Abstract Progress in wound pharmacology is depen-dent on the availability of suitable animal wound mod-els. All animal models try to reflect human wound heal-ing problems. In acute models it is easier to achieve ap-proximations to the clinical situation than in modelsfor the chronic wound.

Introduction

Wound healing in the human has many unique aspectsthat depend on the physiology, aging, and regional char-acteristics of the species, but the opportunity to carry outcontrolled experimentation on the nature and therapy ofwounds is limited. There is also enormous variability inthe standard of care among institutions and clinicians, mak-ing it often difficult to compare treatments or outcomes.During this century investigators turned to other organ-isms in hopes of analyzing the complexities of woundhealing in circumstances having less genetic and environ-mental variability. Although there is considerable biologi-cal variation of the wound healing response even amonginbred animal strains, variance falls to levels that allowlimited numbers of surgeries to yield significant conclu-sions.

In general, animal models (with the exception of sometransgenic and targeted gene deletions) attempt to reflecthuman wound healing problems: dehiscence, ischemia.ulceration, infection, and scarring. Except for acute mod-

els, one must deal with approximations of clinical prob-lems, because of the obvious differences in tissue archi-tecture, immune system function, physiology, and otherhealing responses among species. Often the choice of an-imal model is dictated by practical issues such as cost,procurement, housing costs, and animal husbandry. Forexample, few investigators will jump at the chance to usea full-grown domestic pig as a healing model, even thoughit is better matched chronologically and physiologically tothe patient population most likely to be affected by woundhealing problems. Likewise, age-related differences inwound healing are clearly evident from studies in the rat,mouse, and rabbit, yet many investigators continue to in-vestigate problems of impaired healing in young, rapidlygrowing animals where manipulating the course of a ro-bust healing process is much more difficult.

Although the focus of this presentation is on modelingof the chronic wound, I will provide a broader list of mod-els, especially since some of the pathological models aremodifications of acute wound counterparts.

Acute wound models (Fig. 1)

Acute wound models (Fig.1) are often parallels of au-thentic surgical procedures or traumatic injuries. Thecompilation below is not comprehensive, because of theobvious ability of experimentalists to devise new ways ofinjuring tissue. The term “acute” refers to rapid introduc-tion of the injury and a relatively rapid course of repairthat may be compromised as discussed below.

Incisional

Cutting of the skin or other tissue with a sharp blade re-sults in rapid disruption of tissue integrity with minimalcollateral damage. There is rapid extravasation of plasmaand blood cells into the new tissue space, and, dependingon the extent and rapidity of hemostasis, the formation ofa fibrin clot that bridges the injury margins. Other cutting

J. M. Davidson

Animal models for wound repair

Arch Dermatol Res (1998) 290 (Suppl) :S1–S11 © Springer-Verlag 1998

Work presented at the 7th Annual Meeting of the European TissueRepair Society, Special Symposium “Proteolysis and Tissue Re-pair”, 23–26 August 1997, Cologne, Germany

J. M. DavidsonDepartment of Pathology, Vanderbilt University School of Medicine and Research Service, Department of Veterans Affairs Medical Center, Nashville, TN, USAe-mail: jeff.davidson@vanderbilt.edu, Tel.: +1-615-327-4751, extension 5488, Fax: +1-615-327-5393

devices such as electrocautery blades and laser surgicaldevices may produce greater collateral damage but lessbleeding. The amount of gape in the incision will dependon a number of factors, including the amount of subcuta-neous fat, the tensional forces on the wound site, and thespecies. The skin of loose-skinned animals such as ro-dents, lagomorphs, and dogs can slide and retract oversubcutaneous fascia to produce initially a large gape,while the skin of the pig, like that of humans, is firmly at-tached to underlying structures and gapes little unlessthose fascial planes or subcutaneous structures are them-selves incised.

Primary closure. Wounds closed by mechanical meansheal rapidly with minimal scar tissue formation. Whetherbandaged, sutured, or clipped, the principle is always toreduce the tissue gap to a minimum to allow rapid and ef-ficient bridging of the wounded edges by granulation tis-sue and new epithelium. As a consequence, this type ofwound is excellent for biomechanical analysis of woundstrength. It is less adequate for histological assessment ofhealing because of the limited volume/area of woundhealing activity; for the same reasons, it is poor for evalu-ation of tissue biochemistry or epithelialization.

Secondary closure. Incisions that are deliberately leftopen are said to heal by secondary intention. In this

process, a much more extensive fibrin clot gives way tomore ample granulation tissue and a significant gap to bebridged by epithelialization. In loose-skinned animals,wound closure is facilitated by the contraction of the mar-gins to approximate the edges. Nevertheless, these woundsbegin with several millimeters of gape and usually resolvewith several tens of microns of scar tissue. Healing issomewhat more retarded, especially with regard to the de-velopment of biomechanical properties, and scarring phe-nomena have been evaluated in this model at late (> 65days) time points.

Excisional

As the name implies, such wounds involve the removal ofa significant volume of the target tissue, and the filling ofthe void created allows more ample material for determin-ing biochemical and histological parameters. While theincisional wound is only suitable for in situ techniques,the excision site can be harvested or biopsied to obtaincells, tissue, RNA, exudates, and histological specimensthat have much more ample cross-sectional area and vol-ume. If the excision covers a large enough area, serialbiopsy of the wound site is feasible. Excisional wounds canbe covered with occlusive dressings which retain the exu-date (wound fluid) as a means of assessing the status of vari-ous soluble factors in the wound environment such as nutri-ents, proteinases, cytokines, and tissue degradation products.

Tape stripping. The simplest level of tissue excision in theskin involves partial removal of epidermis with adhesivetape. Depending on the exact adhesive used and the num-ber of times it is applied, one can remove the stratumcorneum and stratum granulosum layers, eventually ex-posing the basal keratinocyte layers. The basement mem-brane is left intact, and there is no blood loss, although thewater barrier of the skin may be temporarily compro-mised. This is a mild injury, yet sufficient to activatemany processes of epidermal repair as evidenced by sub-sequent epidermal hyperplasia. It has served as a usefulmodel when only epidermal phenotype is to be examined.

Blisters. The next level of injury involves rupture of theepithelial basement membrane zone, forcing detachmentof epidermis from underlying dermis. Various forms ofmechanical suction devices can be used, although this canbe difficult on rodent skin. The epidermal structuresabove the blister lose their nutrient supply and becomenecrotic unless rapidly reassociated with the basementmembrane zone. Thus, epidermal healing has to occurfrom the margin. The blister cavity itself is suitable forseeding with grafted epithelial cells. Blister fluid may re-flect interstitial fluid status. Blisters can also be induced witha variety of chemical or biological vesicants or with heat.

Split thickness. The split (partial) thickness injury in-volves the use of a sharp bladed device that is designed tocut parallel to the skin surface at a defined depth. In prac-

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Fig.1 Commonly used acute wound models. This schematic illus-trates the relative proportions of various components of the woundsite. In the incisional, excisional, and burn models, the epidermis(shaded line) and basement membrane (solid line) are disrupted,and all wounds except burns fill with a provisional matrix (cross-hatched fill) composed of fibrin, fibronectin, and other plasma de-rived components. The dead space model segregates the woundspace from surrounding, normal tissue with a biocompatible bar-rier (dashed solid line) that becomes encapsulated (not shown). Inthe burn, epithelial destruction is less complete in first and seconddegree burns, and the key feature is the residual necrotic material(solid black) produced by the injury. As healing progresses (ar-rows), the wound dimensions may be reduced, epithelializationproceeds from the margins and residual epidermal appendages,and the provisional matrix is replaced by granulation tissue thateventually transforms into scar (brick pattern). Healing of burns isretarded by the greater need to eliminate necrotic material. Inburns, the penetration of advancing epidermis beneath the necroticmaterial may be more pronounced than the more superficial elimi-nation of eschar in other transcutaneous models

tice, one usually uses a dermatome, either mechanical orelectrical, to shave off a 100–1500 mm thick layer of theepidermis and upper dermis in one or more passes. Thislesion is the equivalent of the proverbial scraped knee, andthe dermatome is used in clinical practice to prepare skingrafts from donor sites. The “split” refers to the fact that asubstantial amount of dermis, mostly reticular, is left be-hind, and, more importantly, the bases of most epidermalappendages (sebaceous and sweat glands, hair follicles)remain intact. Thus reëpithelialization is rapid and occursnot only from the wound margin, but from epidermal ap-pendages as well. Regeneration of hair follicles is depen-dent on the depth of excision, since progenitor cells residein the shaft, not in the bulb at the base of the follicle.

This type of lesion is difficult to generate in the mousebecause the skin is quite thin. The epidermis is only about50 µ thick, and the dermis is less than 1 mm. Many do-mestic animals are unsuitable for the model because of theextremely high hair density which exaggerates the rate ofreepithelialization. Hairless strains offer an advantage.

This model is useful for determining rates of reepithe-lialization, and, at least in the pig, is a good approxima-tion of the behavior of human graft donor sites. The rapidhealing rate makes it often more difficult to determineclear differences among treatment groups. Evaluation ofhealing is made either by separating the healing epidermisfrom underlying dermis with chaotrope or enzymatic di-gestion, in which case planimetric methods are used, or byhistomorphometric analysis of serial sections. In eithercase, the epithelialization is not continuous. The modelhas proven useful for testing many agents and devices thatpromote reepithelialization, including wound dressings,topical formulations, and growth factors.

As expected, extravasation of lymph and blood occursin this type of lesion, and a typical eschar composed offibrin clot and spent granulocytes will accumulate overthe site. In standardized models in which a test agent isapplied, this type of wound is often dressed with a semi-occlusive dressing or an ointment with moisturizing ormoisture retaining properties to accelerate the healing.However, it may be desirable to impair healing by lettingthe wound become desiccated.

Full thickness. This model involves the complete removalof epidermis and dermis to the depth of fascial planes orsubcutaneous fat. In the loose skinned species, the thinmusculature of the panniculus carnosus, which is firmlyadherent to the base of the dermis, is usually excised aswell. Healing occurs from the margins and the base of thewound by the formation of a fibrin clot that is invaded bygranulation tissue and by the migration of an epidermaltongue along the interface between granulation tissue andclot (eschar). Various devices are used to generate thistype of lesion in a standardized fashion, including biopsypunch, scalpel, and dermatome (set to cut very deeply ormaking several successive passes). The actual wound depth,as in partial thickness wounds, is very dependent onspecies, the mouse having the thinnest skin, while the pigand other large domestic animals having dermis which is

as thick as or thicker than that of humans. Bleeding andfluid loss are more extensive in this model, and there isgreater susceptibility to infection. The model offers theadvantages of significant wound volume, involvement of alldermal components, epithelialization only from the woundmargins, and the ability to analyze chemistry, histologyand cell populations in the wound site. Healing rates areoften monitored on the basis of total excisional volume(or cross-sectional area) filled with granulation tissue(neodermis), extent of reepithelialization, histological or-ganization of connective tissue, angiogenesis, and bio-chemical content of collagen or proteoglycans.

During the course of excisional wound healing, granu-lation tissue growth can be exuberant enough to raise thewound above the plane of the skin. This hypertrophicphase subsides rapidly in animals, but it may be the clos-est approximation of cutaneous hypertrophic scarring asseen in humans.

Splinting. In full thickness excision, the mechanical struc-ture of the dermis is completely disrupted, and variousforces in the surrounding dermis and the wound site canchange the dimensions of the wound without actually fill-ing the site with new tissue. Two distinct phenomena areassociated with this type of wound closure, contractionand contracture, and they can be counteracted by physicalsplinting of the wound with retaining rings, biopolymer(e.g., collagen) plugs, or other mechanical devices.

Contraction

This is a phenomenon predominantly seen in rodent andrabbit models, in which the excision of loose skin resultsin rapid shrinkage of the surrounding skin to reducewound dimensions in a presumably adaptive fashion. Thisprocess, although perhaps distantly related to excisionalhealing in humans, is termed “wound contraction,” and itsrates can be readily measured by noninvasive, morphome-tric techniques. In our laboratory, images are transferredfrom a video camera and thence to a frame grabber andthe NIH Image 1.62 software for measurement. Commer-cial and clinical devices are also available. There are cleardifferences in contraction rates in healing impaired ani-mals, and this measure is often one of choice when animalnumbers are limited, as in transgenic animals or targetedgene deletions in mice.

If contraction is not desirable, one has limited choices:splinting by the methods mentioned above, with the draw-back that material is usually introduced into the woundsite, or use of a tight-skinned animal, i.e., the domestic pig.Advantages of the rabbit ear lesion are discussed below.

Contracture

Wound contracture is a pathological process often associ-ated with hypertrophic scarring, and it seems to be pro-duced by contractile forces within wound granulation and

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scar tissue. The myofibroblast is often implicated as theculprit, although fibroblasts without organized bundles ofα-smooth muscle actin can certainly generate contractileforces. This phenomenon can be modeled in many speciesby administration of second and third degree burns. Exci-sional wounds in tight-skinned species such as the pig willalso exhibit some degree of contracture, although residualscarring is minimal.

Dead-space models

Connective tissue formation can be isolated from otherevents of tissue repair such as epithelialization and con-traction by employing porous subcutaneous implants.Though they differ somewhat in design, all such modelsfunction by creating an artificial tissue space into whichplasma infuses. This leads to development of a fibrin clotand subsequent formation of granulation tissue. Depend-ing on the implant material, further maturation into scarmay occur, and the implant is usually surrounded by aconnective tissue capsule comprised of several collage-nous fascia.

Implant materials vary widely: viscose/cellulose spon-ges, polyvinyl alcohol sponges, chambers of stainlesssteel or nylon mesh, porous Teflon tubing, perforated sili-cone tubing with sponge material within, or merely an airbubble. In general, implant materials are chosen to be rel-atively inert and nonbiodegradable, so that the implantsite has minimal inflammation, the site is well marked,and the implant is readily removed and dissected awayfrom surrounding connective tissue. These models areideal for biochemical assessment because of the well-de-fined volume enclosed, and many of the implant materialsare soft enough to be suitable for embedding in paraffinand sectioning. These implants generally have a symmet-rical organization, with the least mature portion at thecore, and tissue organization may be assessed simply bythe histological progression of granulation tissue into thecenter of the implant.

Because implants have defined dimensions, they areexcellent for biochemical determinations. Indeed, param-eters such as collagen content, DNA content, and quanti-ties of various biochemicals can be normalized on a per-implant basis or to implant weight. At early time points(3–5 days) the interstitial fluid that accumulates within theimplant may be harvested with a syringe and needle, pro-viding status information on metabolites, cytokines,growth factors, and nonadherent cell populations. Thiscavity can also be used as a reservoir for injectable or im-plantable material such as growth-stimulating substances,chemoattractants, or neutralizing antibodies. When long-term exposure is necessary, our lab frequently uses thepolyvinyl alcohol sponge model into which is implanted asmall, slow-release material containing reagent [1].

Limitations of the implant models include the interfer-ence of the implant with normal scar maturation, probablyby the uncoupling of physical interactions among cells,the lack of epithelial components, and the likelihood of an

eventual foreign body response. This latter response istaken to the extreme when material such as cotton plugsor carageenan are implanted, but even polyvinyl alcoholinduces a giant cell reaction and eventual ectopic calcifi-cation. Thus, studies with most implant materials are onlyrelevant for the first 3–4 weeks of the repair process.

Wound chambers

Some of the earliest modern studies of wound progressionwere performed in a punch wound in the rabbit ear thatwas occluded on both sides by cover slips, permitting themicroscopic examination of tissue reorganization. Exci-sional wounds may be enclosed with occlusive materialsto permit the collection of wound fluid/exudate or to con-centrate the administration of topical materials [2].

Burns

Chemical, thermal, or radiation burns to the skin or othertissues produce a remarkably different healing responsedue to their effects on the viability of cells and tissue.Thermal burns in particular create an extensive zone offrank necrosis that includes dead cells and denatured oreven charred connective tissue. Beyond the area of totaldestruction, a zone of coagulation necrosis exists, inwhich denaturation of plasma and cellular proteins leadsto the obstruction of blood vessels and lymphatics. Thiseffect, in turn, induces nutrient starvation of the involvedtissue.

Thermal

In general, burn lesions are confined to small areas by us-ing instruments or devices that have a limited contact sur-face with the skin or another target tissue. Although mostburn models have used the skin, burn models on internalorgans or peritoneal surfaces have been used to create sur-gical adhesions. As in medical practice, the depth of theburn may be designated by first, second, or third degree.This type of injury is always inflicted under general anes-thesia, and considerable postoperative care may be neces-sary, including analgesics and antibiotics.

Cold. The transient freezing of tissue causes local necro-sis without extensive protein denaturation or coagulationnecrosis. Cell death occurs by formation of internal icecrystals that rupture the plasma membrane. This injury isa mimic of frostbite, although it is usually used to injuretrunk skin rather than extremities. Cold may be applied tothe skin/tissue surface using a solid (metal) object that hasbeen cooled in dry ice or liquid nitrogen. The mass of theobject and the length of time it is held against the targettissue will determine, to a large extent, the depth of the in-jury. This should be confirmed by histological examina-tion of the tissue at 1–3 days after injury.

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Hot. Thermal burns are readily induced on the shaved skinsurface by placing a heated, conductive object against theskin with constant, calibrated pressure. One convenientmethod has been the use of brass rods that are placed in awater bath [3, 4]. These are generally used to produce second degree burns. The weight of the brass rod controlsthe pressure applied. If third degree burns are desired, therods may be heated in an oil bath, heated anywhere be-tween 100° and 400 °C. The metal is quite conductive, soit may be reheated quite rapidly for producing successivelesions.

Our laboratory has devised an electrical device basedon an electronically controlled soldering iron. Instead ofthe conventional soldering tip, brass or copper disksmounted on a threaded stud can replace the normal tip.These disks may be of any desired diameter up to 3 cm.Equivalent pressure is maintained by using a series ofweighted, slotted brass collars that can be slipped onto theback portion of the soldering iron, giving it a substantialmass. Since the standard instrument has a low heat rangeof about 400°C, the device must be modified to give alower limit of 150°C. Obviously, lesions of any shapecould be designed, and the degree of tissue damage isconsistent and readily controlled by a combination ofweight, time, and temperature. As with any of these de-vices, the extent of tissue damage must be confirmed withhistological cross-sections.

The (pulsed) CO2 laser may also be used as a preciseburning device. Depending on the energy input, substan-tial collateral thermal damage can occur. Computer assistedbeam control systems allow the investigator to createburns of precise dimensions and in a wide variety of sites.

A harsh but effective method of introducing extensiveburns involves the partial immersion of deeply anes-thetized small animal into a 95–100°C water or ethanolbath for several seconds [5]. This is probably the only ef-fective way of producing an extensive, third degree burnwithout charring, realizing that it produces much greaterphysiologic stress than a series of small lesions.

Caustic and chemical

A number of caustic agents, particularly alkalis, havebeen used to produce skin lesions in various species. Suchagents can be environmentally or occupationally hazardousmaterials whose treatment can only be standardized byanimal modeling. Such agents have the experimental ad-vantage of being applied in pure form and standardizeddoses.

Impaired healing models

Much of current research in the field of wound repair isconcerned with understanding and treating conditions ofdefective wound repair. At the outset, it should be madeclear that the etiology of human chronic wounds is con-siderably different from that which can be mimicked in

animal species. Human wound healing defects are often acombination of impaired circulation, poor nutrition,chronological age, restricted physical activity, and chronicphysiologic imbalance. Developing animal models ofsuch complexity is a formidable challenge, and most in-vestigations have attempted to isolate segments of theproblem in a reproducible, cost effective fashion, first todemonstrate that healing is impaired and second to deter-mine what agents or actions will remedy the condition.

Metabolic defects (Table 1)

A classical approach to impaired wound healing is the in-duction of a metabolic defect that represents a similar hu-man condition. Many drugs and therapies, especiallythose that affect the immune system, have a clear impacton wound healing end points (Table 1).

Chemical diabetes

Diabetes mellitus is one of the most prevalent causes ofimpaired wound healing in humans. The most commonlesions formed are those in the lower extremity and thefoot, and they are the result of a chronic diabetic state andpoor glucose control. This results in a combination of un-favorable conditions including poor peripheral circulationand mirocirculation due to progressive arteriosclerosis;neuropathic changes that result in an insensate extremityprone to trauma; intrinsic defects in the wound healingprocess that may include reduced abundance and responseto cellular growth factors.

Chemical agents produce a rapid development of dia-betes mellitus by selective destruction of Langerhans cellsin the pancreatic islet. Irreversible hypoinsulinema andhyperglycemia (defined as blood glucose levels > 300µg/dl) ensue within a few hours of treatment. Both ro-dents and lagomorphs are remarkably tolerant of thisstress. Rats survive up to 6 months without insulin ther-apy, although cachexia resulting in 25% body weight losseventually dictates their euthanasia. Rabbits can be main-

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Table 1 Models of systemicimpairment Immunosuppression

GlucocorticoidsAdriamycinTotal body irradiation

DiabetesGeneticChemical

Aging

NutritionVitamin deficiencies (A, C)Protein/caloric restriction

Zn deficiency

Renal Failure

tained in the diabetic state for periods up to 1 year. In bothspecies, water consumption and urinary output are severaltimes higher than normal, and adequate drinking watersupplies and dry bedding must be assured.

Streptozotocin. In appropriate doses (55 mg/kg, i.m.),streptozotocin sulfate is highly effective in rodents. Athigher doses, this drug is known to affect macrophagefunction. Animals should be fasted for 12 h prior to treat-ment and observed for several hours afterwards. Injectionsites should be chosen carefully to avoid accidental dam-age to nerves. Diabetic rats have many distinctive fea-tures, including poor coat condition, a yellowish pallor tothe pelt (in albino species), and reduced physical activity.Although not consistent, diabetic rats held long term maydevelop ulcerations on their hind limbs. These sponta-neous lesions have not been exploited experimentally asthe location is difficult to treat and to dress. Many woundhealing parameters are defective in diabetic rats, includingbiomechanical strength of incisional wounds, closure ofexcisional wounds, formation of granulation tissue inporous implants, and formation of new blood vessels inseveral angiogenic assays.

Alloxan. Streptozotocin is toxic to rabbits, requiring thesubstitution of alloxan. This is administered intravenously,usually through a marginal ear vein. As with rodents, ani-mals must be fasted prior to treatment, and pretreatmentwith atropine (75 mg/kg, i.v.) is also necessary. Hyper-glycemia develops rapidly, although weight loss and othersigns, at least in New Zealand White rabbits are muchmore subtle. Diabetic rabbits exhibit clear healing defects,however; closure rate of dermal excisional wounds in theear (see below) is reduced by 50%.

Genetic diabetes

Several rodent strains exhibit the diabetic phenotype, al-though it is often associated with obesity. For this reason,closure of large, excisional wounds is doubly impeded bythe physiologic state and the restriction of skin contrac-tion imposed by the excess fat. In essence, this results in asplinted wound that must heal by granulation tissue for-mation.

db/db mouse. This animal model has been used widely todemonstrate wound healing defects and possible mecha-nisms [6, 7]. For example, this strain expresses lower levels of several growth factors and receptors, accounting,at least in part, for the reduced rate of healing. [8].

db/db rat. This animal has a very similar phenotype. Be-cause of its size, it has the potential advantage of produc-ing larger or multiple wounds in a single animal as well ashaving a thicker skin.

Obesity. The ob/ob mouse and other related strains shouldexhibit a similar restriction of skin mobility and contrac-

tion that would allow them to serve as a splinted woundmodel in which granulation tissue dominates [9]. This isnot a commonly used system, however.

Malnutrition

Some of the earliest healing impairment models were nu-tritionally based. These include the vitamin A-deficientrat, the zinc-deficient rat, and the scorbutic (vitamin C-de-ficient) guinea pig [10]. Similar vitamin and mineral defi-ciencies can be created in larger species, but the cost andtime involved is often prohibitive. Vitamin C is only es-sential in the guinea pig and primates. Conditions whichreduce nutrient consumption or utilization will similarlyaffect wound repair status. Pair feeding is generally un-necessary in healing studies if the objective is simply toattenuate the repair process.

Immune system

The immune system, particularly granulocyte and macro-phage function, is a critical element of acute wound re-pair, and many different immunosuppressive strategies ul-timately manifest themselves in impaired wound healing.In some cases, impairment may be great enough to extendwound healing into a chronic phase.

Glucocorticoids

Members of this drug family are widely known for theiranti-inflammatory activity with collateral effects that in-clude thinning of the skin and diminished healing capac-ity. Experimentally, various species may be dosed withshort-acting agents such as hydrocortisone or dexametha-sone, or, for long-term inhibition, with intramuscularmethylprednisolone. The effects are demonstrable inmany different wound healing models in many species.Effects undoubtedly include the diminished capacity ofmonocytes to differentiate into growth factor-expressingmacrophage and the reduced capacity of many fibroblaststo produce collagen and other connective tissue compo-nents. Since glucocorticoid use is clinically widespread, itis felt that these models have reasonable clinical rele-vance.

SCID/Nude mouse/rat

Genetically immunodeficient rodent strains are not notedfor their inability to heal acute wounds; indeed, T cell-de-ficient mice are reported to heal more efficiently [11].These strains have been used in several interesting waysto dissect wound healing mechanisms. These animals willaccept human xenografts [12, 13], and one can examinethe relative contributions of murine and human cells to thereconstitution of microcirculation. Likewise, once en-

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graftment is complete, the graft sites may be wounded todetermine: (1) the role of circulating host cells and (2) therole of resident tissue fibroblasts, epithelium, and endo-thelium. This is now facilitated by genetic marking tech-niques that can be used in the host.

Chemotherapeutics

Antiproliferative agents have strong effects on wound re-pair because they deplete critical bone marrow stem cellpopulations. If these agents are present during the surgicalintervention, there is likely to be some direct action on therapidly proliferating cell populations in the repair site.

Adriamycin. This is one of many systemic chemothera-peutics that can impair wound healing by indirect actionon the propagation of immune cells. The adriamycin-treated rat has been used to demonstrate the efficacy ofgrowth factors in an impaired wound healing environment[14]. Although adriamycin is not a commonly used com-pound now, it is a suitable prototype for the many otherDNA intercalating agents in the pharmacopoeia. As withthe corticosteroids, there is a clinical population that is af-fected by these drugs, although their periodic administra-tion is designed, at least in part, to minimize loss of hostdefenses.

Nitrogen mustard. This compound and other vesicantsproduce severe chemical burns in topical application.There is a strong relevance to military medical applica-tions, and sulfur mustard has been used to develop an an-imal model [15].

Hydroxyurea. This antimitotic agent can be used to distin-guish betwen the roles of cell proliferation and cell migra-tion during healing [20] and to deplete neutrophils selec-tively from the circulating pool [21, 22]. In theory, thisdrug may be used to develop models with a markedly re-duced capacity for infection resistance and debridementof foreign material.

Antibody depletion

Experimental depletion of specific elements of the repairsystem with neutralizing antibody has been most notablyapplied in the case of antimacrophage serum as developedby Leibovitch and Ross [16]. This has never been pro-posed as a specific impairment model, but given the avail-ability of large quantities of antibody, this could certainlybe done in principle. Neutralizing antibody to specificmolecules such as transforming growth factor-β (TGF-β),platelet-derived growth factor (PDGF) and basic fibro-blast growth factor (bFGF) has also been shown to retardwound repair, at least at the local level [1, 17, 18]. Con-versely, such antibodies have been used systemically toanalyze excessive wound healing. [19].

Radiation

Like chemotherapy, radiation is a highly effective if un-specific means of reducing local or systemic propagationof cell populations critical to tissue repair.

Local radiation. Localized radiation dosing will distin-guish the importance of local cell proliferation from re-cruitment of peripheral, circulating cell populations.Doses may be chosen to minimize the proliferative capac-ity of connective tissue and epithelial cells. A local heal-ing impairment is created that can then be overridden withappropriate therapies [23]. If dosing is high, the local ra-diation burn (necrosis) may be followed by a prolongedfibrotic response [24, 25].

Total body radiation. In contrast to local irradiation, sys-temic radiation addresses issues of global distribution ofmarrow precursors that are critical to efficient repair. Intandem with local therapy, this model can be used to dis-tinguish local versus systemic effects of wound therapyagents.

Local infection

Infectious burden is one of the most crucial issues inchronic wound care. Current dogma dictates that woundswill fail to heal if contamination is > 105 organisms/g oftissue [26]. Impairment is likely to be due to a combina-tion of the exaggeration and exhaustion of host defensestogether with the elaboration of bacterial endotoxins. Re-producible models have been developed with many aero-bic strains including Pseudomonas spp., Staphylococcusspp., and Leishmania spp. (Table 2). Bacterial suspen-sions may be introduced either topically or by local injec-tion. A range of 106–109 organisms has been used to re-tard wound healing in incisional and excisional models[27]. Bacterial counts should be confirmed by subsequentbiopsy of target tissue.

Aging

Chronologic aging is an important factor in repairprocesses. Age related healing impairment is seen in therabbit, rat and mouse, particularly in last decile of thelifespan. Between juvenile and adult (8 vs 16 weeks in theSprague-Dawley rat) there is a marked decline in the rate

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Table 2 Biological impair-ment wound models Infections

PseudomonasStaphylococcusLeishmania

Brown Recluse spider venom

TNF-α overexpression

of healing, which argues against the use of younger ani-mals for the study of impaired wound healing.

Chronic wound models

The treatment and analysis of chronic wounds remainsone of the greatest challenges to the wound healing com-munity. It is critical to recognize that these wounds,though commonly involving skin, are highly diverse intheir etiology and therapy. Furthermore, the chronicwound is uncommon in both domestic and laboratory an-imals, and extraordinary measures are required to begin toreproduce authentic lesions in an accessible animalmodel. At least three important clinical entities have notbeen reproduced in animal models: venous stasis ulcers,diabetic foot ulcers, and pilonidal sinus. These conditionscertainly reflect anatomical, cultural, and postural predis-posing factors peculiar to humans, keeping in mind thatall of the conditions are compounded by advancing chro-nological age and collateral, systemic disease processes.Banal issues such as improper shoe fit, lack of physicalactivity, and restrictive clothing are not conditions easilyextrapolated to the vivarium. In addition, human cuta-neous anatomy is dissimilar from most common labspecies except the pig. It is very important from the pointof view of therapeutic development to understand howwell present models reflect, at least in a segmental way,the ultimate clinical target, and how they are limited byspecies and age differences.

Ischemia

Reduced blood supply and fluid drainage is a critical ele-ment in wound healing. Chronic ischemia in the limbs is akey factor in ulcer development, and prognosis is poor un-less blood flow and tissue perfusion are corrected. Acutecutaneous ischemia causes the decubitus ulcer, but it issurprisingly difficult to produce in most laboratory ani-mals. Simple application of pressure is difficult to stan-dardize and maintain. Nevertheless models that can ap-proximate this condition are very important for under-standing the etiology and treatment of this relativelyprevalent medical problem (Table 3).

Skin banding

A controlled cutaneous ischemia has been demonstratedin a guinea pig model [28, 29]. The model involves thesubcutaneous placement of the tip of the plunger from adisposable plastic syringe, closure of the wound used forinsertion, and subsequent ligation of the base of theplunger with a nylon fastening strap. In the experimentsdescribed, 9 h of ligation produced a necrotic lesion thatwas suitable for wound debridement studies. Undoubt-edly, other procedures could be tested with this technique.

Vessel ligation

Ischemia can be assured by the ligation of the blood sup-ply to a region of skin or other tissue. This type of proce-dure requires good surgical skills and anatomical knowl-edge.

Rabbit ear. This model has been extensively used in thelast decade for growth factor studies. It is sometimes re-ferred to as the rabbit ear “ulcer” model, although in itsunmodified form healing is quite efficient. It is producedby creating excisional wounds (4–6 per ear) with a biopsypunch on the inner aspect of the ear to the depth of the au-ricular cartilage, followed by scraping away of any under-lying perichondrium. Since the dermis of the rabbit ear isfirmly attached to underlying cartilage, this procedure cre-ates a full-thickness excisional wound with an avascularbase. Great care must be taken not to disrupt the cartilage,because new granulation tissue will rapidly stream infrom the contralateral surface. Several advantages recom-mend its use over other excisional wound models: woundcontraction is absent; animals may be treated, in mostcases, with restraint rather than anesthesia; many replicatewounds are available within a single animal.

The rabbit ear full thickness lesion can be compro-mised by ligation of two of the three supplying arteries[30]. This slows the rate of healing considerably, and itprovides a wider window for therapeutic application. Theloss of blood supply is not irreversible, and collateral cir-culation develops in about 2 weeks. A similar model canbe set up in the smaller mouse ear.

Lower limb. Several surgical models of lower limb isch-emia have been developed [31]. One recent applicationhas been the demonstration of efficacy of gene therapy byintramuscular injection of vascular endothelial growthfactor and the subsequent formation of new collateral cir-culation [32].

Flap surgery

There are many modifications of skin flaps, usually on thedorsal skin, in which the raised flap has a compromisedcirculatory pattern, and injuries within the circumscribedarea show necrosis and retarded rates of repair [33]. A re-

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Table 3 Models with localhealing impairment Ischemia-surgical or mechanical

restriction of blood flowFlapPedicleArterial ligationSkin ligation

PresureGreyhound dogDenervated limb

Local Radiation

cent modification is the so-called H-shaped incision, inwhich blood flow from three lateral directions is inter-rupted [34, 35]. Of course, if the flap is allowed to reat-tach at the dermal base, revascularization ensues. A mod-ification that ensures stable low perfusion rates is thetubular pedicle skin flap, in which a strip of tissue israised from the dorsal surface and sutured or clippedalong its length to form a closed tube [36]. The incisedarea below is closed by sutures as well. After healing iscomplete, the pedicle exhibits a perfusion gradient fromeach end towards the central portion of the “suitcase han-dle”. Thus, wounds of graded healing capacity can be pro-duced along the length of the pedicle. Perfusion impair-ment can be determined by laser Doppler flow techniquesor measurement of transcutaneous pO2.

Other pressure sore models

Several investigators have used the greyhound dog as amodel for decubitus ulcer treatment, since this strain hasparticularly thin skin and little subcutaneous fat [37]. Thelesion is standardized to some extent by placing a limb ina cast which is designed to apply pressure or abrasion tothe site of a bony prominence. This model may meet withobjections in some locales, although it may be no moreharmful to the animals than the rigors of racing for whichthey are bred. In the pig, limb denervation followed bycasting has also been used to develop model decubiti [31].

Calcium treatment

Senk et al. observed that epithelialization is retarded bytreatment of excisional wounds with agents containing el-evated calcium concentrations [38]. This may be a usefulmodel to exploit for development of agents or devices toaccelerate wound resurfacing.

Dermonecrotic lesions

In addition to the necrosis induced by chemical and ther-mal burns and by local ischemia, other agents can causelocal necrosis and ulceration of the skin (Table 4). Twoexamples are the chemotherapeutic agent, adriamycin(doxorubicin) and the venom of the Brown Recluse spider(Loxosceles spp.).

Adriamycin

This is a chemotherapeutic agent that had been morewidely used for tumor therapy by parenteral administra-tion. It acts as a potent DNA intercalating agent. Acciden-tal infusion into the dermis during phlebotomy was knowto cause severe local dermonecrosis with subsequent ul-ceration. Rudolph [39] took advantage of this property tostandardize the lesion. The injury is felt to be due to a free

radical mechanism, since rapid administration of scav-engers such as DMSO can quench the severity of the in-jury. More recently, this laboratory has calibrated themodel in rat and rabbit systems. In the rat, intradermaldoses of adriamycin (25 mg/ml), ranging from 50 to 500µg per site, can be injected in 50–100 µl into four to sixsites per animal. A dermonecrotic lesion develops overthe course of 14 days that is accompanied by a mild butpersistent inflammatory infiltrate dominated by granulo-cytes. A thick eschar is formed by the necrotic skin. Thelesion persists for > 50 days. During that prolonged pe-riod, we have measured the expression of several matrixmetalloproteinases, including collagenase (MMP-1),stromelysin (MMP-3), and gelatinase A (MMP-9). Ex-pression of each enzyme is very prolonged and elevated,peaking between 14 and 28 days after injury and persist-ing to at least 35 days. At the same time, collagen III ex-pression, a marker of early wound repair processes, isstrongly suppressed and delayed. One drawback of the ratmodel is that higher doses do produce a systemic effectwhen total body burden passes about 5 mg/kg. This maybe an advantage if one desires further, systemic impair-ment of the healing process.

The rabbit model performs similarly to the rat, but thelesions formed (doses of 100–2000 mg/site) do not resultin systemic effect. Six lesions are easily produced, andthey persist for 45–65 days. In both species, this type ofchronic lesion is a challenging test for the efficacy of de-briding agents and procedures, since there is a heavy bur-den of necrotic material. These lesions will also be a chal-lenging target for therapeutic treatments that stimulategrowth and for agents that control proteinase activity.

Spider venom

The bite of the Brown Recluse spider injects a venomcontaining sphingomyelinase, resulting in a local hemor-rhagic lesion that, if left untreated, can develop into gan-

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Table 4 Chemically impairedwound healing models Cytotoxic Dermonecrosis

AdriamycinMitomycin C5-Fluorouracil

Vesicants and Caustic AgentsNitrogen mustardAlkalai burns

Cacium – retarded epithelialization

Corneal ulcersNaOHAbrasion

Gastric ulcersAcetic AcidEthanolCysteamine

grene [40, 41]. It is reported that the initiating reaction isthe induction of E-selectin expression on the luminal sur-face of the microvasculature, causing the dramatic andrapid accumulation and autoactivation of neutrophils [42].The local discharge of neutrophil proteinases, togetherwith the free radical generating mechanisms, causes ex-tensive local tissue lysis and necrosis. This ulcerative le-sion can be reproduced in several animal species using ex-tract of the spiders’ venom sacs. We have calibrated thelesions in the rat and rabbit by intradermal injection ofpartially purified material (10–50 µg). A typical blanch-ing and wheal reaction is followed within a few hours byclear signs of hemorrhage, resulting in a black lesion withinflamed margins. This lesion gradually develops into afrank ulceration, dominated by neutrophil activation, andpersists for 25–40 days, depending on venom dose. Thismodel may be very useful for developing strategies tocontrol excess activity of serine proteinases (elastase,cathepsin G, and proteinase 3) released by neutrophils.

Nonunion fracture

Nonhealing wounds are certainly not confined to the skin.An important area of investigation in musculoskeletal re-pair is the nonunion fracture. This is modeled in a numberof animal species, usually by the mechanical distractionof a fracture site or a frank osteotomy [43, 44]. In additionto long bones, the mandible has been used as an experi-mental site [45].

Gastric, duodenal ulcer

Chronic lesions of the aerodigestive tract are clinicallyimportant and mechanistically distinct from cutaneous ul-ceration. A wide variety of irritants such as acetic acidand cholestyramine can be used to induce gastric lesionsin the rat and other species in a fashion that is suitable forexperimental intervention [46, 47, 48]. As with other ani-mal chronic wound models, healing occurs in a delayedfashion.

Concluding remarks

Animal models of wound repair can provide reliable andreproducible information on the behavior and response ofwounds to experimental therapy. They cannot replace theultimate verification of agents and actions in humanwounds, because of the substantial differences in tissuearchitecture and immune response. The experimentalistmust consider the merits and disadvantages of each typeof model and species in the context of the data beingsought. Impaired healing is relatively easy to achieve, butthe true chronic wound, an important clinical target, israrely attained in laboratory animals. Given the remark-able rate of advancement in genetic manipulation of miceand other animal species, we may be able to develop new,

more incisive models of the repair process and its keysteps. (Table 5) Models should strive for reproducibility,clinical relevance, humane treatment, and quantitative in-terpretation.

Acknowledgements Supported by funds from the National Insti-tute on Aging and the Department of Veterans Affairs.

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