrate the pixel size in

cm? we can simply have the computer add up the number of pixels in a given T2

range and hence determine the areas represented by that tissue. Limited

repeat imaging was performed in both subjects 1 and 2. The results given

below are for slices which are from approximately the same anatomical area in

the two subjects. Subject 1 is a swimmer while subject 2 is a jogger/limited

marathoner. The repeat images of subject 1 occurred on the same day as the

original while those of subject 2 were separated by about 3 weeks.

Tissue Area from T;? Reconstructed Images

Tissue Area (cm2)

Subject #1 (AL) Subject #2 (HE)

Total Area

Muscle

Fat

Tibia Bone

Tibia Marrow

Circulation

Scan 1

Area

cm2

86.4

43.5

18.6

4.20

2.43

2.2

T2

msec

39

109

*

132

172

Scan 2

"Area

-cm^

85.2

41.4

21.4

3.27

3.03

1.1

T2

msec

38

107

*

128

167

area %

1.4

4.8

14

24

22

58

Scan 1

Area

cm?

87.4

59.3

14.4

5.05

2.38

.181

T2

msec

38

98

*

132

155

Scan 2

~Area

cm?

89.0

59.1

14.6

5.17

2.43

.181

T2

msec

39

98

*

130

155

area %

1.8

0.4

1.4

2.3

2.1

0

(*) Low value; difficult to measure.
The total limb area was reproducible within about 1.5 % using T2- Using cpl

images however precision is about 0.2%. Despite a machine problem with the

acquisition of scan 2 of Subject 1, precision of muscle fat, bone and marrow

appear promising. As expected in the adult, the marrow of the extremity is

mostly yellow/fatty marrow as reflected in the ?2 value which is close to

subcu fat.

iWe have an approved human use protocol for performing NMR imaging in bedrest

^subjects. A copy of this protocol is contained in the appendix. To date 6

'subjects have been imaged pre and post 5 weeks of bedrest. Each subject was

repeated twice before and after bedrest. These results are now being analyzed

at JSC. Table 1 and Table 2 summarize some of these early results.

We have shown that leg area measurements using NMR are possible with a precision of about 0.2%; fat and muscle cross-sectional area may be accurately

measured in a given anatomical section; differential muscle atrophy can be

quantitated. Table 1 gives the total and regional muscle area in mm^ through

the belly of the gastrocnemius pre and post 5 weeks of bedrest in 6 normal

subjects. Each pre and post bedrest value represents the average of 2 slices

separated by two cm with each slice measurement repeated. The mean and standard deviation is obtained from 4 area measurements. This region of the limb

which includes both soleus and gastrocnemius varies less in area with limb

position and therefore shows very good reproducibility, i.e., about 2 - 5%.

Measurement of the soleus alone, which appears to demonstrate even larger percentage changes, are in progress. The mean loss of gastrocnemius and soleus

cross-sectional area for all 6 subjects was 9% while the total muscle area

loss was 6%.

The values in Table 2 are the average of 5 slices separated by 2 cm starting

at a position through the belly of the soleus. Each series of slices is

repeated therefore the mean and standard deviation are calculated from 10 NMR

limb measurements. The reproducibility is within 4%. Data in Table 2 demonstrates a significant decrease in T? compatible with -an increase in marrow

fat.~.Slowly, moving or stagnant blood has T^ values considerably higher than

fat. Table 2 also shows that no change in subcutaneous fat T2 relaxation time

occurred. It has been reported that the muscle water content of biopsied soleus muscles decreased following 8 hours of head down tilt bedrest. Tissue Tj

and T2 values are very sensitive to tissue water. For example, a 2% change in

tissue water may produce a 16% change in the relaxation time of tissue. Our

studies with 5 weeks of horizontal bedrest indicate no change in tissue hydration since muscle T2 relaxation times were not altered despite significant

muscle atrophy; see Table 2.
TABLE I

Total and regional muscle area (mm2) after 5 weeks of bedrest

-4cm section at level of gastrocnemius center

i

GASTROCNEMIUS + SOLEUS MEAN ± SD

Subject Pre-Bedrest

36

37

39

42

46

47

*

36

37

39

42

46

47

6085 ± 446

*3970

4548 ± 138

4517 ± 79

3558 ± 137

4456 i 73

One measurement because of

TOTAL

8980 ± 472

6374

6731 ± 281

7053 ± 70

5326 ± 85

6643 ± 9

Post-Bedrest

5005 ± 328

4163 ± 57

3697 ± 108

4291 ± 80

3384 ± 168

3956 ± 12

acquisition artifacts

MUSCLE MEAN ± SD .

8121 ± 384 -

6519 ± 197

6004 ± 211

6514 ± 140

5307 ± 71

6186 ± 6

A %

-18

5

-19

-5

-5

-11

-10

2

-11

-8

0

-7

P

<0.001

0.04

<0.001

NS

NS

<0.001

<0.001

NS

<0.001

0.03

NS

<0.001
TABLE 2

Preliminary Results of NMR limb imaging of bedrest subjects

(n=6) - Tissue T2, pre vs post-bedrest

i

MARROW, msec ± SD MUSCLE, msec ± SD FAT, msec ± SD

Pre Post A% Pre Post Pre Post

135 ±

147 ±

130 ±

132 ±

149 ±

145 ±

6

11

4

2

8

7

114

129

128

124

115

116

±

±

±

+

+

±

7

17

4

3

2

2

-16

-12

-2

-6

-23

-20

44

53

43

48

51

48

± 1

i 2

± 2

± 2

± 3

± 1

52

53

46

47

48

50

± 2

± 4

± 2

± 1

± 1

± 2

93t

96t

102t

103t

TBA*

TBA*

93t

104t

loot

94t

x±SE 140 ± 3 ' 121 ± 3 48 ± 2 49 ± 1 98 ± 2 98 ± 2

A% -14 +2 0

P 0.001 NS NS

*TBA - to be analyzed

t average of 2 measurements only

13
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