Efficacies Of Chitosan And Hyaluronidase On Skin Damage Caused By Docetaxel Extravasation In Rats

Background and objective:Malignant tumor is severily threatening to human life.Chemotherapy is one ofthe principal methods for synthetically treating the tumor.Most chemotherapeuticagents are given by intravenous administration clinically, although some drugs areavailable orally.However, when they escape from a vessel into the surroundingtissues by leakage or are injected involuntarily into the tissues,a severe tissuereaction may arise,varying from erythema or tenderness to nerves and tendonsdamage,even to tissue necrosis or disability.The frequency of chemotherapeuticagents extravasation is considered to be between 0.6％and 6％of all their adverseeffect.Therefore,the extravasation is one of the most dreaded complications whenadiminstering chemotherapy.The type of treatment for extravasation is dependent on the chemotherapeuticdrugs.The topical application of dimethylsulfoxide(DMSO) has been advocated forthe treatment of extravasation of anthracyclines,or the application of intravenousdexrazoxane in combination with intermittent cooling.Topical DMSO has also beenused for the treatment of mitomycin C extravasation.An immediate subcutaneousadministration of sodium thiosulfate solution is recommended to treat the nitrogen mustard extravasation.Hyaluronidase(HAase)(150U-1500U subcutaneously insurrounding tissues) in combination with hot packs is the treatment of extravasationof vina alkaloids.Docetaxel,with more and more reports about the tissues damage by itsextravasation clinically, has be defined as a vesicant,but no guideline for treatingdocetaxel eatravasation has been proposed.Several case reports indicate thatdocetaxel may cause pain,erythema,blistering and ulceration,and the treatment oflocal hypothermia,topical DMSO,dilution with subcutaneous saline,corticosteroidsor diclofenac were reported to be effective in restricting inflammation.In animalmodels,a treatment with intradermal hyaluronidase was effective in preventingdocetaxel-induced ulceration.However, there were some people keeping an anotherviewpoint which consider topical treatment as topical DMSO,cooling or heating didnot have a beneficial effect.The purpose of this study is 1)to explore the efficacies of topicalchitosan,intralesional hyaluronidase on skin damage caused by docetaxelextravasation in a rat model,2)to compare the efficacies with intralesionalhyaluronidase in combination with topical chitosan and monotherapy of topicalchitosan or monotherapy of intralesional hyaluronidase on skin damage caused bydocetaxel extravasation in a rat model.Materials and methods:Materials20 mg/ml medical chitosan,1500 u hyaluronidase powder,10 mg/ml doce-taxel,saline,10％chloral hydrate, SPF(Specific Pathogen Animal) Sprague-Dawley(SD),electrical hair clipper, equi-armbalance,ruler, compasses,Canon-610 type digitalcamera, SHANDON AS325 type paraffin microtome, OLYMPUS BX40 type micro-scope, NAPCO MODEL5410 type electrothermostat. MethodsThe SPF Sprague-Dawley(SD) rats(Department of Animal Laboratory,Southern Medical University, Guangzhou, Guangdong, P.R.China) weighing (180±5)g were caged in 6 groups(8 of each group) in SPF environment.1)The 48 rats were randomly divided 6 groups averagely and received chitosanembrocation,hyaluronidase injection, hyaluronidase injection plus chitosanembrocation,saline embrocation,or saline injection,or received no treatment ascontrol.An animal model with docetaxel extravasation was planned to be establishedin each lower extremity of each rat.There were 16 cases in each group when defininga lower extremity as a case.Each rat of all groups would be marked with2,4,6-trinitrophenol to distinguish each other.2)The each lower extremity of 48 rats was shaved with an electrical hair clipper,with the diameter of skin lay bare larger than 4cm.The experiment would notcontinue if inflammatory reaction such as red swelling or hyperemia appeared onshaved skin in 24 hours.3)Based on previous experiments,docetaxel 1.5 mg/ml was injectedsubcutaneous with 0.4 ml in each lower extremity of rats after anesthesia in anintraperitoneal.Then adjuvant treatments with topical or intralesional antidote wereimmediately given,while the control group received no treatment.4)Injection sites were observed daily.The occurrence rate,the extent of skindamage,and the healing time were observed and compared.A wound was defined asloss of hair,dermal disruption or ulceration of at least 2 mm~2.Healing was defined ascomplete healing of ulceration and regrowth of hair in wound area without redswelling and hyperenia. The observation period ended when all lesions had healed.5)A rat picked out randomly from the wound rats in each group was killed onthe 3rd day, 11th day and 19th day respectively, and the wound skin was dissected and prepared for histological evaluation.6)Sizes of lesions were measured every day with a compasses and a ruler.Thewound area were calculated from the product of the two longest perpendiculardiameters.For each case,the wound area in square millimeter was plotted againsttime in a day, and the area under the ulceration curve(AUC) wascalculated.Simmilarly,the mean area of wounds in each group was calculated andplotted against time,and the mean AUC was calculated.Then we can compare theextent of skin damage by comparing the mean AUC.7)Data handling and statistical methods were performed in SPSS softwareversion 10.0. Chi-Square Test was applied to analyze the incidence rate of skindamage.The mean AUCs of wound area and the time of healing were comparedusing the One-Way ANOVA.The comparation between two groups was used LSD-twith aqual variances assumed while Tambane's T2 Test with equal variances notassumed.Results:1)Each rat of all groups occurred edema and erythema in 3～5 minutes afterinjecting docetaxel and the symptoms were aggravating gradually.The major wounddeveloped erosion and ulcer. The blister not appeared in whole period of this study.2)The occurrence rates of skin damage in chitosan group, hyaluronidase group,hyaluronidase plus chitosan group,saline embrocation group,saline injectiongroup,and control group were 93.75％,50.0％,37.5％,100％,93.75％,and 100％respectively. Significant difference about the occurrence rate was found among thesix groups(x~2=37.642, P＜0.001).Moreover the occurrence rate was lower inhyaluronidase group and hyaluronidase plus chitosan group than that in other groups.3)Significant difference about the AUC was found among the six groups(F=22.411, P＜0.001). The AUC was significantly less in hyaluronidase group and hyaluronidase plus chitosan group than that in saline injection group and controlgroup(P＜0.01).The AUC was significantly shorter in chitosan group than that insaline embrocation group and control group(P＜0.01). The AUC in hyaluronidaseplus chitosan group was not significantly shorter than that in hyaluronidasegroup(P=0.205),while the skin damage of the former was more gently.4)Significant difference about the healing time was found among the sixgroups(F=36.486, P＜0.001).The healing time was significantly shorter in chitosangroup than that in saline embrocation group and control group[(18.33±2.00)d vs.(23.70±2.41)d and (25.70±2.26)d,P＜0.01].The healing time in hyaluronidase group(12.00±3.00)d or hyaluronidase plus chitosan group (9.50±2.12)d was significantlyshorter than that in other 4 groups(P＜0.01).The healing time in hyaluronidase pluschitosan group was not significantly shorter than that in hyaluronidase group(P=0.228),while the healing time of the former was shorter.5) Significant difference about histological evaluation was found in six groupson the 3rd day, 11th day,19th day.On the 3rd day after skin damage,loss of partepidermis with little ulcer,gentle necrosis,inflammatory cell infiltrating and edemawas found in hyaluronidase group and hyaluronidase plus chitosan group,but in other4 groups edema was severe with large ulcer, generous necrosis and inflammatory cellinfiltrating.On the 11th day,edema and ulcer lessened with a great quantityinflammatory cell infiltrating,a small quantity blood capillary,epidermic cell growingand fibrous tissue proliferating in hyaluronidase group and hyaluronidase pluschitosan group,but in other 4 groups edema was still severe with large ulcer,generousnecrosis and inflammatory cell infiltrating and all of blood capillary,epidermic celland fibrous tissue proliferating were not found.On the 19th day,epidermis healingcompletely and scar forming with a small quantity inflammatory cell infiltratingwere found in hyaluronidase group and hyaluronidase plus chitosan group,but in other 4 groups was still severe with a great quantity inflammatory cellinfiltrating,epidermic cell growing and fibrous tissue proliferating slightly.Conclusions:1)Docetaxel is a vesicant with the symptom offlare,pain,induration,hyperpigmentation,tissue necrosis and ulcer whenextravasation.2)Both the monotherapy with hyaluronidase injection and hyaluronidaseinjection plus chitosan embrocation can decrease the occurrence rate of skin damagecaused by docetaxel extravasation,while chitosan embrocation can't decrease theoccurrence rate.3)Both the monotherapy with chitosan embrocation or hyaluronidase injectionand hyaluronidase injection plus chitosan embrocation can improve the healing ofskin damage caused by docetaxel extravasation4)Hyaluronidase injection plus chitosan embrocation can decrease theoccurrence rate and the extent of skin damage caused by docetaxel extravasation,andshorten the healing time,but there is no significant difference between it andhyaluronidase injection.