14 December 2019 |       Share
 
 
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CASE STUDIES
Myelotoxicity
Skin Wound Healing
Skin Toxicity Study
Endocrine Disruptors
Hepato-Toxicology
Lipid Metabolism
Carcinogenicity
Neuroinflammation
Lung Inflammation
Oxidative Stress in NDs
Estrogenic Drugs Profile

Skin Wound Healing assay 

inexpensive, direct and accurate in vivo quantification of proliferation in wound healing


The wound healing process consists of four highly integrated and overlapping phases: hemostasis, inflammation, proliferation and tissue remodeling. For a wound to heal successfully, all four phases must occur in the proper sequence and time frame. The conventional wound healing assay includes:

- in vitro
analysis encompassing three processes of tissue repair: cellular migration, proliferation and differentiation
- in vivo studies mainly on mini pig model

A complete analysis is costly, requires time, qualified technical skills and the use of a large number of animals.

TOP srl offers an accurate, highly reproducible, direct measurement of biological process of wound healing in vivo at the same cost of an in vitro assay with the possibility to screen a large number of compounds for the lead identification.

The assay developed by TOP srl is based on the measurement of the cellular proliferation associated to the wound healing by
in vivo imaging in the repTOPTMmitoIRE mice reporter mouse, a transgenic engineered to express in all cells the luciferase reporter under the control of a promoter activated in mitotic cells (Goeman et al, Molecular Biology of the Cell, 2012). In this mouse, photon emission has been demonstrated to be directly proportional to cell proliferation.


Rapid, accurate and highly reproducible measurement of the proliferative activity of a compound in vivo and in time.

In the
repTOPTMmitoIRE mice, proliferation in the wound area can be visualized and measured in vivo simply by taking a picture of the anaesthetized mouse with a CCD camera.  The extent of the regenerative effect of the treatment is directly proportional to the photon emission measured.



In vivo imaging and photon emission quantification of wound area. The repTOPTMmitoIRE mice were shaved with depilatory cream a day before the treatment. The round full-thickness wound was made cutting out a section of full-thickness skin (around 9 mm in diameter) on the dorsal skin of the animals. repTOPTMmitoIRE mice were treated with cycatrene compound (neomycin sulfate + bacitracin zinc) daily for 13 days. The CCD camera pseudoimage shows the intensity of photon emission in whole body (dorsal view) and in punch areas of repTOPTMmitoIRE mice at 1, 3, 8 and 13 days. Each point represents the mean ± SEM of photon emission measured in 4 animals/group (*p<0.001 vs 0h).

The major advantages of TOP srl assay versus conventional procedures are:

- excellent sensitivity and increased reproducibility

This assay is highly reproducible and accurate: results obtained from the bioluminescent signals have a SEM of about 3-5% compared to the signal obtained through the measurement of the wound area where the SEM revolves around 30-35%. 
The high reproducibility and the great accuracy allow the reduction of experimental animals number and the in vivo compounds screening at the same cost of in vitro assay.

- direct, in vivo quantification of the proliferative effect of the drug in the wound area and in whole body

The bioluminescence of wound area of
repTOP™mitoIRE is the measure of proliferative activity of cells which, following treatment, are responsible of the repair and healing process.
Furthermore one of the main advantages of repTOP™mitoIRE model is the possibility to use its bioluminescent signal in order to establish with a rapid analysis the occurrence of proliferative systemic effects of a compound.

- accurate measurement of the time necessary for wound healing

Through the use of our reporter system it is possible to observe the effect of compound much earlier than the traditional procedures. In fact the measurement of photon emission in wound area every day clearly shows the increase of regenerative cells proliferating in treated animals already at day 3. 
Proliferative bioluminescent signal is in accordance with wound healing: in the treated animals we effectively observe   a more rapid closure of wound than the vehicle animals.

- full compliance of the 3R regulations with a significant reduction of the number of animals to be used and of their distress

Thanks to the
repTOP™mitoIRE model it is possible a more efficient randomization of experimental groups and  to evaluate long term analysis in the same animal importantly reducing animals number.


Applications:

Wound healing activity of 
- new Chemical/Biological Entities
- new combinations of therapeutics
- novel formulation and drug delivery systems 

Types of assay available:

Short  and long term evaluation
Single and multiple treatment
Drug Combination Study