Rabbit Ear Skin Research Articles

Post-iontophoresis transport of ibuprofen lysine across rabbit ear skin

The aim of this work was to study in vitro the post-iontophoresis transport of ibuprofen lysine across rabbit ear skin, from ibuprofen lysine water solutions (20–200 mg/ml and pH 6.8–7.8). Current densities of 0.125, 0.25, and 0.5 mA/cm 2 were applied either continuously or for 15, 30, and 60 min followed by passive diffusion for up to 5 h. The results showed a significantly higher cathodal transport compared to passive flux. Anodal iontophoresis also increased ibuprofen permeation, even though the drug is negatively charged. The application of an electric current for a limited period of time, followed by passive diffusion from the reservoir in contact with the skin, produced much higher post-iontophoresis fluxes of ibuprofen than passive diffusion. Post-iontophoresis transport of ibuprofen from lysine salt solutions linearly depended on the total amount of current applied during iontophoresis, and in the absence of background ions was independent of donor drug concentration. The reason for this behavior was the creation of a drug reservoir in the skin owing to the short period of current application.

Characterization of the permselective properties of rabbit skin during transdermal iontophoresis

The use of iontophoresis to enhance transdermal transport of drugs necessitates further investigations on the membrane used to simulate human skin because one of the transport mechanisms involved, electroosmosis, strictly depends on the properties of the skin (i.e., its isoelectric point, IP). The aim of this work was to characterize rabbit ear skin permselectivity by measuring the sodium transport number at different pH values. This method allowed us to estimate the skin IP. To confirm the validity of the method, mannitol flux was measured. In addition, the sodium transport number method was applied to the study of human skin and a model drug iontophoretic flux through rabbit and human skin was evaluated. The results indicate that rabbit ear skin behaves as a permselective membrane, with an IP between 2 and 3. The same result was obtained using human skin. The mannitol flux data confirm that the direction of electroosmotic flow at physiological pH is in the anode-to-cathode direction. Finally, permeation experiments performed with a model drug showed that the relative electroosmotic and electrorepulsive contributions to the total flux are the same for human and rabbit skin. It can be concluded that rabbit ear skin is a suitable model for the study of iontophoretic permeation of drugs.

Transdermal application of bupivacaine-loaded poly(acrylamide(A)-co-monomethyl itaconate) hydrogels

Bupivacaine, an amide local anaesthetic agent of long-acting and intense anaesthesia, was incorporated into poly(acrylamide(A)-co-monomethyl itaconate (MMI)) hydrogels. The swelling behaviour of two gel compositions, without drug, 75A/25MMI and 60A/40MMI, through rabbit ear skin, mounted on a modified Franz diffusion cell, was studied. Both gel compositions reach the equilibrium swelling degree (88.9±0.7 wt.% for 75A/25MMI and 92.5±0.1 wt.% for 60A/40MMI). The swelling kinetics was in accordance with the second Fick’s Law; diffusion coefficients indicate faster swelling for gels with lower amount of monomethyl itaconic acid. The skin flux of bupivacaine solution through rabbit ear skin was 105±24 μg/cm 2/h, the effective permeability coefficient was 26×10 −3±9×10 −3 cm/h, and 77±15% of bupivacaine was permeated. Bupivacaine-loaded gels allow the drug was permeated through the skin. 47±4% and 36±3% of the drug amount included in 75A/25MMI and 60A/40MMI hydrogels, respectively, was permeated. The skin flux of the drug was between 90±5 and 16±7 μg/cm 2/h depending on the amount of bupivacaine included in the gel and the gel composition. Skin flux increases with the drug load of the gels. Furthermore, as more MMI in the gel slower skin flux of the drug due to bupivacaine-gel interactions.

Skin Permeation of 5-Methoxypsoralen from Topical Dosage Forms

The topical photochemotherapy of dermatoses with psoralens (PUVA therapy) requires an adequate drug level at the target site (basal epidermis) at the time of UVA radiation. The aim of this work was to enhance 5-methoxypsoralen transport to the basal epidermis, with the goal to shorten the delay between drug application and UVA irradiation. 5-Methoxypsoralen transport through rabbit skin was studied in vitro from topical formulations (water solution, gel, and emulsion). The results obtained show that the use of the emulsion increased the flux through rabbit ear skin, even if partitioning was not favorable. Additionally, the time lag was sensibly reduced, compared with the gel and solution. Furthermore, drug accumulation in human skin in vitro was determined using the thin slicing technique. Human skin accumulation profile was significantly higher for the emulsion, compared with the gel, indicating that the delay between psoralen application and UVA irradiation can be shortened.

Rabbit and pig ear skin sample cryobanking: effects of storage time and temperature of the whole ear extirpated immediately after death

The post-mortem temporal and thermal limits within which there will be ample guarantees of rescuing living skin cells from dead specimens of two species, rabbit and pig, were studied. Post-mortem extirpated whole ears were stored (in non-aseptic conditions) either at 4 °C or at room temperature (from 22 to 25 °C) or at 35 °C for different time lapses after animal death. In both species, the post-mortem maximum time lapses where cell viability was not significantly reduced were 240, 72, and 24 h post-mortem (hpm) for 4, 22–25 and 35 °C, respectively. Once the post-mortem temporal limits for each tested thermal level at which cells from skin samples are able to grow in culture were defined, the survival ability of skin samples submitted to these temporal limits and cryopreserved were tested. In the pig, skin samples stored at the three tested thermal levels survived after vitrification-warming, reaching confluence in culture. In rabbit, only tissue samples from ears stored at 35 °C for 24 hpm did not survive after vitrification-warming. In conclusion, we should remark that cell survival rates obtained according to the assayed post-mortem time lapses and thermal levels are sufficient to collect and to cryopreserve skin samples from the majority of dead specimens.

Changes in the levels of glutathione after cellular and cutaneous damage induced by squalene monohydroperoxide

Squalene monohydroperoxide (Sq-OOH), the initial product of ultraviolet-peroxidated squalene, was used to investigate the effect of peroxidative challenge upon the glutathione contents in rabbit ear skin and primary-cultured fibroblasts derived from rabbit ear skin. The cellular reduced glutathione (GSH) contents decreased during 30-minute incubations in vitro with Sq-OOH, and oxidized glutathione (GSSG) was formed concomitantly, indicating that Sq-OOH had a potential for GSH-depleting activity in vitro. When Sq-OOH was applied topically to the skin in vivo, only GSSG contents increased significantly within 30 minutes. Moreover, pretreatment with the GSH depletors, DL-buthionine sulfoximine (BSO) and diethyl maleate (DEM), could potentiate the cytotoxicity and comedogenicity induced by Sq-OOH. These findings suggest that the endogenous antioxidant, glutathione, is quite sensitive to Sq-OOH and may be an important material for protecting cells and/or tissues against the oxidative stress induced by Sq-OOH treatment.

Evaluation of transcutol as a clonazepam transdermal permeation enhancer from hydrophilic gel formulations

The influence of diethyleneglycol monoethyl ether (transcutol), alone or in combination with propylene glycol, on clonazepam permeation through an artificial membrane and excised rabbit ear skin from Carbopol hydrogels was investigated. Drug kinetic permeation parameters were determined for both series of experiments and compared. Rheological characteristics, drug solubility and membrane/vehicle partition coefficient for each gel formulation were also determined, and their role in the formulation performance was investigated. Both series of experiments showed an increase of drug permeation as a function of transcutol content in the formulation. The combination of transcutol and propylene glycol resulted in a synergistic enhancement of clonazepam flux. A different trend was found in experiments with gels containing mixtures of the two enhancers, where an increase (in the case of artificial membrane) or a decrease (in the case of rabbit ear skin) of drug permeation was found by increasing the transcutol/propylene glycol ratio in the mixture. Such a result is explained on the basis of the particular mechanism of action demonstrated for transcutol which associates the increase of drug solubility to the potent effect of a depot in the skin.

Comedogenicity of squalene monohydroperoxide in the skin after topical application.

The comedogenicity of squalene peroxides was examined on the rabbit ear skin after topical application of squalene-monohydroperoxide (Sq-OOH), the initial product when squalene was irradiated with UV-A. Since comedogenic products from UV-irradiated squalene were extracted with methanol solution, we isolated Sq-OOH by reverse-phased HPLC with a methanol mobile phase solvent. The degree of comedogenic reaction induced by Sq-OOH was higher than that of well-known comedogenic cosmetic ingredients. Unlike two other mono-peroxides, tert-butyl hydroperoxide and cumene-mono-hydroperoxide, Sq-OOH induced comedo-formation in the rabbit ear skin. However, the comedogenicity of reduced Sq-OOH, squalene-hydroxide (Sq-OH) and squalene itself was lower than that of Sq-OOH. These results indicate that Sq-OOH is a potent comedogenic mono-hydroperoxide chemical to rabbit skin.

The effects of Azone and capsaicin on the permeation of naproxen through human skin

The permeation of naproxen through excised human skin and isolated perfused rabbit ear skin has been determined. It was found that both Azone and capsaicin enhanced the permeation with an enhancement ratio of up to 4-fold. The magnitudes of the effect were similar in human and rabbit skin. The permeation of naproxen from a saturated solution of the drug through skin pre-treated with Azone was similar to that from a commercial preparation (Naprosyn). In the perfused rabbit ear experiments the presence of capsaicin had no effect on the vasodilatation of the blood vessels, inferring that the penetration enhancement was a direct result of capsaicin influencing the barrier function of the skin. Structural similarities between Azone and capsaicin were seen using molecular graphics.

Therapeutic evaluation of compounds in the SCID-RA papillomavirus model

A previous study by Kreider (Kreider et al., 1979) indicated that rabbit skin, which had been transplanted to immunodeficient nude mice, could be successfully infected with cottontail rabbit papillomavirus (CRPV). We have extended this observation in developing a rodent model for evaluation of compounds for activity against the papillomaviruses. In this model (called the SCID-Ra model), rabbit ear skin is transplanted to the dorsum of SCID mice and allowed to heal for 3 weeks. Infection with CRPV by scarification leads to the growth of warty lesions within 2–3 weeks in>95% of the animals. Topical and/or systemic therapy can be initiated at various times post infection (PI). Weekly lesion scores are recorded and compounds are evaluated for their ability to suppress wart growth when compared to untreated control mice. Ribavirin, which has had a suppressive effect both in the clinic for the treatment of respiratory papillomatosis and on the growth of warts in the rabbit back model, was evaluated and showed significant anti-proliferative activity with oral dosing. Both antiviral and antiproliferative compounds including podophyllin and 5-fluorouracil, which have been used clinically for the treatment of human papillomavirus (HPV) infections, were evaluated in this model. The anti-mitotic compound, Navelbine™ (vinorelbine tartrate), which is used for the treatment of non-small cell lung carcinoma was evaluated in this system and showed significant inhibition of wart growth with somewhat less topical cytotoxicity when compared to podophyllotoxin.

Influence of vehicle composition variations on the in vitro and ex vivo clonazepam diffusion from hydrophilic ointment bases

A systematic study of formulation factors influencing the release of clonazepam from hydrophilic ointment bases was performed. Diffusion experiments were carried out using both artificial membranes (cellulose nitrate membrane impregnated with lauryl alcohol or isopropylmiristate or vaseline oil) and natural ones (rabbit ear skin). The formulation variables were the percentage of polyethylene glycol 400, polyethylene glycol 6000 and water, and the type of lipophilic component (lauryl alcohol, isopropylmyristate or vaseline oil) added to the vehicle. In vitro and ex vivo results were compared and the best formulation was found, even if it was not possible to establish a precise correlation between the in vitro and ex vivo flux values.

Toxicity testing of organic solvent extracts from Annona squamosa: Effects on rabbit eyes and ear skin

AbstractSome organic solvent extracts of seeds and leaves from Annona squamosa exhibit insecticidal activity. They were studied for the management of human lice. However, toxicological data of these extracts have not yet been widely reported. In this experiment, the extracts of active compounds of leaves and seeds from A. squamosa in four organic solvents, i.e. petroleum ether, ether, chloroform and ethanol were tested for toxicity to the eyes and ear skin of rabbits, using 1:10 (w/v) dilutions of dried extracts in propylene glycol as test substances. The results revealed that some extracts caused conjunctival redness, chemosis, rugged cornea, skin erythema and oedema. The organic solvent extracts that produced adverse effects on rabbit eyes were in the following order of severity: ether > petroleum ether > chloroform > ethanol, and those on rabbit ear skin were petroleum ether > ether > chloroform > ethanol. Ethanol extracts of leaves and seeds seemed to produce mildest toxicity to rabbit eyes and no toxicity to rabbit ear skin.

Changes in adrenergic nerves and tissue perfusion after freezing injury to the ear skin of rabbits.

The aim of the present study was to investigate by a combined trypan blue and catecholamine fluorescence technique the sequential changes in cutaneous adrenergic nerves, the distribution of blood flow, and vascular permeability at the demarcation line that occurred after experimental cold injury. Nine New Zealand white rabbits weighing 4.0-4.4 kg had frostbite induced by pressing the bottom of a glass bottle 2 cm in diameter filled with liquid nitrogen against the shaved skin of the ear. All the rabbits were anaesthetised with ketamine hydrochloride (Ketamine) and xylazine hydrochloride (Rompun). Specimens were taken one and three days, and two weeks after frostbite. Control samples were taken from the opposite normal ear. Trypan blue was injected into a saphenous vein just before the specimens were taken. The specimens were always taken in the same way and selected so that the probable demarcation line of the frostbite ran across the middle. The glyoxylic acid-induced fluorescence method was used to show the adrenergic nerves. There were no adrenergic nerves around the vessels and no arteriovenous anastomoses in the central area of the injury after one day, but catecholamines had started to accumulate in the adrenergic nerve endings at the margins of the injured area. This accumulation was still more obvious three days after frostbite. Some fluorescent regenerating adrenergic nerves could already be seen at the probable demarcation line two weeks after frostbite. There was increased trypan blue fluorescence near the margin of the injured area after one day, indicating extravasation and vascular damage.(ABSTRACT TRUNCATED AT 250 WORDS)

The arterialised venous flap: experimental studies and a clinical case

It is accepted that small arterialised venous flaps (AVF) can survive on a recipient bed with poor blood supply but survival of larger flaps is not always satisfactory. Possible reasons for this include the flap anatomy, particularly the pattern of the venous network, or factors at the recipient site. To investigate the possible factors in the flap design and the recipient site of an arterialised venous flap, we studied the relationship between (1) size of the artery used to arterialise the flap at the recipient site and the survival rate and (2) the number of draining veins and the survival rate, using rabbit ear skin flap models. Our results suggest that AVFs may become necrotic in the presence of a relative excess of arterial blood inflow, and that two exit veins are more effective than one. We also report a case where a 10 × 15 cm sized free AVF harvested from the lower extremity survived on the forearm.

Trypanosoma vivax in Glossina palpalis gambiensis do not appear to affect feeding behaviour, longevity or reproductive performance of the vector

Feeding behaviour of Glossina palpalis gambiensis Vanderplank infected with Trypanosoma vivax Ziemann was studied and compared with that of uninfected control tsetse. The parameters measured were: total number of probes into the ear-skin of rabbits; rate of bloodmeal engorgement; weight of freshly ingested blood; survival; and mean weight of pupae. The results showed that the rosettes of T.vivax parasites in the labrum did not interfere with the feeding behaviour of the vectors. Furthermore, mean survival of T. vivax-infected males was significantly higher (82.2 +/- 4.2 days) compared with that of uninfected ones (70.5 +/- 3.1 days). However, with the female tsetse, mean survival of those infected was lower (98.8 +/- 4.0 days) compared to the uninfected controls (102.2 +/- 5.6 days), but the difference was not significant. A few infected males and females lived a little longer than the uninfected ones. Fecundity of the female tsetse remained unaffected by the infection, and furthermore the mean weight of pupae from the infected females was not significantly different from that of pupae from the uninfected control group. Thus the physiology of pregnant female tsetse in terms of nourishment of intra-uterine larva was unaffected by T.vivax infection. Two successive probes into the skin of two different goats followed by feeding on a third goat by each of four infected tsetse resulted in successful transmission of the infection to eleven out of twelve goats. Thus probing alone into the skin of this host can result in the transmission of T.vivax infection.

Failure of Perivascular Sympathectomy to Remove Adrenergic Nerves from Peripheral Vessels of the Rabbit Ear Skin

A perivascular sympathectomy was performed at the base of the ear artery in 11 New Zealand white rabbits. Two days later three were killed, and the central nerve was cut in the remaining eight. The contralateral ear served as a control. Specimens were taken from the distal parts of both ears two days later and the glyoxylic acid-induced fluorescence method was used to show the adrenergic nerves. The arteries of the normal, control ear were surrounded by a dense plexus of fluorescent adrenergic nerves, which were abolished by perivascular sympathectomy only in the segment from which the adventitia had been removed. The adrenergic innervation was normal proximal to the site of perivascular sympathectomy, but there was a short segment of the central vessel distally in which it was diminished. No changes in adrenergic innervation were found in the distal third of the rabbit ear.

Long-Term Organ Culture of Rabbit Skin: Effect of EGF on Epidermal Structure In Vitro

A method is described for maintaining the epidermal structure of normal rabbit ear skin explants in organ culture for up to 12 weeks. Split-thickness skin specimens were put in diffusion chambers made of either millipore filters or bovine collagen membranes, and then submitted to a roller tube culture at 15 rpm and 36 degrees C. The culture medium was Dulbecco's modified Eagle's medium (DMEM) supplemented with 20% fetal calf serum (FCS) + 0.4 micrograms/ml hydrocortisone. The gas used in the culture tube was air + 5% CO2. Autoradiography revealed the incorporation of [3H]-glycine into the 68-kD keratin band of explants for up to 12 weeks, indicating that normal keratinization was maintained throughout the entire culture period. The turnover time of the epidermis from basal layer to granular layer was around 7 d in both the early and late stages of culture. The addition of epidermal growth factor (EGF) to the culture caused the epidermis to become acanthotic with orthokeratosis, but with high concentrations of EGF (greater than or equal to 10 ng/ml) parakeratosis and increased proliferation of the epidermis occurred. Dexamethasone (DMS) strongly inhibited the EGF effect.

Antibody-mediated neutralization in vivo of infectious papillomaviruses

Specific antibody-mediated neutralization of infectious human papillomavirus type 11 (HPV-11) was achieved in the athymic mouse xenograft system, in which HPV-11 induced morphological transformation of human foreskin. Virus-specific neutralization was demonstrated by the ability of an HPV-11-specific polyclonal antiserum to neutralize HPV-11 infectivity and not bovine papillomavirus type 1 (BPV-1) or cottontail rabbit papillomavirus (CRPV) infectivity. In all three virus infectivity systems, neutralization was detected by the failure of the virus suspension to induce morphological transformation of the appropriate skin xenografts placed under the renal capsule of athymic mice. Rabbit polyclonal antisera were also generated against intact virions of both BPV-1 and CRPV, and neutralizing activity was tested in the xenograft system with BPV-1 and fetal bovine skin and with CRPV and rabbit ear skin. The three polyclonal antisera contained virus-specific neutralizing antibodies, demonstrating that neutralizing epitopes existed on all three papillomaviruses and that these epitopes were antigenically non-cross-reactive. The athymic mouse xenograft system was a useful model for detecting papillomavirus-specific neutralizing antibodies and offers the only opportunity for the analysis of neutralizing antibodies to human papillomaviruses.

Improved Procedure for Assessing Acnegenicity (Comedogenicity) in the Rabbit Ear Model

AbstractThe various procedures for assessing the comedogenicity of cosmetics, toiletries, and drugs using the rabbit ear model have produced conflicting results. The relevance and value of the rabbit ear model have been seriously questioned.This paper describes a method based on histopathologic evaluation of the ear skin of male rabbits, treated once daily for 3 weeks. Identifying substances capable of creating follicular horny impactions (comedones) is not the only goal of the test. It is equally important to be able to detect irritating formulations that can provoke an inflammatory folliculitis. These can cause papulopustular, acneform eruptions in humans.

Morphological appearance of epidermal cells cultured on fibroblast-reorganized collagen gels

Human foreskin fibroblasts were used to reorganize hydrated collagen gels into a dermal-like matrix, after which freshly isolated keratinocytes isolated from rabbit ear skin were placed on the surfaces of the matrices and cultured for up to 12 days. Transmission electron microscopy revealed 8-12 cell layers of epidermal cells organized in three distinct strata. The basal stratum consisted of cuboidal to columnar cells with typical complement of organelles, oval nuclei, and prominent tonofilaments inserting into desmosomes. Mitotic cells often were found at this level. There was no well-defined basement membrane region; rather, many of the cells appeared to be in close contact with collagen fibrils. The intermediate stratum of suprabasal cells consisted of elongated cells that had reduced organelles, but still were connected to each other by desmosomes. Finally, the superficial stratum of suprabasal cells contained cells that were completely flattened and often appeared to be sloughing off the apical surfaces of the cultures. Indirect immunofluorescence studies carried out on frozen sections revealed bullous pemphigoid antigen associated with basal epidermal cells; pemphigus vulgaris antigen around the epidermal cells of all strata, and keratin present in the epidermal cells of all strata. Filaggrin was observed in punctate and fibrillar arrangements in suprabasal cells. Fibronectin was found in a linear deposit at the dermal-epidermal junction and around the fibroblasts in the reorganized collagen gels. Type-IV collagen and laminin, however, were not detected.