A Comparative Study on the Protection Profile of …cancerres.aacrjournals.org/content/canres/65/22/10486.full.pdf · A Comparative Study on the Protection Profile of Lidocaine, Amifostine,

A Comparative Study on the Protection Profile of Lidocaine,

Amifostine, and Pilocarpin on the Parotid Gland

during Radiotherapy

Samer G. Hakim,1Hartwig Kosmehl,

4Isabel Lauer,

2Roger Nadrowitz,

2

Thilo Wedel,3and Peter Sieg

1

Departments of 1Maxillofacial Surgery and 2Radiotherapy and Nuclear Medicine, University Hospital Schleswig-Holstein; 3Instituteof Anatomy, University of Luebeck, Luebeck, Germany; and 4Institute of Pathology, Helios Medical Centre, Erfurt, Germany

Abstract

The aim of this study was to evaluate the individual and thesynergetic radioprotective effect of lidocaine, amifostine, andpilocarpin on the parotid gland. Forty-nine rabbits wererandomized into seven groups (n = 7)—control, irradiatedsham-treated, irradiated/lidocaine–pretreated, irradiated/amifostine–pretreated, irradiated/pilocarpin–pretreated, ir-radiated/lidocaine + pilocarpin–pretreated, and irradiated/amifostine + pilocarpin–pretreated groups. One week beforeirradiation (15 Gy) and 72 hours as well as 1 month afterward,the parotid gland was investigated morphologically, sialoscin-tigraphically, and immunohistochemically with the use oftenascin-C and A smooth muscle actin. Compared with con-trol animals, there was a significant reduction of the salivaryejection fraction in the irradiated untreated group 72 hoursfollowing radiation. Only animals pretreated with lidocaine oramifostine (alone or combined with pilocarpin) showed aslight nonsignificant reduction of salivary ejection fraction.Immunohistochemically, we observed a significant loss of Asmooth muscle actin and an up-regulation of tenascin-Cexpression in irradiated/untreated glands. These changeswere less evident in animals pretreated with lidocaine orlidocaine + pilocarpin. Amifostine and pilocarpin did notshow any influence on tenascin-C or A smooth muscle actinexpression. Ultrastructural damage was observed in irradiateduntreated and pilocarpin–pretreated glands. In contrast,lidocaine and amifostine could largely preserve the glandularultrastructure. One month postradiation, all changes wereregressive regardless of treatment protocol. Potential radio-protective agents show different effects on both morphologyand function of the parotid gland. Associated immunohisto-chemical and ultrastructural findings could prove the pre-vailed protection profile of lidocaine. This may provide aprophylactic approach in the field of radioprotection ofsalivary glands. (Cancer Res 2005; 65(22): 10486-93)

Introduction

Xerostomia (dry mouth) is a common symptom followingradiotherapy of head and neck malignancies, which indicatesdiminished salivary gland output. Although the degree of salivary

gland dysfunction and related p.o. complications is influenced byindividual patient characteristics, such as inherent salivary glandactivity, age, and sex (1, 2), the most important role is played by thevolume of irradiated salivary gland tissue (3). It is known that theparotid gland represents the most radiosensitive salivary gland,causing significant xerosomia if more than half of both glands wereirradiated (4). Noteworthy is that saliva reduction becomesmanifest during the first week of conventional fractionatedradiotherapy (usually after 10-15 Gy) and persists during the restof the patient’s life (5, 6).To avoid this distressing side effect, many therapeutic proce-

dures have been introduced to increase the tolerance of the parotidglands during radiotherapy, including either depletion of secretorygranula by muscarinic M3 receptors agonists (7) and isoproterenol(8) or recently by pretreatment with the free radical scavengeramifostine (9). A single clinical trial on a further protection ofradiogenic sialadenitis and mucositis by coumarin/troxerutine wasalso reported by Grotz et al. (10). Among the so-called preemptivedrugs, pilocarpin has gained ground, not only in experimentalstudies (11) but in clinical trials as well (12, 13). Postulating thehypothesis of basolateral membrane damage as an initial target siteof radiation, Stephens et al. (14) suggested a further prophylacticapproach, which has been proven to reduce this damage in cellculture, namely the membrane stabilization agent lidocaine.Besides the sialoscintigraphic changes (15), early immunohisto-chemical changes such as a smooth muscle actin reduction (loss ofmyoepithelial differentiation) and tenascin-C matrix remodelinghave been described recently following radiation and have provencorrelation with functional impairment (16, 17), indicating thatinitial damage of endpiece epithelium could be identifiedimmunohistochemically.The aim of the current study was to investigate the potential

radioprotective effect of lidocaine, amifostine, and pilocarpin and thesynergetic effect of lidocaine/pilocarpin as well as amifostine/pilocarpin on the parotid gland. As variables to evaluate the gradeof damage or protection, we used functional sialoscintigraphy,histologic, immunohistochemical, and ultrastructural investigations.

Materials and Methods

Animals and study design. Forty-nine healthy female New Zealandrabbits of 2.5 to 3.5 kg weight were used for the study. They were purchasedfrom Charles-River-Wiga (Sulzfeld, Germany) and kept under laboratoryconditions and alternating 12 hours day/night rhythm. All animals wereacclimatized for at least 2 weeks before starting the study and thenrandomized into seven groups (n = 7)—control (sham-treated/unirradiated),irradiated/sham-treated, irradiated/lidocaine–pretreated, irradiated/ami-fostine–pretreated, irradiated/pilocarpin–pretreated, irradiated/lidocaine +pilocarpin–pretreated, and irradiated/amifostine + pilocarpin–pretreated

Requests for reprints: Samer G. Hakim, Department of Maxillofacial Surgery,University Hospital Schleswig-Holstein, Campus Luebeck, Ratzeburger Allee 160, 23538Luebeck, Germany. Phone: 49-451-500-2266; Fax: 49-451-500-4188; E-mail:[email protected].

I2005 American Association for Cancer Research.doi:10.1158/0008-5472.CAN-05-0023

Cancer Res 2005; 65: (22). November 15, 2005 10486 www.aacrjournals.org

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groups. All experimental procedures were approved by the local authorityaccording to the current German law on the protection of animals.

Animals underwent a first scintigraphy as described below. Except forthe control group, all rabbits were irradiated 1 week later with a single dose

of 15 Gy. Pretreatment groups received the drug before irradiation

according to the related pharmaceutical features of each substance. Both

procedures were done under general anesthesia using a combination of3 mg/kg (S)-ketamine hydrochloride (Ketanest-S) and 0.1 mg/kg xylazine

hydrochloride (Rompun). Seventy-two hours after irradiation, a second

scintigraphy was done with a subsequent biopsy of the unilateral parotid

gland for immunohistologic and ultrastructural examination. The secondscintigraphy was carried out 30 days after irradiation along with a

subsequent biopsy of the contralateral parotid gland.

Drugs. Lidocaine hydrochloride (Xylocain, 2%, 1 mg/kg) was slowly

administered i.v. 10 minutes before irradiation via a marginal ear vein.

Amifostine (Ethyol) was administered according to the recommended

dose of 200 mg/m2 15 minutes before irradiation via a marginal ear vein

as well. Pilocarpin hydrochloride (Pilomann, 1% EDO) was administered

i.p. (4 mg/kg) 90 minutes before radiation.

Irradiation. Under general anesthesia, X-ray irradiation was done usingMEVATRON 74-Siemens teletherapy unit (photon energy) operated at

10 MeVwith a dose rate of 3 Gy/min. The output of accelerator was 1 Gy = 80

MU at source-to-skin distance of 98 cm. The rabbit was placed laterally andcovered with a 1-cm-thick tissue equivalent bolus material. An axial beam

was directed toward the head of the rabbit, extending from the retroauricular

region into the tip of the nose ( field size 7.5 � 10 cm), thus including all

potential localization of salivary gland tissue. A single dose of 15 Gy wasapplied by two opposing X-ray tubes. Dose rate was determined using a

thermoluminescence dosimeter.

Tissue preparation and immunohistochemistry. The biopsy was

always provided from the mediocaudal portion of the parotid gland as

previously described (15, 16); the volume of tissue was kept at a minimum

(5 � 5 � 5 mm) to avoid injury of major blood vessels, nerves, or excretory

ducts, which may, in turn, manipulate subsequent scintigraphic results due

to neuronal and vascular impairment or secretory retention. Samples were

then fixed in neutral phosphate-buffered 4% formalin. After a minimum of

48 hours of fixation, the tissue was trimmed and processed by standard

paraffin-embedding methods. Sections were cut at 4 Am, deparaffinized,

and then stained with H&E to obtain conventional histologic sections.

For immunohistochemical staining, the alkaline phosphatase–antialka-

line phosphatase (APAAP) technique was used to visualize the primaryantibodies, namely monoclonal mouse antibodies directed against

a-smooth muscle actin (clone 1A4, diluted 1:40, DakoCytomation, Glostrup,

Denmark) and monoclonal mouse anti-human tenascin (BC4; Dr. L. Zardi,

University of Genoa, Genoa, Italy). The APAAP technique was used forvisualization of the bound primary antibodies. The secondary rabbit anti-

mouse antibody and the APAAP complex were diluted 1:50 (both from

DakoCytomation). Naphtol-AS-biphosphate (Sigma, Seelze, Germany) and

new fuchsin (Merck, Darmstadt, Germany) were used as substrate anddeveloper, respectively. As negative control, the primary antibody was

replaced by a nonimmune serum.

To evaluate the degree of a smooth muscle actin loss per acinus in

irradiated glands, a semiquantitative score was introduced to describe the

relative decrease of immunostaining around the acini compared with the

labeling pattern in the control group. The score was defined as follows: 0, no

a smooth muscle actin loss (no difference to control glands); +, 0% to 25%

loss of a smooth muscle actin; ++, significant a smooth muscle actin loss

>50%; +++, >75% a smooth muscle actin loss.

For the evaluation of tenascin-C reaction, 10 view fields of each sample in

every group were chosen at random and the area of positive reaction in

relation to the whole view field was calculated using the professional Soft

Imaging System software ANALYSIS. Values were expressed as mean F SE.

Areas without acinar cells ( fatty tissue) were not considered for evaluation.Transmission electron microscopy. Samples were immediately fixed

by immersion in 0.1 mol/L cacodylate buffer containing 2.5% glutaraldehyde

and 2% paraformaldehyde (pH 7.4) for 24 hours. The specimens were

postfixed in 1% OsO4 and stained en bloc with 2% uranylacetate. After

dehydration in graded alcohols, the specimens were embedded in Araldite.Semithin sections were stained with methylene blue and azure II to

visualize the regions of interest containing acinar and myoepithelial cells as

well as glandular ducts. Ultrathin sections were cut and stained with lead

citrate and examined under an electron microscope (EM 109, Zeiss,Oberkochen, Germany). The findings were recorded both by conventional

films and a digital imaging system.

To evaluate the ultrastructural alterations induced by radiation, the

following variables were assessed: intracellular edema, nuclear alterations(size and distribution pattern of chromatine), alterations of cytoplasmic

organelles (swelling, disintegration, and dissolution), alterations of secretory

vesicles (size, shape, and electron density), and the degree of secretion

congestion. The findings were recorded by a semiquantitative score asfollows: 0, normal; +, minor alterations; ++, moderate alterations; +++, major

alterations.

Salivary gland scintigraphy. After i.v. application of 100 MBq 99mTcO4�,

the rabbits underwent a sequential scintigraphy in a prone position and

basal projection of the head, using a single-head g camera (Picker CX 250

compact, LEHR collimator, field-of-view 25 cm). At the 20 minutes, 0.01 mg/

kg Carachol was applied s.c. to stimulate saliva secretion and thescintigraphy was continued for a further 25 minutes. Dynamic studies

were acquired with 90 frames and 30 seconds per frame in 256 � 256 matrix

with zoom 4. Percentage uptake of the administered activity was calculated

for the 10 to 19 and 36 to 45 minutes by summation of the appropriateframes and regions of interest of the parotid glands. Time-activity curves

were registered and analyzed.

The second scintigraphy was always done 1 week after the first one to avoidany interaction or uptake disturbance by the radiotracer injected previously.

The salivary ejection fraction has been defined as the activity excretedwith

the radiolabeled saliva of the parotid gland after Carbachol stimulation

expressed as percent: salivary ejection fraction (%) = maximal uptake (till 20minutes) � remaining activity (after 45 minutes) / maximal uptake of the

gland.

Data and statistical analysis. Both parotid glands were evaluated in

every animal scintigraphically and immunohistochemically. When provedto follow a normal distribution, values of salivary ejection fraction and

tenascin-C reaction were expressed as mean F SE and the two-sided t test

for paired/unpaired samples was used to compare data obtained prior and72 hours as well as 1 month after irradiation. The Kruskal-Wallis test for

unpaired samples was used to evaluate the difference of immunohisto-

chemical reaction of a smooth muscle actin among groups. An a level of

P < 0.05 was considered to be significant. Electron microscopic evaluationwas only descriptive and underwent no statistical test.

Results

Scintigraphic data. Before irradiation, the initial tracer uptakeof the parotid gland in all groups ranged from 0.08% to 0.16% ofapplied activity. After secretion stimulation with carbachol, theremaining activity decreased significantly from 0.03% to 0.14%. Thecalculated salivary ejection fraction for the parotid gland beforeirradiation was 38.28 F 11.7%.Three days following radiation of control glands, the primary

tracer uptake decreased and the ability of excretion after carbacholadministration and thus salivary ejection fraction underwent asignificant reduction up to 19.11F 12.93% (irradiated sham-treatedgroup; P = 0.03). No significant difference could be observedbetween the salivary ejection fraction prior and 72 hours followingirradiation in animals pretreated with amifostine alone (P = 0.78),indicating intact salivary gland function. Compared with theamifostine group, glands pretreated with lidocaine showed a lowergrade of protection after 72 hours (salivary ejection fractionpreradiation = 34.38 F 10.65%; salivary ejection fraction post-radiation = 28.05 F 18.34%). However, there was no significantreduction of salivary ejection fraction (P = 0.16). Animals

Radioprotection of Salivary Glands

www.aacrjournals.org 10487 Cancer Res 2005; 65: (22). November 15, 2005



pretreated with pilocarpin alone displayed a similar reduction ofsalivary ejection fraction like irradiated sham-treated animals (P =0.02). In the combination groups, lidocaine/pilocarpin as well asamifostine/pilocarpin, we could observed a preservation of thesalivary ejection fraction (P = 0.77 and P = 0.15, respectively). Therewere no significant differences of the primary tracer uptake after72 hours among irradiated pretreated glands regardless of thedrug(s) administered (data not shown).One month after radiation, the primary uptake and the salivary

ejection fraction of all groups, including the irradiated sham-treated group, met normalization and values measured beforeirradiation were not significantly different (Table 1).Overall histomorphology. Compared with control glands, the

main feature observed in irradiated groups was a secretoryretention and an intracellular edema with a consequent ruptureof cell membrane especially on the luminal side. The cellularborders seemed diminished and it was not possible to delimitadjacent cells. Scattered vacuolopathy and anisonucleosis wasnoticed in acinar and ductal cells as well. The secretory retentionand vacuolopathy underwent a slight regression throughout theobserving period in control and experimental groups; however, noevaluable differences could be assessed among the differenttherapy groups neither 72 hours nor 30 days following irradiation.The myoepithelial cells were not always distinguishable and theirevaluation was not reliable.Tenascin-C. Tenascin-C distribution in unirradiated control

parotid gland displayed a circular pattern around blood vessels; inthe parenchyma, it was limited to the basal membrane of theintercalated and secretory ducts and some scattered acinar cells.The staining evaluated digitally amounted to 17 F 8% of view field(Fig. 1A). Irradiated/sham-treated glands investigated 72 hours later

showed a remarkable redistribution of the tenascin-C staining. Asignificant increase in the expression of tenascin-C in the acinarcells was noticed, reaching 82 F 31% of view field investigated(Fig. 1B). This up-regulation was reduced in irradiated/lidocaine–pretreated as well as irradiated/lidocaine + pilocarpin–pretreatedglands (21 F 13% and 26 F 10%, respectively; Fig. 2A). Amifostine-,amifostine + pilocarpin–, and pilocarpin–pretreated glands showedno significant difference to the irradiated sham-treated glands(76 F 19%, 80 F 23%, and 77 F 19%; Figs. 2B and C).The combination with pilocarpin had no significant effect on the

tenascin-C redistribution.We could not notice any difference among samples examined

1 month after irradiation. The glands in all groups displayednormalization and no longer significant differences to unirradiatedparotid gland tissue (Table 1).A Smooth muscle actin. In control parotid glands, myoepithe-

lial cells were stained around the acini forming a hem-like figure,whereas the myoepithelial cells of the intercalated and secretoryducts as well as blood vessels displayed a circular distribution(Fig. 1C). Irradiated/sham–treated parotid glands investigated72 hours after irradiation displayed a special pattern of staining:The myoepithelial cells of the endpieces were irregularly stainedand a lot of acinar myoepithelial cells were not stained at all(Fig. 1D); there was a significant loss of a smooth muscle actinstaining in all samples. On the contrary, the myoepithelial cellsof the intercalated and secretory ducts remained eitherunaffected or showed less impairment. Irradiated/lidocaine–pretreated glands showed no significant difference in theexpression pattern of a smooth muscle actin to unirradiatedglands (Fig. 2D). In contrast, pretreatment with amifostine orpilocarpin did not hinder massive a smooth muscle actin staining

Table 1. Scintigraphic and immunohistochemical evaluation of the parotid gland of all study groups before irradiation 72 hoursand 1 month following irradiation with 15 Gy

Preradiation After 72 h P After 1 min P

SEF (%)

Only irradiated 38.28 F 11.7 19.11 F 12.93 0.03 37.23 F 6.91 0.42

Lidocaine 34.38 F 10.65 28.05 F 18.34 0.16 26.08 F 16.39 0.19Amifostine 22.67 F 11.26 21.52 F 6.65 0.78 23.06 F 12.09 0.24

Pilocarpin 32.25 F 11.54 18.46 F 8.83 0.02 31.01 F 9.31 0.34

Lidocaine/pilocarpin 21.92 F 9.41 20.85 F 12.98 0.77 21.32 F 9.81 0.24

Amifostine/pilocarpin 24.57 F 12.14 11.70 F 3.50 0.15 16.10 F 3.45 0.32TN-C (%)

Only irradiated 17 F 05% 82 F 19% 0.002 18 F 06% 0.09

Lidocaine 19 F 02% 21 F 08% 0.33 20 F 06% 0.57

Amifostine 15 F 09% 76 F 19% 0.02 17 F 04% 0.52Pilocarpin 19 F 07% 77 F 17% 0.05 19 F 10% 0.55

Lidocaine/pilocarpin 24 F 06% 26 F 10% 0.56 26 F 08% 0.71

Amifostine/pilocarpin 18 F 07% 80 F 20% 0.01 20 F 02% 0.63ASMA (score)

Only irradiated 0 +++ <0.001 0 (+) >0.05

Lidocaine 0 0 (+) >0.05 0 (+) >0.05

Amifostine 0 ++ <0.05 + >0.05Pilocarpin 0 ++ (+) <0.001 + >0.05

Lidocaine/pilocarpin 0 0 (+) >0.05 0 (+) >0.05

Amifostine/pilocarpin 0 ++ (+) <0.001 + >0.05

Abbreviations: SEF, salivary ejection fraction; TN-C, tenascin-C; ASMA, a smooth muscle actin.

Cancer Research




loss (Figs. 2E and F). The protective effect of combined therapywas only evident for the lidocaine/pilocarpin group. One monthlater, no significant changes could be noticed in the parenchymaof various groups; however, loss of acinar cells and associatedstructures influenced the total labeled areas with a smoothmuscle actin (data not shown).Transmission electron microscopy. The ultrastructural inves-

tigations were done selectively in two glands of each monotherapygroup because drug combinations did not influence the outcome ofpretreatment.The glandular acini of untreated-irradiated animals showed a

remarkable intracellular edema, electron lucent cytoplasm, and areduction of cytoplasmic organelles. The secretory vesicles displayeda heterogeneous ultrastructure characterized by the concomitantpresence of either electron dense or electron lucent granularmaterial. In addition, several vesicles changed from round to angularshapes and frequently showed multiple intravesicular vacuolations(Fig. 3A).

In contrast, irradiated/lidocaine–pretreated glands revealed arather normal morphology at ultrastructural level. Neither relevantcellular swelling nor decrease of intracellular organelles wasobserved. The densely distributed secretory vesicles showed auniform appearance with round shapes and were filled homoge-neously with electron dense secretion material (Fig. 3B).Glandular acini derived from irradiated/amifostine–pretreated

animals basically resembled the ultrastructural morphology en-countered in the lidocaine-pretreated group. However, several acinarcells reproducibly showed intracellular edema and were poorlyequipped with cytoplasmic organelles. Moreover, a subset of secre-tory vesicles lost its round shape, became larger in size, and formedirregular borders (Fig. 3C). These alterations were most likely due toan overproduction of secretion of individual vesicles rather than to aconfluence of neighboring vesicles, as different stages ranging fromsmall- to large-sized vesicles could be observed.In comparison with both the lidocaine- and amifostine-pre-

treated groups, irradiated animals pretreated with pilocarpin

Figure 1. Immunohistologic expression pattern of tenascin-C and a-smooth-muscle actin in control unirradiated glands (A and C , respectively).Arrows, labeling of acinar cells; arrowheads, circular staining of ductal cells. B and D, changes in the expression of both antibodies in irradiated-untreatedgroup 72 hours after external beam irradiation with 15 Gy. Notice up-regulation of intracellular tenascin-C expression and massive loss of a smooth muscle actin stainingof myoepithelial cells.





showed the most severe ultrastructural alterations similar to thoseobserved in untreated radiated animals (Fig. 3A). The acinar glandswere characterized by intracellular swelling, paucity of organelles,and secretory vesicles of highly varying electron density. Whereas

some areas were filled with normally shaped vesicles of electrondense secretion material, most regions displayed clusters of denselypacked vesicles containing granular material of either minor orvery poor electron density (Fig. 3D). All findings underwent a

Figure 2. Pretreatment with lidocaine prevents overexpression of tenascin-C (A ) as well as a smooth muscle actin loss (D ). In contrast, significant changes in theexpression of both antibodies are still evident after treatment with amifostine (B and E) and pilocarpin (C and F ).

Cancer Research




semiquantitative analysis of the ultrastructural changes describedabove. The results are summarized in Table 2 and confirm thedifferent degrees of ultrastructural damage characteristics for eachexperimental group.

Discussion

Salivary gland damage associated with structural alteration andfunctional restriction is a well-known sequela of radiotherapy inthe head and neck region. Apart from partial sparing of parotidgland through three-dimensional planning of radiotherapy (18)and saliva substitution after radiotherapy, many pharmacologicalapproaches have been used, including prophylactic agents orsialogogues. The prophylactic approach became importantbecause of improving compliance of patients and its economicadvantage.To study the effect of potential radioprotective drugs on salivary

gland function, reliable variables are required. Besides the evaluationof scintigraphic findings and flow rates, previous studies alsoconsidered morphologic aspects (19–21). However, the morphologiccriteria applied to estimate the degree of damage or protection werevariable. Studies suggesting the use of amifostine in the radiopro-tection of salivary glands have indicated the grade of subsequentlipomatosis of the parotid gland as a protection variable (19). Thisfinding is, however, attributed to the late regeneration process andthus could not serve as a reliable indicator of radiation damage,

particularly when considering that the parotid gland in thisexperimental model displays naturally extensive fatty tissuedistribution. Other studies have indicated glandular weight gainand reduction of p.o. complications as a sign of radioprotection(22). Morphologic signs reported as an indicator of salivary glandprotection using pilocarpin were also rare and unspecific. Theyranged from weight loss of the studied gland to acinar cell loss(23, 24). The only report regarding the use of lidocaine for salivarygland protection was derived from Stephens et al. (14), who have

Figure 3. Transmission electronmicroscopy of glandular acini fromirradiated parotid glands withoutpretreatment (A) compared with irradiatedglands pretreated with lidocaine(B), amifostine (C ), and pilocarpin (D ).A, acinar cells are characterized byintracellular swelling, paucity ofcytoplasmic organelles (*), and secretoryvesicles containing secretion material ofdifferent electron density (arrows ). Severalvesicles display angular shapes andmultiple vacuolations (arrowheads ).B, acinar cells are densely packed withnormally configured secretory vesiclesand reveal no major morphologicalterations at ultrastructural level. C, acinarcells show moderate intracellular edema(asterisk ). Although most of the secretoryvesicles are normally formed, a subset ofvesicles is irregularly shaped and largerin size (arrows ). D, acinar cells arecharacterized by remarkable intracellularswelling, decrease of intracellularorganelles, and secretory vesicles of highlyvarying electron density ranging fromelectron dense to electron lucent granularsecretion material (arrows ). Bar, 3 Am.

Table 2. Semiquantitative evaluation of the ultrastructuralchanges of the parotid gland tissue in the monotherapygroups 72 hours following single dose irradiation with15 Gy

Cellular

edema

Nucleus Cytoplasmic

organelles

Secretory

vesicles

Secretion

congestion

Lidocaine 0 0 + 0 +

Amifostine ++ + + + +Pilocarpin +++ ++ +++ ++ +++

NOTE: 0, normal; +, minor alteration; ++, moderate alteration; +++,

major alteration.





considered the low grade of nuclear aberration as the goal effect ofpretreatment. Apart from earlier investigation assessing thecapability of local anesthetics from the amid group to stabilizeand protect the cell membrane during radiation in cell cultures(25, 26), the use of lidocaine as a radioprotective agent of the salivarygland is uncommon and has only been used in a single study onacinar cell culture (14).In the present study, we investigated the radioprotective effect of

amifostine, lidocaine, and pilocarpin as well as the combination ofamifostine + pilocarpin and lidocaine + pilocarpin in an establishedrabbit model. The aimwas to evaluate the potential synergic effect ofsubstances with direct influence on the stabilization of cellmembrane and structures like amifostine or lidocaine (8, 9, 22,25, 26) with the widely examined muscarinic alkaloid pilocarpin,which induces depletion of secretory granules in acinar cells, aprocess that was considered radioprotective in previous studies(7, 11, 12, 20). To provide comparable data, we used a single-doseirradiation of 15 Gy, the radiation modality used by the majority ofexperimental studies regarding radiation-induced impairment andprotection of salivary glands (7, 11, 15, 20, 21, 24). Further studiesdealing with the role of fractionation at the histomorphologic andfunctional level gave evidence on the negligible protective effect ofthis modification on the morphology of acinar cells at this stage.According to these studies, irradiation with 15 Gy given in a singledose would implicate acinar cell impairment similar to thatobserved using conventional or accelerated fractionation (6, 21, 27).Using this experimental model, we could show that the use of

lidocaine in the dosage recommended for interrupting ventricularfibrillation (1 mg/kg) applied 15 minutes before irradiation couldreduce the functional impairment and avoid reduction of salivaryejection fraction. The limited expression of tenascin-C observed insamples investigated 72 hours after irradiation could be attributed tothe direct stabilization effect of lidocaine on the acinar cellmembrane and extracellular matrix interaction. Immunohistologicdata derived from previous studies may explain the relationshipbetween cell membrane damage and up-regulated tenascin-Cexpression in both acinar and myoepithelial cells (16, 17, 28, 29),an aspect that was evident in our results through a smooth muscleactin preservation. The most efficient preservation of the ultra-structural morphology of irradiated glandular tissue was alsoachieved by administration of lidocaine. Both the general cellularintegrity and the structural configuration of the secretory apparatuswere not essentially altered when compared with unirradiatedglands. The fact that local anesthetics are able to sensitize the cell toradiation damage when present after irradiation indicates themembrane binding effect (30). The concentration reached intracel-lularly must, therefore, be kept at a minimum after the radiationperiod. The short half-life period of lidocaine provides anappropriate feature to avoid high postirradiation concentrations,thereby preventing permanent damage of acinar cells.Pertechnetate used as a radiotracer shares the Na+/K+/Cl�

cotransporter by the basolateral acinar cell membrane (31), which,consequently, influences the initial tracer uptake of the gland. Thisexplains the sialoscintigraphic alteration following irradiation onthe one hand and the radioprotective effect of the membranestabilization agent lidocaine and the expected suppression oftenascin-C expression on the other. Similar results were obtainedfor amifostine; only minor alterations concerning the shape and sizeof vesicles and the number of cytoplasmic organelles were observedin our study. This is in accordance with the results of other early aswell as recent experimental studies on rat and rabbit models (32)

and clinical trials, which provided encouraging results (33).Scintigraphic investigations as shown above have also proven theintact primary tracer uptake and excretion of radiolabeled saliva inpretreated acinar cells. Because tenascin-C was up-regulated in theacinar cells of amifostine-pretreated glands, a kind of cell membranealteration should be expected besides the moderate loss ofmyofilaments within myoepithelial cells. These minor cellularchanges had, however, no effect on the secretory function of thegland. To explain these results, we suggest a threshold of minimalcell membrane damage that may induce a smooth muscle actin andtenacin-C remodeling without implicating salivary function damage.The use of pilocarpin to improve the secretory function of impaired

salivary glands after radiotherapy is a common approach and alreadyin clinical use (34). Clinical and experimental trials on the prophylacticeffect of muscarinic receptor agonists, however, provided controver-sial data. Whereas pilocarpin reduced structural damage and weightloss in a rat model (20, 21), clinical trials showed no significantprotective effect (12). Our immunohistologic and ultrastructuralresults showed moderate cell damage and signs of myoepithelial celldisintegration after application of pilocarpin. The cellular alterationsweremore pronounced at ultrastructural level, in particular comparedwith both lidocaine and amifostine groups. The evidence of electronlucent granules, such as those observed in sialadenitis, most likelyreflects alterations in the protein consistence of secretion (35, 36). Theup-regulation of tenascin-C in the acinar cells 72 hours followingradiation may also indicate early subbasement membrane alteration.This hypothesis is strongly supported by our scintigraphic results,which displayed disturbance of primary 99mTcO4

� uptake as well asreduction of salivary ejection fraction. Normalization of scintigraphicand immunohistologic findings observed at 1 month including allgroups may indicate functional recovery. These results are inaccordance with several clinical trials, which registered a thresholdof 22 to 26 Gy as critical dose for the parotid gland (37–39). Potentialchanges between the two investigation time points are possible andcould be revealed by an alternative study design. The interval chosen,however, provides comparable data and showed significant changesthat indicate the overall course of such histologic and functionalchanges of salivary glands.Considering all this, the prophylactic application of lidocaine and

amifostine seems to reduce functional, immunohistochemical, andstructural damage of the parotid gland during radiotherapy.Pilocarpin may represent an adjuvant approach postradiation tostimulate survived acinar cells. Taking into consideration thatlidocaine has not yet been used clinically for this purpose and thepharmacokinetic of amifostine is still widely unknown, furtherexperimental studies are needed to rule out potential protection oftumor cells. Keeping in mind that the encouraging results presentedabove, although being evidential are valid for the parotid gland in arabbit model, clinical trials are crucial to introduce a comprehensiveand effective procedure for protecting salivary glands duringradiotherapy of the head and neck region.

Acknowledgments

Received 1/5/2005; revised 8/19/2005; accepted 9/13/2005.Grant support: German Research Foundation (Deutsche Forschungsgemeinschaft)

and the European Union FP6, LSHC-CT-2003-5032, Selective Targeting of Angiogenesisand of Tumor Stroma.

The costs of publication of this article were defrayed in part by the payment of pagecharges. This article must therefore be hereby marked advertisement in accordancewith 18 U.S.C. Section 1734 solely to indicate this fact.

We thank Dr. L. Zardi for providing antibodies and G. Knebel (Institute of Anatomy,University of Luebeck, Germany) for her painstaking assistance in the transmissionelectron microscopy.

Cancer Research






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2005;65:10486-10493. Cancer Res Samer G. Hakim, Hartwig Kosmehl, Isabel Lauer, et al. RadiotherapyAmifostine, and Pilocarpin on the Parotid Gland during A Comparative Study on the Protection Profile of Lidocaine,

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