Int J Clin Exp Pathol 2019;12(4):1468-1477
www.ijcep.com /ISSN:1936-2625/IJCEP0091860

Original Article
Effect of cold and hot compress on neutrophilic
migration to the site of doxorubicin extravasation
Natsuko O Miura1,2, Naing Ye Aung2, Mitsunori Yamakawa2
Department of Basic Nursing, Iwate Prefectural University Faculty of Nursing, Takizawa, Iwate Japan; 2Department of Diagnostic Pathology, Yamagata University Faculty of Medicine, Yamagata, Japan
1

Received January 26, 2019; Accepted February 20, 2019; Epub April 1, 2019; Published April 15, 2019
Abstract: In the present study, we investigated a part of the mechanism responsible for the effects of hot and cold
compresses for extravasation of doxorubicin. We injected 20 μl of doxorubicin (DOX) (1 μg/μl) subcutaneously into
the dorsal area in mice and observed the resulting skin lesions macroscopically and histologically from day 1 to day
14 thereafter in groups treated with a cold pack (18-20°C) and a hot pack (38-40°C) or left untreated (control).
Immunofluorescence and RT-PCR for C5a receptor (CD88), interleukin-8 receptor (IL-8RA), and transient receptor
potential cation channel subfamily V member 1 (TRPV1) were also performed. Macroscopic observation showedthat the area of the skin lesion was significantly smaller in the cold group than in the control group, but was significantly larger in the hot group. The neutrophil count in the lesion was significantly higher in the hot group than in thecold (3 hrs) and control groups. The numbers of inflammatory cells expressing CD88 and IL-8RA were significantly
lower in the cold group than that in the other groups at almost time points and in the hot groups at later time points,
respectively. The number of nerve fascicles expressing TRPV1 was higher in the hot group than in the cold groupon days 1, 3 and 14. mRNA for CD88, IL-8RA and TRPV1 was detectable by reverse transcription-polymerase chain
reaction in both the cold and hot pack groups. Consequently, these results suggested that the cold pack for the
extravasation of DOX might reduce inflammation.
Keywords: Hot and cold compresses, doxorubicin, C5a receptor (CD88), interleukin-8 receptor, transient receptor
potential cation channel subfamily V member 1

Introduction
Doxorubicin (DOX) is a commonly used anthracycline anticancer drug. Since its introductionin 1975, DOX has been used in rituximab plus
cyclophosphamide, DOX, vincristine, and prednisolone therapy [1], adriamycin, bleomycin,
vinblastine and dacarbazine therapy, and modified cyclophosphamide, vincristine, doxorubicinand methotrexate therapy for lymphomas. The
reported frequency of extravasation of infused
anticancer drugs is 0.01-6.5% in Europe andAmerica [2]. DOX is classified in a vesicant drugthat can cause serious injury to skin tissue
when it leaks out of blood vessels. DOX extravasation causes pain and necessitates cessationof treatment for wound healing, sometimes fora long period, and skin grafting and flap surgery
may be necessary [3]. Application of a hot compress is also recommended for treatment of
extravasation [4, 5]. Other reported therapies

have included infusion of saline and hyaluronidase [6] or wash-out at the leakage site [7].
Local injection of steroid and application ofdexrazoxane have been also recommended forextravasation of DOX [8, 9]. Furthermore, local
cooling reduces the area that needs to be surgically resected, and decreases a recall phenomenon [10]. Animal experiments have demonstrated that cooling decreases ulceration [11],but other reports have indicated that neither
cooling nor warming reduces the ulcer size or
healing period [12].
Cooling causes local vasoconstriction, thus limiting diffusion of the drug into tissue, whereas
warming causes local vasodilatation, and promotes absorption of the drug [13]. For drugs
that bind to DNA, local cooling has been reported to reduce absorption of the drug by skin tissue after leakage. However, the period of cooling has varied, with sometimes the cold com-

Effect of compress on extravasation of doxorubicinpress being applied for 15-30 min every 1-2
days, or sometimes continuously for 1-3 hrs[13, 14], and in fact the mechanism of theeffect of such compresses remains unclear.
The purpose of the present study was to determine a part of the mechanism responsible forthe effect of compress application by examining the migration of neutrophils to the site of
DOX leakage, and also the intracellular expression of C5a receptor 1 (CD88), interleukin-8
receptor (IL-8RA), and transient receptor potential vanilloid type 1 (TRPV1) at the leakage site.
Material and methods
Mice
Thirty-nine BALB/c mice (male, 6 weeks, 22-25g) were obtained from Nippon Clea (Tokyo,
Japan) and housed at constant temperature(22-23°C) under a 12: 12-hrs light-dark cycle insawdust floor cages with free access to standard laboratory chow and tap water. The mice
were anesthetized with isoflurane (Abbott
Japan, Tokyo, Japan) and their backs were
depilated with a commercial cream (Kanebo,
Tokyo, Japan).
This study was approved by the Research Ethics
Committee (22091) of Yamagata University
Faculty of Medicine, Yamagata, Japan.
Hot and cold compress application
Using a syringe (Nipro, Osaka, Japan) with a
30-gauge needle, 20 μl of DOX (Kyowa Hakkou Kirin, Tokyo, Japan) (1 μg/μl) was injected subcutaneously into the backs of the mice.
Using a Cold/Hot Pack (3 M Healthcare, Tokyo,
Japan), the site of injection was then either
cooled (18-20°C) or warmed (38-40°C) whilemeasuring the skin surface temperature witha digital thermometer (CE-309, Sato Shoji,
Kawasaki, Japan). The mice were placed in asupine position. The compress was applied 3
hrs or 20 min, four times every 6 hrs [13, 14].
The mice were divided into a control group (n =15) not subjected to compress application after
DOX injection, a cold pack group (n = 15), and a
hot pack group (n = 15). The lesioned skin was
photographed before treatment and its area
was measured using Image J (Texel Studio,Tokyo, Japan).
A picture of the lesion, including skin swellingaround redness considered as the greatest
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dimension of inflammation was taken on thefirst and third days after DOX administration,and its area was measured using the image
processing software Image J-Win Japanese edition. After photography on the third day, we
resected the skin tissue and fixed it in 10%
formaldehyde for histological examination. Skin
specimens were cut into parasagittal slices,
dehydrated, and embedded in paraffin using
standard procedures to prepare sections 5 μm
thick. The paraffin sections were stained withhematoxylin and eosin and assessed by lightmicroscopy. We photographed 6 fields (×400)of the specimen, confirmed the presence ofsegmented leukocytes, and measured the neutrophil count per 1 mm2.
Measurement of vascular permeability
On the basis of a report by Yamaki [15], we
examined vascular permeability. A caudal vein
was injected with 100 mg/kg body weight offluorescein isothiocyanate (FITC)-labeled bovine serum albumin (Inter-Cell Technologies,
Jupiter, FL), and then 20 μl of DOX was injected
into six sites in the panniculus carnosus muscle. We subsequently applied one compress for
30 min (n = 15) or 3 hrs (n = 6). We divided
these mice into a control group (n = 7) untreated after DOX injection, a cold pack group (n =7), and a hot pack group (n = 7). After application of the compress, we collected blood fromthe abdominal vena cava and centrifuged it(3,000 rpm, 10 min) to obtain serum after leaving it at room temperature for 60 min. We
resected the skin tissue using an 8-mm biopsy
punch (Kai Industries, Gifu, Japan), and placed
it in 24-well tissue culture plates (Falcon, 3047,
Beckton Dickinson Japan, Tokyo, Japan), followed by incubation with 1 ml of formamide(Kanto Chemical, Tokyo, Japan) at 50°C for 2
hrs for measurement of the fluorescence intensity using a fluorescence plate reader (Gemini
EM, Japan Molecular Devices, Tokyo, Japan). In
addition, we added 50 μl of serum to 24-wellplates and added 950 μl of formamide to measure the intensity of fluorescence after warming treatment, similar to that for the skintissue.
Immunofluorescence for CD88, IL-8RA andTRPV1For this experiment 75 mice were used. We
injected 20 μl of DOX (1 μg/μl) into the panniculus carnosus muscle, and then treated the
Int J Clin Exp Pathol 2019;12(4):1468-1477

Effect of compress on extravasation of doxorubicin
injection site with a compress for 3 hrs using anappliance. After DOX injection, we divided themice into an untreated control group (n = 15), a
cold pack group (n = 15), and a hot pack group(n = 15) as described previously.
We took pictures of the injection site on days 1,
3, 5, 7 and 14 after DOX administration andmeasured the area of the lesion using Image
J-Win Japanese edition. We then resected thetissue on days 1, 3, 5, 7 and 14 after DOX
administration. Formalin-fixed tissue was used
for histologic examination and fresh frozen tissue for immunofluorescence for CD88, IL-8RA
and TRPV1. Frozen tissue samples were mounted in Tissue Tek OCT compound (Sakura,Tokyo, Japan), quickly frozen in liquid nitrogen,and stored at -80°C. We obtained frozen sections 5-10 µm thick using a Cryostat (CM1850, Leica, Tokyo) and mounted them on MAS
glass cover slips (Matsunami Glass Industry,
Tokyo, Japan), followed by air-drying for 1 hr. We
then fixed the sections with cold acetone (4°C)
for 10 min, followed by addition of 3% skim milk (Morinaga Milk Industry, Tokyo, Japan) dissolved in PBS after washing in 0.01 M phosphate-buffered saline, pH 7.4 (PBS), for 15 min.
For double immunofluorescence, slide-mounted tissue sections were incubated with a primary antibody [anti-CD88 (C5aR) antibody (rabbit, polyclonal, Santa Cruz Biotechnology,
Santa Cruz, CA), anti-rat IL-8RA antibody (recognizing mouse IL-8RA, goat polyclonal, Santa
Cruz Biotechnology), anti-rat TRPV1 (recognizing mouse TRPV1, rabbit, polyclonal, Alomone
Labs, Jerusalem, Israel) and mouse monoclonal anti-α-SMA antibody (Clone 1A4, mouse
IgG2a, Dako, Glostrup, Denmark)] overnight at
4°C. The tissue sections were washed in PBS,
and then incubated with the secondary antibody [FITC-conjugated goat anti-rabbit IgG(H+L) antibody (Beckman Coulter, Brea, CA),
FITC-conjugated donkey anti-goat IgG (H+L)
antibody (Beckman Coulter) and tetramethylrhodamine (TRITC)-conjugated donkey antimouse IgG (H+L) antibody (Jackson Immuno
Research, West Grove, PA) at room temperature for 3 hrs. Finally, the tissues were washedin PBS, and mounted in Fluoromount (Diagnostic
BioSystems, Pleasanton, CA). As a negative
control, we used PBS, non-immune rabbit IgG,
mouse IgG, and goat IgG (Dako) in place of the
primary antibody.
After mounting, we observed CD88 and IL-8RA
in the specimen using a fluorescence micro1470

scope and took photographs of 6 fields (400×).
In addition, we performed double staining using
TRITC and FITC for TRPV1 and α-SMA to exclude
the erector pili muscle, which includes TRPV1.
Subsequently, we photographed 6 fields (400×)including the TRPV1-positive funiculus, andmeasured the number of cells positive for CD88
and IL-8RA per 1 mm2.
Reverse transcription-polymerase chain reaction (RT-PCR) for CD88, IL-8RA and TRPV1
A fresh frozen section of the lesion tissues witha compress for 3 hrs on days 1, 3, 5, 7 and 14
after DOX was also obtained for RT-PCR. mRNA
was purified using WaxFreeTM RNA (TrimGen,
Sparks, MD). cDNA was synthesized with PrimeScript reverse transcriptase (TaKaRa Bio,
Tokyo, Japan) and Random 6 mers. RT-PCR was
performed using EmeraldAmpTM PCR MasterMix (TaKaRa Bio) and a Veriti thermal cycler(Applied Biosystems, Foster City, CA). The cDNA
was amplified using the following PCR primers:
CD88 [16], sense: 5’-TATAGTCCTGCCCTCGCTCAT-3’, anti-sense: 5’-TCACCACTTTGAGCGTCTTGG-3’, IL-8RA [17], sense: 5’-GACACTCTCTTAGGAGCCCACTTG-3’, anti-sense: 5’-CAGGGCCTGGTCAATGTCA-3’, TRPV1 [18], sense: 5’-TTGGATTTTCCACAGCCGTAGT-3’, anti-sense: 5’-GAACTTGAACAGCTCCAGACATGT-3’, glyceraldehyde-3phosphate dehydrogenase (GAPDH) [19], sense: 5’-ATCAAGAAGGTGGTGAAGCAG-3’, anti-sense: 5’-GAAGGTGGAAGAGTGGGAGTT-3’.
PCR amplification was performed as follows:denaturation for 10 min at 95°C; followed by
40 cycles of PCR amplification, with each cycle
consisting of 40 s of denaturation at 95°C, 1
min of annealing, and 40 s of elongation at
72°C; and with one final cycle of elongation at
72°C for 7 min. The annealing temperature wasset at 60°C for CD88, IL-8RA and GAPDH, and
65°C for TRPV1. The amplified PCR products
were separated by electrophoresis on a 4.0%
agarose gel containing ethidium bromide, andthen visualized with UV illumination.
Statistical analysis
The data of vascular permeability are presented as the mean ± standard deviation (SD). For
the other data, the median of each group is
shown as a box plot with whiskers from minimum to maximum. The interquartile range
shows as box with the median marked as ahorizontal line, and minimum and maximum
Int J Clin Exp Pathol 2019;12(4):1468-1477

Effect of compress on extravasation of doxorubicinfrom lower and upper quartile
represent error bar. The Kruskal-Wallis and Bonferroni significant difference tests wereused to analyze the results,
and differences at P < 0.05
were considered significant.
All tests were performed using
SPSS 16.0 for Windows.
Results
Effects of compress application on the size of lesioned
area, neutrophilic infiltrate,
and vascular permeability
We compared the area of swelling after application of each type of compress on thefirst and third days following
administration of DOX. This
showed that, relative to thecontrol group, the area of thelesion in the cold pack group was significantly smaller
at both time points, whereas that in the hot pack groupwas significantly larger (Figure1A). There was an evident tendency to have higher neutrophil count in the hot packgroup on the third of 3 days
than in the other groups (Figure 1B). Vascular permeability was significantly increased at 3 hrs of hot pack application relative to the other groups (Figure 1C).
Effects of compress application to the prolongation of
inflammation
Figure 1. Effects of compress application on the size of lesion area, neutrophilic infiltrate, and vascular permeability. Both cold and hot compresses
were applied 20 min or 3 hrs four times every 6 hrs. Box plot explanation:
The median for each group is shown as a box plot with whiskers from minimum to maximum. The interquartile range is shownas a box with the medianmarked as a horizontal line; minimum and maximum from lower and upper
quartile represent error bar. A. The macroscopic lesioned area after cold andhot compresses. After administering of doxorubicin into the dorsal skin of
the mouse, the lesioned areas (cm2) on the 1st and 3rd days were measured
and compared in each group. B. Relationship between neutrophilic infiltration and cold or hot compression time. After administering of doxorubicin
into the dorsal skin of the mouse, followed by cold or hot compression during
20 min or 3 hrs (4 times), the number of neutrophils (/mm2) was counted on
the third day. C. Vascular permeability: After doxorubicin administering into
mouse dorsal skin followed by 30-min or 3-hrs compress, the intensity offluorescein isothiocyanate-labeled bovine serum albumin was measured ina fluorescence plate reader. *: P < 0.05.

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Next, we evaluated the prolongation of inflammation on days 1 to 14 after DOX administration followed by a cold or
hot compress. Because thebacks of mice had resumed
hair regrowth by day 14, thearea of the lesioned skin wasmeasured up to day 7. The
lesions in the cold pack groupwere significantly smaller thanthose in the other groups ondays 1, 5 and 7 (Figure 2A,
2B).

Int J Clin Exp Pathol 2019;12(4):1468-1477

Effect of compress on extravasation of doxorubicin

Figure 2. Histologic examination of prolongation of inflammation after administration of doxorubicin into mousedorsal skin followed by 3-hrs compress. (A) Macroscopic pictures. Greatest dimensions of lesions were measured
including edema. Bar = 10 mm. (B) Extent of the lesioned area after doxorubicin administration and 3-hrs cold or
hot compresses. Box plot explanation: The median for each group is shown as a box plot with whiskers from minimum to maximum. The interquartile range is shown as a box with the median marked as a horizontal line, minimum
and maximum from lower and upper quartile represent error bar. *: P < 0.05. (C) Histologic findings (Hematoxylin
and eosin stain). (a) The dorsal skin of healthy mouse: epidermis (E), dermis (D), and subcutaneous tissue (Sc),sebaceous glands (S), the hair follicle (H). (b) Control group: neutrophils (arrowheads). (c) Cold group: neutrophils(arrowheads). (d) Hot group: neutrophils (arrowheads). Bar = 50 μm. (D) The number (per mm2) of neutrophils after
3-hrs cold or hot compresses. Box plot explanation: The median for each group is shown as a box plot with whiskers
from minimum to maximum. The interquartile range is shown as a box with the median marked as a horizontal line;
minimum and maximum from lower and upper quartile represent error bar. *: P < 0.05.

The epidermis, dermis, subcutaneous tissue
and panniculus carnosus muscle were evident
in the dorsal skin tissue of the healthy mouse,
and folliculus pili and sebaceous glands appeared in the dermis (Figure 2C). Infiltration ofneutrophils into areas of epidermal thinningand flattening, the dermis, and subcutaneous
tissue was evident on the first day after DOX

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administration (Figure 2E). An ulcer had formed by the third day, and with local epidermal
hypertrophy and extension of the rete ridge.
Collagen fibers in the deep dermis showed finetears, and the panniculus carnosus muscle
showed edematous injury with inflammatory
cell infiltration (Figure 2C). Neighboring epidermal growth with sloughing of the skin was

Int J Clin Exp Pathol 2019;12(4):1468-1477

Effect of compress on extravasation of doxorubicinfound on the fifth day (Figure 2E), with many
lymphocytes and macrophages other than neutrophils in the dermis and subcutaneous tissue. Re-epithelialization of the epidermis around the lesion was evident on the seventh day,but many inflammatory cells were still present
in the dermis. Re-epithelialization under thecrust was advanced on day 14, and the epidermis continued, and it was localized, and subepidermal bullae including lymphocytes and red
blood cells were found. The lesion became
fibroblast-rich, and granulation tissue was
reduced.
The neutrophil count was maximal on the third
day and decreased gradually thereafter. It had
an evident tendency to be lower in the cold
pack group in comparison with the othergroups, and there was a significant difference
between the cold and hot groups on days 1, 3,
5 and 14 (Figure 2D).
Immunofluorescence for CD88, IL-8RA, and
TRPV1
We observed autologous fluorescence of hairson the dorsal skin of mice. Because positivity
was found in the stratum corneum and folliculus pili in the negative controls, these structures were omitted in subsequent examinations. The number of inflammatory cells showing positivity for CD88 and IL-8RA was a tendency to be lower in the cold pack group thanthe hot pack groups on any days and days 5, 7
and 14, respectively (Figure 3A-D). There wasalso a tendency to observe less positivity for
TRPV1 in the funiculus in the cold pack groupthan in the other groups, and there was a significant difference between the cold and hotgroups on days 1, 3 and 14 (Figure 3E, 3F).
RT-PCR for CD88, IL-8RA, and TRPV1
RT-PCR of frozen tissue revealed the expression of mRNA for CD88, IL-8RA, TRPV1 and
GAPDH in the positive control. Similarly, in skintissue on the first day after DOX administration,
expression of mRNA for CD88, IL-8RA, TRPV1and GAPDH was detected, but was absent in
the negative control (Figure 4).
Discussion
Currently there is no consensus regarding the
optimal period of treatment with hot or cold

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compresses for anticancer drug extravasation[4, 20, 21]. A method involving repeated application of compresses for 20 min has been
adopted empirically [12], but this was originallybased on PRICE therapy for sprains [22] andcryotherapy for ankle strain [23]. In the present cold pack group, the neutrophil count at 20
min did not differ significantly from that in thecontrol group, but was decreased at 3 hrs; also,
the neutrophil count in the hot pack group was
found to be increased regardless of the period of compress application. Repeated application of a cold pack every 20 min inhibits neutrophil infiltration relative to application of ahot pack, but it is thought that continuous cold
pack application for 3 hrs is better. Furthermore,
application of a hot pack for 3 hrs increases
vascular permeability. A 30-min compress application has been performed empirically, but
in the present study there was no significant
difference in vascular permeability between
the cold pack and hot pack groups. With regard
to the effect of 30-min cooling, a laboratory
study of rats has shown that inflammatory cell
infiltration and edema resulting from extravasation of an antibiotic agent were inhibited [24].
However, no study prior to the present one has
investigated leakage of an anticancer drug.
Because anticancer drugs are more injurious toskin tissue and the resulting inflammation
tends to be protracted, brief cooling or warming
seems to have little effect. In addition, we followed the protocol from preliminary research in
the injury of the skin with the doxorubicin
administration by this experiment andchose
suitable animal models [25]. Based upon theseprevious findings, we studied the subcutaneous leakage of 20 μl of DOX (1 μg/μl) on thepanniculus carnosus muscle in the backs of
mice, and examined the mechanism of theeffect of 3-hrs compress application.
We examined the effects of compresses applied for 3 hrs on injury resulting from leakageof doxorubicin into subcutaneous tissue. We
found that, in comparison with the cold packgroup, the lesions were exacerbated in the hot
pack group, and that the neutrophil count wasincreased. In the cold pack group, inflammatory
cells positive for CD88 and IL-8RA were significantly decreased on any days and days 1, 5, 7
and 14, respectively, as were funiculi positive
for TRPV1 and TRPV1 in the epidermis, relative
to the hot pack group. Furthermore, we were

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Effect of compress on extravasation of doxorubicin

Figure 3. Immunofluorescence for CD88, interleukin-8 RA (IL-8RA) and transient receptor potential cation channel
subfamily V member 1 (TRPV1) on 3rd day after administration of doxorubicin into mouse dorsal skin followed by
3-hrs compress. Box plot explanation: The median for each group is shown as a box plot with whiskers from minimum to maximum in (B, D and F). The interquartile range is shown as a box with the median marked as a horizontal
line, minimum and maximum from lower and upper quartile represent error bar. (A & B) Immunofluorescence for
CD88 (A) and IL-8RA (C), and the number (per mm2) of CD88 (B) and IL-8RA (D)-positive cells after 3-hrs cold or hot
compresses. Immunofluorescence of TRPV1-positive cells (E) and the number (per mm2) of TRPV1-positive nerve
fascicles (F) after 3-hrs cold or hot compresses. [(a) TRPV1 (fluorescein isothiocyanate; FITC), (d) α-smooth muscle
actin (α-SMA) (tetramethylrhodamine; TRITC), (g) merge TRPV1 (arrowheads); (b) TRPV1 (FITC), (e) α-SMA (TRITC),(h) merge TRPV1 (arrowheads); (c) TRPV1 (FITC), (f) α-SMA (TRITC), (i) merge TRPV1 (arrowheads)]. (a) control group.
(b) cold compress group. (c) hot compress group in (A, C and E). Bar = 50 μm, *: P < 0.05.

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Effect of compress on extravasation of doxorubicinby high fever (> 43°C) andnitric monoxide, but when inflammation occurs, it is activated within the normal temperature range and is associated with inflammatory pain[29]. In addition, in colitis, TRPV1 increases the number ofneutrophils migrating into areas of inflammation, and increases myeloperoxidase activity [30]. In the present study,
expression of TRPV1 was found in peripheral nerves fromthe first day after DOX extravasation. Expression of epidermal TRPV1 was also observed from day 1. Expression of
Figure 4. Reverse transcription-polymerase chain reaction (RT-PCR) for
CD88 was found from the firCD88, interleukin-8 RA (IL-8RA), and transient receptor potential cation
st day of inflammation espechannel subfamily V member 1 (TRPV1). mRNAs of CD88, IL-8RA, andcially with the hot compress,
TRPV1 including glyceraldehyde-3-phosphate dehydrogenase (GAPDH) wereexpressed in skin tissue on the first day after doxorubicin administration.
but a marked inflammatory
MM: molecular marker, Lane 1; positive control (brain), Lane 2; control (noreaction was promoted by macompress), Lane 3; cold compress, Lane 4; hot compress, Lane 5; negative
ny neutrophils that migrated
control.
into the area of tissue injury,
and these were thought toable to determine the expression of mRNA for
express TRPV1. In addition, the expression of
CD88, IL-8RA and TRPV1 by RT-PCR. ChemoTRPV1 and CD88 continued after the first day,kines and C5a, including IL-8, are closely asand skin injury caused by DOX continued. DOX
sociated with migration of neutrophils intothat leaks into skin tissue binds to DNA, andinflammatory tissue. IL-8 is classified as a CXC
not only causes local cell death, but also
chemokine and is a chemotactic factor that
release of DNA-anthracycline complexes fromcauses neutrophils to migrate into inflammatodead cells. These are taken up by neighboringry tissue after activation of its receptor, IL-8R
normal cells by endocytosis, thus increasing[26]. C3a and C5a stimulate mast cells by actithe degree of cytotoxicity. However, in thisvation of the complement system, bringing
study, the expression of CD88 mRNA did notabout vascular hyperpermeability, followed byseem to be consistent with the expression levelmigration of neutrophils and macrophages intoof immunofluorescence. Future work should
inflammatory tissue [27]. It inhibits the migraquantify these factors by real-time PCR andtion of neutrophils into skin tissue and decreasquantify the amount of protein by westernes their expression of CD88 and IL-8R, thus
blotting.
decreasing any unnecessary injury.
In this study, it was suggested that CD88 andIn the present study, application of a cold pack
IL-8RA were expressed on neutrophils, andfor 3 hrs tended to decrease the expression of
TRPV1 was expressed on peripheral nerves,
TRPV1 in peripheral nerves at the injury site on
and these were associated with expansion ofdays 1, 3 and 14, relative to that in the hot packthe injury. Three-hrs cooling inhibited the exgroup. TRPV1 belongs to the TRP superfamilypression of these molecules, suggesting thatand is activated by capsaicin, being a channel
this would have been effective for inhibition of
having high Ca2+ permeability. It is a thermosenthe inflammatory reaction, whereas warming
sor expressed on dorsal root ganglia andworsened the lesion. In addition, application of
C-fibers, which are sensory nerves releasing
a hot pack for 3 hrs significantly enhanced vasneuropeptides such as substance P and calcicular permeability. These results suggest that
tonin gene-related peptide [28]. It is activatedas well as inducing local vasodilatation [13], a1475

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Effect of compress on extravasation of doxorubicin
hot pack does not promote healing, but rather
causes sudden edema.
In the present study, we were able to reveal thefollowing using mouse model for the first time:(1) Application of a cold pack (20°C) for 3 hrs
inhibited the expression of CD88 and IL-8RA,
and decreased the neutrophil count. In addition, it significantly reduced the extent of thelesion evident macroscopically until day 7. (2)Application of a hot pack (40°C) aggravatedvascular permeability and increased the degreeof swelling. In addition, it increased the expression of CD88 and IL-8RA and raised the neutrophil count, significantly exacerbating the lesion.
In relation to risk management, it is important
to monitor the occurrence of cytostatic drug
extravasation in order to improve the quality ofnursing care provided to cancer patients.
Extravasation of anticancer drugs can cause
severe skin lesions, and is usually detectedclinically during routine patient care.
When infused anticancer agents leak fromblood vessels, both cold and hot packs arecommonly used to treat the skin surface.
However, application of a hot pack to damaged
skin tissue results in further injury, and our findings suggest that such lesions might be aggravated. In addition, increased expression of
TR-PV1 causes pain, and the excessive inflammatory reaction may prolong the healing period. We think that the present findings haveshed some light on the effects of application ofcold and hot packs that are used routinely inclinical practice, and the underlying mechanisms involved.
Acknowledgements
We would like to thank Hiromi Murata andJunko Takeda (Department of Pathological Diagnostics, Yamagata University Faculty of
Medicine) for their valuable assistance during
this study.

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Disclosure of conflict of interest
None.
Address correspondence to: Natsuko O Miura, Department of Basic Nursing, Iwate Prefectural University Faculty of Nursing, 152-52, Takizawa, Iwate
020-0693, Japan. Tel: +81-19-694-2236; Fax: +8119-694-2236; E-mail: natsuko@iwate-pu.ac.jp

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