In Vivo Lipid Profiling Using ProtonMagnetic Resonance Spectroscopy

in an Experimental Liver FibrosisModel

Jerry S. Cheung, PhD, Shu Juan Fan, MSc, Darwin S. Gao, BEng, April M. Chow, PhD,Jian Yang, MD, PhD, Kwan Man, PhD, Ed X. Wu, PhD

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FrS.El(KKoDeMDiXiSeKoE.

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Rationale and Objectives: The aim of this study was to characterize early hepatic lipid changes in an experimental model of liver fibrosisusing proton (1H) magnetic resonance spectroscopy (MRS) at high magnetic field in vivo.

Materials andMethods: Liver fibrosis was induced in 12 Sprague-Dawley rats by twice-weekly carbon tetrachloride (CCl4) administration

up to 4 weeks. Eight normal rats were used as controls. Single-voxel 1H MRS experiments were performed at 7 Tesla to measure signalintegrals of various lipid peaks including –CH3, (–CH2–)n, –CH2–C=C–CH2–, =C–CH2–C= and –CH=CH– at 0.9, 1.3, 2.0, 2.8, and 5.3 ppm,

respectively, and peak from choline-containing compounds (CCC) at 3.2 ppm. Total lipid, total saturated fatty acid, total unsaturated fatty

acid, total unsaturated bond, polyunsaturated bond, and CCC indices were quantified.

Results: Significant increases (P < .01) in total lipid and total saturated fatty acid indices were found in animals with CCl4-induced fibrosis

as compared with normal animals. In addition, total unsaturated bond and polyunsaturated bond indices of animals at 4 weeks after CCl4insult were significantly higher than (P < .01 and P < .05, respectively) those of normal animals and animals at 2 weeks following insult;

whereas there was only significant increase (P < .01) in total unsaturated fatty acid index in animals with 4-week CCl4 insult as comparedwith normal animals.

Conclusion: The hepatic lipid changes in CCl4-induced experimental fibrosis model were documented in vivo and longitudinally using1HMRS at 7 Tesla. The experimental findings suggested that total saturated fatty acid increase contributedmainly to the total lipid increasein animals with CCl4 insult. This study also demonstrated the potential value of high fieldMRS to resolve lipid composition and alterations in

liver fibrosis.

Key Words: Proton magnetic resonance spectroscopy (1H MRS); liver fibrosis; lipid; saturated fatty acid; unsaturated fatty acid; carbon

tetrachloride (CCl4).

ªAUR, 2011

iver fibrosis associated with chronic liver injury can progression or regression in response to treatment (3). Because

L progress to cirrhosis and ultimately hepatocellular

carcinoma (HCC) (1,2). Percutaneous liver biopsy

has been considered the standard technique for diagnosis

and staging of liver fibrosis. However, liver biopsy is highly

invasive and associated with risk of complications, limiting

its applicability in longitudinal monitoring of fibrosis

ad Radiol 2011; 18:377–383

om the Laboratory of Biomedical Imaging and Signal Processing (J.S.C.,J.F., D.S.G., A.M.C., J.Y., E.X.W.), Departments of Electrical andectronic Engineering (J.S.C., S.J.F., D.S.G., A.M.C., J.Y., E.X.W.), Surgery.M.), and Anatomy (E.X.W.), The University of Hong Kong, Pokfulam, Hongng SAR, China; Athinoula A. Martinos Center for Biomedical Imaging,partment of Radiology, Massachusetts General Hospital and Harvardedical School, Charlestown, MA 02129, USA (J.S.C); Department ofagnostic Radiology of the First Affiliated Hospital, School of Medicine of’an Jiaotong University, Xi’an, Shannxi Province, China (J.Y.). Receivedptember 12, 2010; accepted October 29, 2010. Supported by the Hongng Grant Council (GRF HKU7808/09M). Address correspondence to:X.W. e-mail: ewu@eee.hku.hk

AUR, 2011i:10.1016/j.acra.2010.10.012

disease progression to liver cirrhosis can be prevented by early

interventions and treatments (4–6), there has been a great

interest in the development of noninvasive techniques for

early diagnosis and characterization of liver fibrosis. Proton

magnetic resonance spectroscopy (1H MRS) allows the

study of cellular biochemistry and metabolism, and provides

a noninvasive means to determine disease abnormalities and

progression in vivo and longitudinally. Liver lipid content,

which has been suggested to play an important pathogenic

role in the development of liver fibrosis and cirrhosis in

patients with chronic hepatitis C (7,8) and nonalcoholic

steatohepatitis (9–11), can be measured by 1H MRS

noninvasively. Most importantly, specific lipid changes

related to saturated and unsaturated fatty acids can be

monitored in vivo by high resolution 1H MRS.

Carbon tetrachloride (CCl4) intoxication is a well-

characterized, reproducible and the most commonly used

experimental animal model of liver fibrosis. It has been widely

377

Figure 1. Schedule of carbon tetrachloride (CCl4) twice-weekly

administration for induction of liver fibrosis in adult Sprague-

Dawley rats, 1H proton magnetic resonance spectroscopy (MRS)experiments, and liver histology.

CHEUNG ET AL Academic Radiology, Vol 18, No 3, March 2011

studied with respect to the histological, biochemical, cellular,

andmolecular changes associated with development of fibrosis

(12,13). By interfering hepatic energy metabolism and

protein synthesis, CCl4-induced hepatotoxicity can lead to

triglyceride accumulation, mitochondrial injury, and

necrosis (14). With the increased availability of high-field

($3.0 Tesla) magnetic resonance (MR) systems for clinical

and preclinical studies, both signal-to-noise ratio (SNR) and

spectral resolution of metabolites in the MR spectra can be

improved significantly (15), allowing more accurate metabo-

lite identification and quantification and thus disease charac-

terization. Although MRS can provide insights into liver

metabolism noninvasively, detailed in vivo 1H MRS study of

liver fibrosis with high spectral resolution has been limited

(16–18). The aim of this study was to characterize early

hepatic lipid changes in the experimental CCl4-induced

liver fibrosis model by means of single-voxel 1H MRS at

high magnetic field in vivo.

MATERIALS AND METHODS

Animal Preparation

All animal experiments were approved by the institutional

animal ethics committee. Liver fibrosis was induced in male

adult Sprague-Dawley rats (220 to 260 g; n = 12) by subcuta-

neous injection of 1:1 volume mixture of CCl4 in olive oil at

a dose of 0.2 mL/100 g of body weight twice a week for 4

weeks (13,19). Intermittent administration of CCl4 has been

widely used to experimentally induce liver fibrosis in

rodents by evoking a marked infiltration of inflammatory

cells, thus mimicking the changes in chronic viral

hepatitis‑associated fibrosis in many ways (20,21). The

twice-weekly dosing can induce early stage of liver fibrosis

and established fibrosis after 2 and 4 weeks of CCl4 adminis-

tration, respectively, in rodents (13,22). This well-controlled

CCl4-induced liver fibrosis model allows the study of a homo-

geneous population of liver fibrosis. 1H MRS was performed

in the CCl4-insulted animals at 2 and 4 weeks after the start of

CCl4 administration. The animals were examined 48 hours

after last CCl4 administration to avoid acute inflammatory

effects (18). The overall schedule of the experiment for

378

animals induced with liver fibrosis is shown in Figure 1.

Normal male adult Sprague-Dawley rats (220 to 260 g;

n = 8) were used as controls.

In Vivo Liver 1H MRS Experiments

All 1H MRS experiments were performed on a 7 Tesla MR

imaging scanner with a maximum gradient of 360 mT/m

(70/16 PharmaScan, Bruker Biospin GmbH, Germany). A

60-mm inner diameter quadrature resonator was used for

both radiofrequency (RF) transmission and receiving. During

liver imaging, each animal was anesthetized with isoflurane/

air using 1.0 to 1.5% for maintenance via a nose cone with

respiratory monitoring (23,24). Body temperature was

maintained at about 36.5�C by circulating warm water in

a heating pad. Scout images were first acquired in three

orthogonal planes with a fast low angle shot sequence for

localization of a voxel or volume of interest for MRS. A 5

� 5 � 5 mm3 voxel was chosen within a homogeneous

liver parenchyma to avoid large blood vessels. First- and

second-order localized automatic shimming was first per-

formed within the voxel until a full width at half maximum

<50 Hz was achieved in the water peak. The water signal

was suppressed by variable power RF pulses with optimized

relaxation delays with bandwidth of 200 Hz. Outer volume

suppression combined with respiratory-triggered single-voxel

point-resolved spectroscopic sequence was used for acquiring

liver MR spectrum, with repetition time (TR) = two respira-

tory cycles (∼2.0 to 2.5 seconds), echo time (TE) = 15 ms,

receiver bandwidth = 4 kHz, 2048 data points, 256 averages,

and total scan time of ∼10 minutes. Note that respiratory trig-

gering was used to minimize voxel misregistration in the pres-

ence of respiratory motion, while short TE was chosen to

reduce signal loss due to T2 relaxation to improve SNR (25).

Data and Statistical Analysis

The MRS data were processed using the MR spectroscopic

analysis package provided by the MR imaging vendor

(26,27). MR spectra were zero-filled to 8192 data points,

apodized with a 2-Hz exponential filter, Fourier transformed,

0th- and first-order phase corrected, and baseline corrected.

Signal integrals of lipid methyl protons (–CH3; 0.9 ppm),

methylene protons ((–CH2–)n; 1.3 ppm), allylic protons

(–CH2–C=C–CH2–; 2.0 ppm), diallylic protons (=C–CH2–

C=; 2.8 ppm), methene protons (–CH=CH–; 5.3 ppm),

and protons from choline-containing compounds (CCC;

3.2 ppm) (11,28,29) were measured by integrating areas

under peaks. Spectral noise was calculated from the standard

deviation of the last portion of spectrum from 7.3 to 11.3

ppm, in which no metabolite was observable in liver

(11,28,29). Total lipid and CCC indices were quantified by

dividing peak area of (–CH2–)n and CCC by spectral noise,

respectively, given the fact that SNR was similar among

spectra because of identical voxel size and nearly identical

hardware settings in different measurements. In addition, total

TABLE 1. Peak Area Ratios of Various Metabolite IndicesMeasured by Proton Magnetic Resonance Spectroscopy(1H MRS)

Index Peak Area Ratio Frequency

Total lipid (–CH2–)n/noise 1.3 ppm

Total saturated fatty acid 3(–CH2–)/2(–CH3) 1.3/0.9 ppm

Total unsaturated fatty

acid

3(–CH2–C=C–CH2–)/4

(–CH3)

2.0/0.9 ppm

Total unsaturated bond 3(–CH=CH–)/2(–CH3) 5.3/0.9 ppm

Polyunsaturated bond 3(=C–CH2–C=)/2(–CH3) 2.8/0.9 ppm

Choline-containing

compound (CCC)

CCC/noise 3.2 ppm

Academic Radiology, Vol 18, No 3, March 2011 1H MRS LIPID PROFILING IN LIVER FIBROSIS

saturated fatty acid, total unsaturated fatty acid, total unsaturated

bond, and polyunsaturated bond indices were estimated by

dividing peak area of (–CH2–)n, –CH2–C=

C–CH2–, –CH=CH– and =C–CH2–C= by peak area of

–CH3, respectively, and scaled to the relative number of

protons contributing to the resonance (11), as shown in Table

1. Note that the signal from (–CH2–)n and –CH2–C=

C–CH2– increases with increasing number of saturated and

unsaturated fatty acids, respectively, whereas the signal from

–CH=CH– and =C–CH2–C= increases with the percentage

of total unsaturated and polyunsaturated double bonds in the

unsaturated fatty acids, respectively (11,30). Furthermore, the

signal contribution of glutamine and glutamate at 2.2 ppm

was assumed to be negligible (11). Results were expressed as

mean � standard deviation. One-way analysis of variance

with Tukey’s multiple comparison test was employed to

compare differences in ratios of peak areas between fibrosis-

induced and control animals, with P values less than .05 consid-

ered statistically significant.

Histology

After theMR examination following 2 weeks of CCl4 admin-

istration, 4 of 12 animals were sacrificed for histological eval-

uation. Furthermore, four of the remaining eight animals

were sacrificed after MR examination after 4 weeks of CCl4insult as shown in Figure 1. One additional normal animal

was sacrificed as a control. Liver specimens were fixed in

formalin, embedded in paraffin, sectioned and examined by

light microscopy after standard hematoxylin-eosin staining

and Masson’s trichrome staining (31,32).

RESULTS

Figure 2 shows the typical liver 1HMRS spectra from a normal

control animal and an animal assessed at 2 and 4 weeks after

start of CCl4 twice-weekly administration, with a typical

voxel placement shown in the anatomical image. It was

consistently observed that all animals with CCl4-induced

liver fibrosis exhibited substantial increase in various lipid

peaks including –CH3, (–CH2–)n, –CH2–C=C–CH2–,

–COO–CH2–, =C–CH2–C= and –CH=CH–, with similar

signals from CCC, as compared with the normal animals.

Water (H2O) signal at 4.7 ppm was effectively suppressed in

the spectra with varying degree. Figure 3 shows the total lipid,

CCC, total saturated fatty acid, total unsaturated fatty acid,

total unsaturated bond and polyunsaturated bond indices in

normal animals (n = 8) and animals with 2-week (n = 12)

and 4-week (n = 8) CCl4 twice-weekly administration. Total

lipid indices of animals at 2 weeks (3.97 � 1.57 � 104) and 4

weeks (5.66� 1.31� 104) after fibrosis induction were found

to be significantly higher than (P < .01) those of normal

control animals (0.72 � 0.17 � 104), along with significant

difference (P < .05) between 2 and 4 weeks. Meanwhile,

a similar trend was observed in total saturated fatty acid index.

Significant increase (P < .01) in total saturated fatty acid index

was found in animals with CCl4-induced liver fibrosis (8.74�1.68 and 10.42 � 1.35 for 2-week and 4-week CCl4 admin-

istration, respectively), as compared with normal animals

(5.91 � 1.23), whereas the difference between 2 and 4 weeks

was also statistically significant (P < .05). On the other hand,

there was only significant increase (P < .01) in total unsatu-

rated fatty acid index in animals with 4-week CCl4 insult as

compared with normal animals. Furthermore, total unsatu-

rated bond index of animals at 4 weeks after CCl4 insult

(1.75 � 0.30) were significantly higher than (P < .01 and P

< .05, respectively) those of normal animals (1.10 � 0.32)

and animals at 2 weeks after insult (1.32 � 0.30). Similarly,

polyunsaturated bond index of animals after 4-week CCl4administration (0.95 � 0.18) were significantly higher than

(P < .01 and P < .05, respectively) those of normal animals

(0.57 � 0.18) and animals with 2-week CCl4 insult (0.73 �0.14). No significant differences were observed in CCC

between normal and diseased animals.

Figure 4 shows the typical hematoxylin-eosin and Masson’s

trichrome staining of normal liver and livers at 2 weeks and 4

weeks after CCl4 insult. Collagen deposition was stained as

blue by Masson’s trichrome staining in fibrotic livers.

Compared with normal liver (Fig 4a), collagen deposition

and intracellular fat vacuoles were consistently observed in

livers with CCl4 insult (Fig 4b, 4c). Similar histological find-

ings were observed in all liver samples collected, and theywere

largely consistent with those from the earlier studies of CCl4-

induced liver fibrosis in rodent models (18). The histological

observations of collagen deposition in the liver samples

collected confirmed the liver fibrogenesis in the animals

studied.

DISCUSSION

As early as 2 weeks after the start of CCl4 administration, total

lipid and total saturated fatty acid indices were found to

increase substantially (P < .01) in animals with liver fibrosis

as compared with control animals (Fig 3a, 3c). Meanwhile,

no increases in total unsaturated fatty acid index were

observed (Fig 3d), suggesting that the total saturated fatty

acid increase contributed mainly to the total lipid increase

in animals with CCl4 insult. The total lipid increase in animals

379

Figure 2. Typical liver 1H protonmagnetic reso-nance spectroscopy (MRS) spectra from

a normal control animal and an animal scanned

at 2 and 4 weeks after start of carbon tetrachlo-ride (CCl4) twice-weekly administration, with

a typical voxel placement (white square) shown

in the anatomical image. Animals with CCl4-

induced liver fibrosis consistently showed mark-edly increases in various lipid peaks including

methyl protons (–CH3; 0.9 ppm), methylene

protons ((–CH2–)n; 1.3 ppm), allylic protons

(–CH2–C=C–CH2–; 2.0 ppm), a-methyleneprotons to carboxyl (–COO–CH2–; 2.2 ppm), dia-

llylic protons (=C–CH2–C=; 2.8 ppm) and meth-

ene protons (–CH=CH–; 5.3 ppm), except the

peak from choline-containing compounds(CCC; 3.2ppm). Thewater (H2O; 4.7ppm) signals

were effectively suppressed in the spectra.

Figure 3. (a) Total lipid, (b) choline-containingcompounds (CCC), (c) total saturated fatty

acid, (d) total unsaturated fatty acid, (e) totalunsaturated bond, and (f) polyunsaturated

bond indices in normal animals and animals

with 2-week and 4-week CCl4 twice-weekly

administration. One-way analysis of variance(ANOVA) with Tukey’s multiple comparison test

was performed with **P < 0.01, *P < 0.05 and

n.s. for insignificance.

CHEUNG ET AL Academic Radiology, Vol 18, No 3, March 2011

with CCl4-induced liver fibrosis was likely due to fatty

infiltration/fatty changes in hepatocytes (Fig 4). During toxic

CCl4 insult, hepatocytes are incapable of synthesizing

380

lipoproteins that are needed for removing triglycerides in

the cytoplasm as a result of the destruction of microsomal

proteins by lipid peroxidation (18,33,34), leading to

Figure 4. Typical hematoxylin-eosin (H&E) staining (400�; left column) and Masson’s trichrome staining (200� and 40�; middle and rightcolumn, respectively) of normal liver (a), and livers subjected to2-week (b)and4-week (c) carbon tetrachloride (CCl4) twice-weeklyadministration.

Collagen deposition (green arrows), fat vacuoles (blue arrows), and cell necrosis/apoptosis (black arrows) were observed in the insulted livers.

Academic Radiology, Vol 18, No 3, March 2011 1H MRS LIPID PROFILING IN LIVER FIBROSIS

increased triglyceride accumulation (35). It is noteworthy that1H MRS can provide valuable information on lipid composi-

tion, which cannot be revealed by histological analysis. The

total saturated fatty acid increase in liver may reflect the

lipid-induced cell toxicity, which has been suggested to be

related with activated apoptosis induced by saturated fatty

acids (9,10,36). In nonalcoholic fatty liver disease,

endoplasmic reticulum stress associated with increased

saturated fatty acids in the liver have been shown to

promote liver injury and partly contribute to the disease

progression from simple steatosis to steatohepatitis (9,10).

Nonetheless, the potential toxic effect of increased saturated

fatty acids in fibrotic livers has yet to be determined.

Although there was no significant change in total unsatu-

rated fatty acid index at week 4 as compared with week 2 after

CCl4 administration, significant increase (P < .05) in total

unsaturated bond index was observed (Fig 3e). These findings

suggested that without a substantial increase in amount of

unsaturated fatty acids, more unsaturated double bonds were

formed within the unsaturated fatty acids at 4 weeks after

the start of fibrosis induction. As such, significant increase in

polyunsaturated bond index (P < .05) was observed (Fig 3f)

as expected. Therefore, the amount of polyunsaturated fatty

acids was likely to increase at the expense of monounsaturated

fatty acids in the animals with 4-week CCl4 insult. Such

increase in degree of polyunsaturation has been observed

and ascribed to increased necrosis/apoptosis in various exper-

imental models (11,37,38), which was also observed in the

current study (Fig 4). Because CCC has been considered to

represent the important constituents in phospholipid metabo-

lism of cell membranes (39), similar CCC levels observed in

the normal and CCl4-insulted animals were largely expected

in the early stage of liver fibrosis because of the similar rate

of cell turnover of hepatocytes prior to the development of

cirrhosis (40).

In vivo phosphorus-31 (31P) MRS has been found prom-

ising in evaluating the cellular turnover and liver energy status

(41–43), but it cannot measure the hepatic lipid content and is

not readily available onmost clinicalMR imaging scanners. By

contrast, in vivo 1H MRS is feasible on most standard MR

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CHEUNG ET AL Academic Radiology, Vol 18, No 3, March 2011

imaging scanners, where high-resolution anatomical images

and functional information such as perfusion can be acquired

in the same imaging session. 1H MRS has been used to

investigate focal liver lesions and found to be useful in

characterizing HCC in humans (44–46) and animal models

(37,47–49) by providing valuable metabolic information.

However, only limited 1H MRS studies have been

performed to investigate liver fibrosis in both humans and

animals (16–18). Note that our study used the highest field

strength to study liver fibrosis compared with previous

studies (16–18). The advantages of performing 1H MRS at

high magnetic field include better SNR and increased

spectral resolution (15), thus providing high-quality spectra

in acceptable scan times. In this regard, 1H MRS at 7 Tesla

in this study can provide precise biochemical information in

liver noninvasively that can be used for characterizing and

monitoring of liver diseases including liver fibrosis. It is worth-

while to note that it is relatively difficult to accurately resolve

and quantify several lipid signals other than (–CH2–)n peak

because of limited spectral resolution and poor SNR at low

field strengths in previous studies (16,18). In contrast, our

current study provided more detailed information about

lipid composition by resolving various lipid peaks (eg,

–CH2–C=C–CH2–, =C–CH2–C= and –CH=CH–) in

fibrotic livers at 7 Tesla. Note that the separation between

methene (5.3 ppm) and water (4.7 ppm) peaks is 180 Hz at

7 Tesla, thus the methene proton peak was minimally

affected by the water suppression RF pulses with bandwidth

of 200 Hz. In addition, the effects of motion on liver MRS

were largely reduced using respiratory gating in the current

study, providing water-suppressed MR spectra with high

quality (Fig 2). It is worth noting that although fat-selective

MR imaging using Dixon methods can provide quantitative

information about hepatic lipid content, these techniques

are only sensitive to one specific lipid proton species (ie, meth-

ylene protons [3.4 ppm apart from water]). In contrast, 1H

MRS can resolve and quantify various lipid proton species

so as to provide insights into lipid composition.

Ratio measurements using spectral noise as internal refer-

ence were employed for quantifying total lipid and CCC

levels in the current study. Note that spectral noise could be

affected by a number of factors such as local sensitivity of

coil and acquisition protocol. Such factors were expected to

be similar among measurements in this study because of the

use of volume RF coil, identical hardware settings and acqui-

sition protocol, yielding similar noise levels in the spectra of

different measurements (Fig 2). The use of lipid peak as

internal reference (16) was avoided, because the amount of

lipid increased markedly in the CCl4-induced liver fibrosis

model (Fig 2). Unsuppressed water signal was not chosen as

internal reference either because the water content may be

affected by pathological conditions (46). Previous human

studies reported inconsistent observations on the lipid levels

in chronic hepatitis (16,17), probably due to varying types

of virus-induced hepatitis investigated. Our results showed

an increase in total lipid levels, consistent with other MR

382

studies in CCl4-induced fibrosis model (18,35). However,

changes in saturated fatty acids, unsaturated fatty acids and

unsaturated double bonds were not investigated in those

studies. Our MRS data at high field allowed better

discrimination and quantification of the –CH2–C=C–CH2–,

–CH=CH– and =C–CH2–C= lipid peaks, thus total

unsaturated fatty acid, total unsaturated bond and

polyunsaturated bond indices can be measured. Moreover,

the elevated total saturated fatty acid measured by 1H MRS

in the current study may suggest its role in the development

of liver fibrosis (9,10). One potential limitation of the

present study is lack of sham controls receiving olive oil

alone. As subcutaneous injection of CCl4 with olive oil is

a well-established protocol to induce liver fibrosis in rodents

(13), effects of olive oil were not examined in the current

study. Nonetheless, previous studies showed that hepatic lipid

changes are associated with metabolic alterations due to intox-

ication of CCl4 (50), whereas no evidences of steatosis

induced by olive oil have been found.

In conclusion, the lipid changes related to saturated and

unsaturated fatty acids in CCl4-induced experimental fibrosis

model were documented in vivo and longitudinally using 1H

MRS at 7 Tesla. Our experimental results demonstrated that1H MRS at high-field was useful in detecting and character-

izing various hepatic lipid alterations as early as 2 weeks from

the start of induction of liver fibrosis in the animal model. 1H

MRSmay be valuable in detecting fatty changes at early phase

in human liver fibrosis prior to the development of cirrhosis,

and potentially useful in determining treatment strategies and

evaluating therapeutic outcomes.

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