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Investigating viability of intestine usingspectroscopy: a pilot study

Barıs‚ R. Karakas‚, MD,a Aslınur Sırcan-Kucuksayan, MS,b

Ozlem G. Elpek, MD,c and Murat Canpolat, PhDb,*aDepartment of General Surgery, Antalya Training and Research Hospital, Antalya, TurkeybDepartment of Biophysics, Faculty of Medicine, Biomedical Optics Research Unit, Akdeniz University, Antalya,

TurkeycDepartment of Pathology, Faculty of Medicine, Akdeniz University, Antalya, Turkey

a r t i c l e i n f o

Article history:

Received 3 October 2013

Received in revised form

21 February 2014

Accepted 18 March 2014

Available online 25 March 2014

Keywords:

Acute mesenteric ischemia

Intestinal tissue viability

Ischemiaereperfusion

Reflectance spectroscopy

Intraoperative

Real time

* Corresponding author. Department of Biop07058, Turkey. Tel.: þ90 242 249 6160; fax: þ

E-mail address: mcanpolat1@msn.com (M0022-4804/$ e see front matter ª 2014 Elsevhttp://dx.doi.org/10.1016/j.jss.2014.03.052

a b s t r a c t

Background: The differentiation of “viable” from “nonviable” bowel remains a challenge in

the treatment of acute mesenteric ischemia. In this study, diffuse reflectance spectroscopy

(DRS) was used to investigate the viability of bowel tissue after ischemia and reperfusion in

an animal model in vivo and in real time.

Methods: A total of 25 females SpragueeDawley rats were divided into five groups based on

different bowel ischemia times. In each study group for four of them, the superior

mesenteric artery was occluded using a vascular clamp for a different period (i.e. 30, 45, 60,

and 90 min; n ¼ 5 for each group). Intestinal reperfusion was accomplished by releasing the

clamps after the given occlusion period for each group. Spectra were acquired by gently

touching the optical fiber probe to the bowel tissue before the induce ischemia, at the end

of the induced ischemia, and after the reperfusion. The data acquired before the ischemia

were used as a control group. Without occluding the superior mesenteric artery, the

spectra were acquired on the bowel with the same time intervals of the experiments were

used as a sham group (n ¼ 5). Subsequently, the same bowel segments were sent for his-

topathologic examination.

Results: Based on the correlation between the spectra acquired from the bowel segments and

the results from the histopathologic investigation, DRS is able to differentiate the histo-

pathologic grading that appearswhen theChiu/Park score�5 (i.e., high-level ischemic injury)

than Chiu/Park score <5. Eight out of nine low-level ischemic injury tissue samples were

correctly definedusing the spectroscopic classification system.All elevenhigh-level ischemic

injury tissues that were histopathologically assigned grade 5 and above were correctly

defined using the spectroscopic classification system in the ischemiaereperfusion groups.

Conclusions: DRS could potentially be used intraoperatively for the assessment of bowel

viability in real time. These preliminary findings suggest that DRS has the potential to

reduce unnecessary resection of viable tissue or insufficient resection of nonviable tissues

may reduce the mortality and morbidity rates of intestinal ischemiaereperfusion as acute

mesenteric ischemia.

ª 2014 Elsevier Inc. All rights reserved.

hysics, Faculty of Medicine, Biomedical Optics Research Unit, Akdeniz University, Antalya90 242 227 4482.. Canpolat).

ier Inc. All rights reserved.

j o u r n a l o f s u r g i c a l r e s e a r c h 1 9 1 ( 2 0 1 4 ) 9 1e9 892

1. Surgical relevance

which requires the intravenous administration of fluorescent

Current approach to mesenteric ischemia comprises the

resection of nonviable intestinal segments. But, clinical

assessment of it is frequently deficient in certain prediction of

bowel viability.

Real time spectroscopic technique has the potential for

comprehensive and intraoperative assessment of tissue

viability based on alteration of tissue oxyhemoglobin and

deooxyhemoglobin contents. This case study was conducted

using rats to assess the effectiveness of spectral data in dif-

ferentiation between necrotic and viable intestine tissues by

comparing the results of histopathology. Our findings show

that spectroscopic measurements are able to assess the

viability of intestinal tissues successfully.

This experiment may be of interest to clinical researchers

as the rat acute mesenteric ischemia model provides a step

forward for clinical studies in humans. In the future, we

believe that diffuse reflectance spectroscopy (DRS) can pro-

vide accurate prediction of bowel viability in the surgical

treatments of acute mesenteric ischemia in real time

intraoperatively.

Fig. 1 e Absorption spectra of oxyhemoglobin and

deoxyhemoglobin. (Color version of figure is available

online.)

2. Introduction

Acute mesenteric ischemia is a relatively rare life-threatening

emergencywithan incidenceof 1 in 1000patient admissions to

hospitals, but this condition has a mortality rate of 60%e80%.

The etiologic factors that contribute to the formation of acute

mesenteric ischemia include emboli (i.e., in 40%e50% of

cases), arterial thrombus (in 25%e30% of cases), nonocclusive

ischemia (i.e., in 20% of cases), and venous thrombus (i.e., in

10% of cases). The overall survival time of patients with acute

mesenteric ischemia is affected by the specific etiologic factors

contributing to thedevelopment of the condition, thepresence

of concomitant diseases, and the characteristics of the sub-

sequent surgical excision [1e4]. Current approaches to

mesenteric ischemia include the resection of necrotic intesti-

nal tissue and anastomosis to reestablish blood flow through

the bowel. However, in some cases, despite the reestablish-

ment of blood flow through the intestinal segments, the

viability of a bowel segment is still unclear [5,6]. The previous

studies suggested that if viability of the bowel is doubtful, it

should not be resected for preserving as much bowel as

possible at the initial operation because of avoiding the short

bowel syndrome. Surgeons should decide the resection of the

bowel segments at the second look within 24e48 h after the

first operation [7,8]. Therefore, accuracy of the assessment of

bowel viability in the first operation is important to avoid

additional surgeries.

Currently, clinical findings (e.g., unaided visual color

inspection, peristaltic activation, and arterial pulsations) are

used to investigate the viability of the intestinal tissues. In

general, two methods have historically been used to intra-

operatively assess the viability of bowel tissue. The first

method is Doppler ultrasound, which has a low accuracy

because of the blood flow that is still present in the ischemic

tissue [9]. The second method is a fluorescence technique,

molecules into the blood stream and should be used within

washout time of injected fluoresce molecule. Both these cur-

rent methods have limitations [10e17]. In addition, near

infrared spectroscopy has been subject of research interest of

several groups to measure tissue oxygen saturation [18e20].

In this study, we investigated correlations between DRS

measurements, and pathology results were used to assess

intestinal ischemicereperfusion injury. DRS data were

acquired from the ischemic-reperfused intestine tissues

without using any extrinsic contrast agent. In the ischemic

tissue, the concentration of oxyhemoglobin decreases,

whereas the concentration of deoxyhemoglobin increases.

The absorption spectrum of oxyhemoglobin differs from that

of deoxyhemoglobin as illustrated in Figure 1. Therefore, the

back reflection spectrum of ischemic tissue should be

different than that of normal tissue. As seen in Figure 1, the

difference between the absorption spectra is highest at

wavelengths 560 nm and 577 nm (i.e., the two black vertical

lines in Fig. 1). As seen in Figure 1, the ratio, R ¼ absorption

(560)/absorption (577) is <1 for oxyhemoglobin and >1 for

deoxyhemoglobin absorption spectra. In this study, the ratio

of the absorption of these two wavelengths in the studied

tissue is compared with the results of histopathology to

determine the potential for using this ratio as a parameter to

assess the viability of intestinal tissues.

3. Materials and methods

This study conformed to the Guide for the Care and Use of

Laboratory Animals as published by the US National Institutes

of Health (NIH Publication No. 85-23 revised, 1985) and was

approved by the local Committee on Animal Research Ethics

at the Akdeniz University (No: 2011.09.01) Twenty-five Spra-

gueeDawley rats with weights of 250 � 25 g were used in this

study. Animals were bred and reared in the Research Animal

Facilities of Akdeniz University, School of Medicine, Turkey.

The rats were kept in polycarbonate cages under the following

conditions: a controlled ambient temperature of 22�Ce25�C,automatically adjusted humidity (i.e., 45%e50%), and 12:12 h

Fig. 3 e Spectra acquisition by gently touching the tip of the

optical fiber probe to the intestinal tissue. (Color version of

figure is available online.)

j o u r n a l o f s u r g i c a l r e s e a r c h 1 9 1 ( 2 0 1 4 ) 9 1e9 8 93

darkelight cycle. The animals were fed standard rat chow and

given tap water ad libitum. The rats were not given food for

12 h before the experiment but were allowed continued access

to water. All the rats were anesthetized via a 1.2 g/kg urethane

intraperitoneally. Then, the abdominal area was shaved and

cleaned with 10% polyvinylpyrrolidone-iodide (Isosol; Merkez

Pharmaceuticals, _Istanbul, Turkey).

3.1. Spectroscopy system and data acquisition

In this study, back reflectance spectroscopy was used to

evaluate the intestinal tissue. The spectroscopy system con-

sisted of a miniature spectrometer (USB200; Ocean Optics,

Dunedin, FL), an optical fiber probe to deliver the light to and

from the tissue (Back reflection probe; Ocean Optics), a white

light source (HD200; Ocean Optics), and a laptop computer.

The optical fiber probe contained seven optical fibers with a

core diameter of 400 nm; the seven optical fibers were ar-

ranged with one at the center with the other six surrounding

it. The six surrounding fibers deliver the light to the tissue, and

the central fiber detects the diffuse light reflected back from

the tissue. Figure 2 depicts a schematic illustration of the

optical fiber system.

The tip of the optical fiber probe gently contacted to the

intestine tissue for the DRS measurements (SR[l]) in vivo as

seen in Figure 3 for 2 s. Diffuse reflectance spectra were ac-

quired from the intestinal tissues before the ischemia, at the

end of the ischemia, and 30 min after reperfusion. Before

acquiring data from the intestine tissues, two spectra were

measured for the system calibration. The first one was back-

ground spectrum (SB[l]) measured from the tissues when the

light source was turned off in ambient light condition. The

second spectrumwas taken to define the spectral distribution

of the light source, where the probe was placed nearly 1 cm

above a diffuse reflectance standard (WS-1-SL; Ocean Optics)

and the spectral distribution of the light source (SB[l]) was

measured. The measured tissue spectra were corrected for

the wavelength dependence of the system components. The

corrected spectrum is

SðlÞ ¼ SRðlÞ � SBðlÞSSðlÞ � SBðlÞ (1)

Fig. 2 e Schematic illustration of the spectroscopic system

used in the study. (Color version of figure is available

online.)

We defined optical density of the corrected spectrum

asdln(S[l]), where, the optical density has two components:

absorption and scattering. Absorption coefficients of light in

the wavelength range between 550 and 600 nm are much

higher than the scattering coefficients because of strong

hemoglobin absorption band. Therefore, we neglect scattering

component of the tissue and define absorption spectra of the

tissues as A(l) ¼ -ln(S[l]).

3.2. Operative procedure

All rats randomly divided into five groups (n ¼ 5 animals per

group). One of them was a sham group, and other four were

ischemic groups. Periods of the ischemia time were 30, 45, 60,

and 90 min. A laparotomy was performed with a 2 cm

midline incision on each rat. After the laparotomy, all

intestinal segments were taken out in the abdominal cavity.

The superior mesenteric artery (SMA) was revealed and

occluded by a bulldog clamp (AA 066/03; Nopa, Tuttlingen,

Germany) as described elsewhere [21]. Intestinal ischemia

was confirmed by absence of the pulsations in SMA and

emerging of pale intestinal segments then the intestinal

segments were returned to the abdominal cavity. Physiolog-

ical saline (1 cc) was administered intraperitoneally, and the

abdomen was closed with continuous 2-0 silk suture to

minimize insensible fluid and heat losses. After the defined

time of ischemia period, the abdomen was reopened and

intestinal perfusion for 30 min was provided by removing the

bulldog clamp on the SMA as described elsewhere [22e26].

Then, 1 cc of physiological saline was administered intra-

peritoneally once more, and the abdomen was closed with

continuous 2-0 silk suture to keep fluid volume and the body

temperature stable during the period of reperfusion time.

After 30 min reperfusion period, the abdomen was reopened

to perform DRS measurements and resection of the 1 cm

region of interest intestinal segment 10 cm away from the

ileocecal valve for histopathology examination [27,28]. In the

sham group, same surgical procedures were performed

without SMA occlusion.

At the end of the study, each rat was sacrificed by cardiac

exsanguination.

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3.3. Histopathologic examination

The tissues were fixed with 10% formalin and processed using

standard histologic techniques, including dehydration and

paraffin embedding. The tissues were then cut into 4-mm

sections and stained with hematoxylin and eosin. Injury

caused by ischemiaereperfusion was evaluated under light

microscopy by a single pathologist in the Department of Pa-

thology, Faculty of Medicine, Akdeniz University. Tissue

injury was graded according to the 8-point Chiu/Park score

[4,5,29] as follows: 0dnormal mucosa, 1dsubepithelial space

at the tips of the villi, 2dextended subepithelial space,

3dmassive epithelial lifting down the sides of the villi, 4dvilli

denuded of the epithelium, 5dloss of villi themselves, 6dthe

intestinal crypt layer also affected, 7dtransmucosal infarc-

tion present, and 8dtransmural infarction. As examples of

this grading score, typical photographs of grades 2, 4, 6, and 8

are shown in Figure 4.

3.4. Statistics

To compare each variable between groups, Student t-test

including Levene test for comparison of variances, and Man-

neWhitney U test were usedwhere appropriate. All tests were

2-sided, and P < 0.05 was considered to be significant.

4. Results

The average absorption spectrum for normal intestinal tissue

was calculated over the five rats DRS measurements just

before the ischemia and used as a control group is shown in

Figure 5A. The absorption spectra of the tissues after 30, 45, 60,

and 90 min of ischemia are given in Figure 5B. Without

occluding the SMA, the spectra were acquired on the bowel

Fig. 4 e Histopathologic changes of the intestinal mucosa and th

under light microscopy. (A) Small bowel biopsy with detachme

indicate the presence of subepithelial space at villus tips (Chiu/

denudation (arrow); denuded villi with erythrocytes in some ca

together with damaged crypts (asterisk) (Chiu/Park grade 6); (D)

[A] 3 200, [B and C] 3 100, [D] 3 50). (Color version of figure is a

with the same time intervals of the experiments were used as

a sham group. There was no difference in the absorption

spectra between the control and sham groups.

The average absorption spectrum of normal intestinal tis-

sues is similar to that of oxyhemoglobin because of the high

oxygen saturation of the tissue. In contrast, the lack of

oxyhemoglobin concentration in the ischemic tissue results in

absorption spectra from the ischemic tissue that is similar to

the absorption spectrum of deoxyhemoglobin as shown in

Figure 1.

Thirty minutes after reperfusion, diffuse reflectance

spectra were acquired from the formerly ischemic intestinal

tissues of the rats. In Figure 6, comparisons between the ab-

sorption spectrum of the normal intestinal tissue and that of

the reperfused tissues after 30, 45, 60, and 90 min of induced

ischemia are shown. Each absorption spectrum in Figure 6

was averaged over the 16 spectra for each intestinal tissue

segment.

As seen in Figure 6A and B, the absorption spectra of the

reperfused tissue after 30min and 45min of induced ischemia

are similar to the absorption spectrum of the nonischemic

intestinal tissues (Fig. 5A). In Figure 6C and D, the absorption

spectra of the reperfused tissues after 60 min and 90 min of

induced ischemia are similar to the spectra of the ischemic

tissues in Figure 5B. As illustrated in Figure 5, the difference in

the spectra of tissues with normal blood flow versus ischemic

tissues is the highest at 560 nm and 577 nm. Therefore, we

chose to use the ratio (R) of the absorption at these two

wavelengths as a parameter to assess tissue viability. If R < 1,

the ischemic injury to the tissue is defined as low, that is, low-

level ischemic injury (LLI). If R � 1, the ischemic injury to the

tissue is defined as high, that is, high-level ischemic injury

(HLI). Correlations between the spectroscopic classification of

the tissues and the histopathologic grading score on the Chiu/

Park score were investigated. The best correlation between

e evaluation of intestinal injury using the Chiu/Park score

nt of the epithelium from the basal membrane; arrows

Park grade 2); (B) Small bowel mucosa with epithelial

pillaries (Chiu/Park grade 4); (C) Digestion of intestinal villi

Transmural infarct (Chiu/Park grade 8). (Magnifications

vailable online.)

Fig. 5 e (A) Average absorption spectrum of normal

intestine tissue in control group, (B) Average spectra of

intestinal tissue after 30, 45, 60, and 90 min of ischemia.

(Color version of figure is available online.)

j o u r n a l o f s u r g i c a l r e s e a r c h 1 9 1 ( 2 0 1 4 ) 9 1e9 8 95

the spectroscopic classification and the histopathologic

grading appears when the Chiu/Park score is �5 (i.e., HLI). As

seen in Table 1, all tissue samples are histopathologically

graded as LLI in the sham and 30 min ischemia groups. After

45 min of induced ischemia, three of the five tissues were

graded as LLI, and the remaining two were classified as HLI. In

contrast, only one out of five of the tissue samples was his-

topathologically graded as LLI, whereas the remaining four

were graded as HLI after 60 min of induced ischemia;

Fig. 6 e Comparison of absorption spectra from normal and isc

ischemic, (C) 60 min ischemic, and (D) 90 min ischemic. After th

tissues, and new spectra were acquired. (Color version of figure

moreover, all the five tissue samples were graded as HLI after

90 min of induced ischemia.

Table 2 compares the spectroscopic classification of the

tissues to the histopathology scoring. Good correlation was

observed between the histopathology and spectroscopy

results. Eight out of nine LLI tissue samples were correctly

defined using the spectroscopic classification system in the

ischemia groups. All 11 HLI tissues that were histopathologi-

cally assigned grade 5 and above were correctly defined using

the spectroscopic classification system. These results showed

a positive correlation between DRS and histopathologic

results (P < 0.001 and r ¼ 0.91).

5. Discussion

The intraoperative prediction of bowel viability is a critical

matter in the treatment of acute mesenteric ischemia. Sur-

geons usually face a very difficult dilemma. An extensive

segment resectionmay result in short bowel syndrome. Yet, if

an insufficient resection is performed, then infarction of the

remaining bowel could lead to sepsis and multiple organ

failure. Therefore, in the event of borderline bowel ischemia,

the resectionmargin is themost important factor contributing

to postoperative mortality and morbidity [1]. Clinical assess-

ments are generally subjective, have often low specificity, and

are thus inaccurate [13]. In the literature, numerous tech-

niques have been described to predict or measure bowel

viability. Some of these techniques, such as fluorescein and

Doppler studies, have gained widespread acceptance and

clinical applicability. Doppler techniques either with ultra-

sound or laser are simple and noninvasive. However, some

disadvantages of Doppler techniques include high false posi-

tive to false negative ratios and false blood flow readings due

to signals from adjacent large vessels. In the intraoperative

assessment of blood flow, laser Doppler flowmetry presents

the problems of being sensitive to motion due to the patient’s

heart beat or respiration, which often results in recording

hemic-reperfused tissues (A) 30 min ischemic, (B) 45 min

e ischemia, 15 min of reperfusion was applied to all the

is available online.)

Table 1 e Histopathologic grading results.

Length of ischemia(min)

Grade (number of samples)

0 1 2 3 4 5 6 7 8

Sham 0 2 2 1 0 0 0 0 0

30 0 0 0 0 5 0 0 0 0

45 0 2 0 0 1 1 1 0 0

60 0 0 1 0 0 3 1 0 0

90 0 0 0 0 0 0 2 1 2

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artifacts [9]. The fluorescein technique ismore predictive than

the Doppler techniques, but this technique requires the

administration of an intravenous fluorescent agent and a UV

light source. Additionally, this technique takes longer because

of fluorescein’s long half-life time [12,13]. Therefore, inputs for

this technique are not reproducible and also impractical for

surgeons. Near infrared spectroscopy has been used to assess

viability of ischemic intestinal tissue based on relative

changes in the absorption spectra of hemoglobin in several

studies [18e20]. However, a correlation between the relative

change in the oxyhemoglobin absorption spectra and the

histopathologic assessment of tissue viability has not been

reported to date.

In this study, a correlation between the DRS spectra of the

tissue and the tissue viability is assessed by histopathology.

That is to say, a spectroscopic parameter is defined to distin-

guish HLI tissues from LLI tissues and compared with the

histopathologic assessment of the tissues. As reported in the

results section, the absorption spectra of the reperfused tissue

after 30 min and 45min of induced ischemia are similar to the

absorption spectrum of the nonischemic intestinal tissues,

and the absorption spectra of the reperfused tissues after 60

and 90 min of induced ischemia are similar to the spectra of

the ischemic tissues (Figs. 5 and 6). These results indicate that

if the time of ischemia is <45 min, the redistribution of blood

within the intestinal tissues is similar to that of the non-

ischemic tissue. However, if the ischemia time is >60 min, the

redistribution of blood within the intestinal tissues is not

similar to nonischemic tissues, and flow in the reperfused

tissue is blocked as the ischemic tissue. Moreover, DRS was

used to correctly predict the viability of the bowel tissue as

Table 2 e Comparison between the histopathologic and spectr

Length of ischemia (min)

Grade Rat 1

Sham Histopathologic LLI

Spectroscopic O

30 Histopathologic LLI

Spectroscopic O

45 Histopathologic HLI

Spectroscopic O

60 Histopathologic HLI

Spectroscopic O

90 Histopathologic HLI

Spectroscopic O

The histopathologic and spectroscopic results are consistent with except

assessed by a histopathologic examination of the tissue

samples.

There are several previous ischemiaereperfusion injury

studies in the rat model with reperfusion times of 60e90 min

[21,23]. However, Boros et al. [24] used 30 min of reperfusion

time to assess the intestinal tissue injury after the ischemia.

In earlier studies, similar designs were also used for assess-

ment of the injury [25,26]. Similarly, we chose to use 30 min

of reperfusion time to assess the intestinal ische-

miaereperfusion injury because it may have practical

importance in the operating room allowing the surgeons to

evaluate the injury in 30min rather than 60e90min during the

operation. The ideal method in the prediction of bowel

viability after an intestinal ischemiaereperfusion injury

would offer the following features: feasibility, easy handling,

accuracy, simplicity, objectivity, reproducibility, and cost

efficiency [30]. In the present study, the use of DRS after

ischemiaereperfusion injury in the rat intestine was evalu-

ated in vivo and real time. It has been shown that there is a

good correlation between the spectroscopic parameter (R) and

histopathologic classification. DRS system has the potential to

define whether the ischemic injury is lower or higher Chiu/

Park score 5. This may help a surgeon in the assessment of

ischemiaereperfusion injury objectively at the time of oper-

ation in vivo.

Consequently, we identified several major advantages.

First of all, DRS is portable and allows the probe to be easily

positioned on the intestinal surface due to its flexibility. Sec-

ond, a very small area (i.e., 1.3 mm2) of bowel surface is

examined by the optical fıber probe. Assessment of local tis-

sue area may be achieved because of small surface area of the

optical probe,without being effected from large vessels. Third,

DRS system is not motion sensitive during data acquisition.

Several studies have reported that ischemia in the anas-

tomotic healing area is an important risk factor for anasto-

motic dehiscence. During the anastomosis, the blood supplies

of anastomotic sides may become ischemic because of over

dissection of marginal arteries. Therefore, a small area of

demarcation at suture line has a more potential risk factor for

anastomotic dehiscence [31,32]. Moreover, this technique has

the potential to assess small demarcation areas of perfusion

and the suture line of anastomosis after resection.

oscopic grading results.

Results

Rat 2 Rat 3 Rat 4 Rat 5

LLI LLI LLI LLI

O O O O

LLI LLI LLI LLI

O O O O

HLI LLI LLI LLI

O O O O

HLI HLI LLI HLI

O O X O

HLI HLI HLI HLI

O O O O

ion to one sample (i.e., 60-mineinduced ischemia) in rat 4.

j o u r n a l o f s u r g i c a l r e s e a r c h 1 9 1 ( 2 0 1 4 ) 9 1e9 8 97

Fourth, the spectroscopic technique developed in this

study has the potential for intraoperative assessment of the

injury of human ischemic intestinal tissues in real time

without the need to inject a contrast agent. The system is

completely safe for the patients because visible light is used in

the measurements. Because the DRS system detects back

reflected light from the tissue, acquired spectra do not depend

on the thickness of the tissues. Lastly, acquiring a spectrum

from intestinal tissue takes <2 s and allows the surgeon to

scan a large portion of intestinal tissue in a short time.

Acknowledgment

Author contributions: B.K. and M.C. contributed to the

conception and design of the study. B.K. and A.S.-K. con-

ducted the experiments and collected the data. O.E. prepared

the histologic slides and evaluated the article. A.S.-K., B.K.,

M.C., and O.E. analyzed and interpreted the results. B.K., A.S.-

K., O.E., and M.C. wrote the article. This work was supported

by Akdeniz University Scientific Research Units, Antalya,

Turkey.

Disclosure

The authors report no proprietary or commercial interest in

any product mentioned or concept discussed in the article.

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