In order to optimize The hamster model for cutaneous Le Moases. Here we describe an approach using an intradermal inoculation of pro magos end dorsal skin. This approach has proven its versatility and easy management at the moment of inoculation follow up and characterization of typical lesions, application of treatments through different ways, obtaining of para clinical samples, obtaining of data of the different promising compounds without it meaning a significant detriment in the model's life quality.
Regarding locomotion, search for food and water play and Social activity with careful movements, sure and Steady, the animal must be restrained by the base of the neck using the fingers, taking advantage of the large amount of skin that the hamster has. The palm of the hand is used to provide the animal a supporting surface in which it feels comfortable. Once the animal feels relaxed, the skin of the neck and pouches can be stretched with the index finger and thumb, and with the rest of the fingers hold the skin of the bag tightly against the palm, so the animal is completely immobilized.
A distinctive feature in the female is a visualization of the Mi Mary line, which is well evidenced from the first week of age. Another characteristic is the short anogenital distance. As for the sexing of the male, the squirrel sac containing the testicles can be observed.
In addition to, and as a consequence of this, there is a greater distance between the anus and the four skin. The animal is grasped holding the base of the neck tightly and placing it in a trap or box that is conditioned on a weighing scale, previously calibrated after a few seconds. When the animal is comfortable in this space, the weight reading is done and is immediately registered in the appropriate format.
The most common methods for the identification of hamsters are the staining of an area of the skin with piri acid and ear piercing for the staining. With piri acid, the procedure is as follows, grasp the animal as mentioned before, and with a swab soaked in piri acid permeate the region to mark usually the anterior or posterior limb left or right with a quantity enough to ensure that the mark will last until the end of the test. For this method of identification, it is recommended to have the animal under sedation or anesthesia.
Although it is not strictly necessary, the U to tag is selected and disinfected with alcohol. After this, the area to perforate is selected, avoiding the region with blood vessels. Then the year is pierced using a ear punch for rodents, the procedure must be done quickly, but in a firm and safe manner.
Given that in a cage there should be two to three animals. Up to two holes can be made per year. To apply the anesthetic to the animal, it should be held as stated in the beginning in such a way as to prevent the movement of the hamster with the animal.
In supine position, the abdominal area in which the drug will be applied is disinfected, using 70%alcohol and dried with a clean cotton. The intraperitoneal route is used with the animal slightly tilted towards the head so that visceral displacement occurs. The needle is completely introduced a bit parallel into the spine.
An initial aspiration is done to confirm that no organs have been punctured, and then the drug is applied to shave the skin area to be inoculated with the animal. Improved position, a point is marked two centimeters from the base of the tail. With the use of scissors, all the hair is removed, clearing an area of about two square centimeters carefully to avoid injuries in a biological safety cabin, and with the hamster anesthetized in a one milliliter syringe, 100 microliters of the known quantity of logarithmic phase.
Pro maus of leishmania is served. The needle must be secured on the syringe as a precaution to avoid accidents. Cotton with sterile saline solution is used to clean the shaved area of the animal in one direction.
Afterwards with the needle bevel upwards, the skin of the hamster is penetrated, making sure the needle enters the sub dermis in a slow and gradual manner. The inoculum is injected to form a subdermal pape to remove the needle rotated slightly and pull. All material used is discarded.
Following biosecurity standards to monitor skin lesions produced by leishmania, initial immobilization of the animal is recommended in a trap to facilitate the process For an accurate measure of a lesion. Palpation of the inoculated skin area is done to delineate the induration and formed ulcer with the help of a digital caliber, the width of the ulcer is covered, and measurement in millimeters is recorded in a format designed for clinical monitoring. Similarly, the length of the ulcer is measured, which must also be registered after the subdermal inoculation.
There are between four to five weeks of waiting time to obtain typical lesions comparable to those presented. In humans. The animal is immobilized in a special tract that enables you to expose the back where the lesion is.
Using a eye dropper, the compound is added on the injury and the liquid is left to dry before returning the animal to its cage. With the animal securely fastened as illustrated above the most skilled hand is used to disinfect the area with alcohol and cotton in one direction. Then the same hand is used to manipulate the syringe containing the drug to be used in a maximum volume of 200 microliters.
The drug is injected through the semi tendus of semi tenderness or semi berness muscles of the hindquarters. If the drug must be applied for several days, a switch in limbs should occur to avoid discomfort and damage to the animal with a less skillful hand, the animal is fastened as previously indicated. The other hand is used to manipulate the or gastric probe.
14 garage that connects to a one milliliter syringe to comfortably provide an amount not greater than 200 microliters. The hamster is securely fastened with an immobilizing device exposing the area to be treated, which is cleaned with sterile cotton and saline solution with a single sweep in one direction with a one milliliter syringe, the bevel down and a volume no greater than 100 microliters, a single subdermal approach by a vertex of the lesions performed and gradually the drug is deposited, covering the entire area of the lesion prior to sacrifice. Animals are anesthetized as described before for the process of euthanasia.
The hamster is taken to a sealed acrylic chamber that is connected to a cylinder or a network of carbon dioxide. The valve is slowly open for several minutes for sampling the sacrificed hamsters fixed onto a dissection surface. Glass lights are needed for imprints of skin and internal organs.
15 milliliter falcon tubes with 10%formin for histopathology samples, and the material includes dissection, scissors, tweezers, scalpel, handle, and blades. Finally, the presence of leishmania in samples from skin lesion or scar, liver, spleen, kidney, and popal lymph nodes is determined directly by examining smears stained with genesa and histopathologic examination of these tissues. A biopsy specimen is fixed in 10%formalin and embedded in paraffin.
Five micron sections of the fixed tissues were stained with hemat, toin eoin, and examine under an optical microscope using 1000 oil immersion. To study the micro architecture, the characteristics of the cellular infiltrate and the presence or absence of parasites. Photo micrographs are taken and digital images captured.
All materials should be discarded following the biosafety regulations for biological waste disposal. The golden hamster Mi has proven particularly useful for studying cutaneous le esis caused by both old and new world leishmania species. Inoculation of pro magos is able to cause cutaneous lesions between one and two months post inoculation.
The hamster displace a predictable disease evolution after experimental infection characterized by development of lesions similar to those observed in human beings due to the chronic nature of cutaneous le esis in the hamster model, the monitoring of therapeutic interventions over long periods is allowed. Traditionally, hamsters are experimentally inoculated in the snout or the foot pad, however, in these sites and notes are not always occurs. Moreover, measurement of lesion size is hard to do and the hamsters show difficulties to eat, breathe, and move.
In summary, this approach demonstrates that the clinical pathologic features of cutaneous le meeses induced in the dorsal skin of hamsters are remarkably similar to the human disease, but strikingly different from the commonly used marine model. Thus, the experimental infection in the dorsal skin of hamsters represents a useful model to validate the potential of compounds that are candidates for an H manual drugs.
