The Role Of Specific Function-Blocking Integrin Antibodies In Burn Scarless In Vivo Healing Mechanism

Functions of Specific Integrin Antibodies in Wound Healing

Discuss about the Fluorescent and Nonfluorescent Cytosolic.

Wounds are common symptoms in skin caused by surgery or traumas. The coordinated process of wound healing restores functional barrier and epithelium integrity through blood coagulation, inflammation, in re-epithelialization caused by migration of keratinocytes, tissue formation by granulation and finally remodeling of the tissue. Integrins receptors play an essential role in all events of wound and scar healing by the forming and regulation cell adhesion (Weber et al., 2012). In this following report, we will design an experimental model in order to discuss the role of specific function-blocking integrin antibodies in healing and reducing the scars formed by burns through scarless invivo mechanism.

During the process of wound healing the, cell interaction occurs with ECM molecules in the wound comprising the integrin receptors. Evidence has showed that integrins bind with various ECM molecules and alternately the various integrin heterodimers recognizes the ECM molecules management. Therefore, based on their specific overlapping and compensating functions, many integrin based knockout animals have displayed phenotypes of wound healing. Many extensive interactions are found in the different cells of wound healing. The major components that play a key role during the interactions are ECM proteins and integrins (Olczyk, Mencner & Komosinska-Vassev, 2014). The functions of some of the specific integrin antibodies that play a vital role in wound healing are provided below:

Source: (Koivisto et al., 2014)

Thus, in order to understand the mechanism of the specific integrins tabulated above in the process of wound healing, an experimental model based on scarless in vivo is designed involved in healing the wounds due to burn. To experimental model was conducted through evaluation of three processes for proposing the animal model successfully in vivo such as indirect Immunofluorescence microscopy, Light microscopy and Western blot analysis management. In indirect immunofluorescence microscopy, light microscope combined with fluorescence microscope is used to identify the target molecules by visualizing the specificity of antibodies towards their antigen by marking with fluorescent dye such as fluorescein isothiocyanate (FITC) (Atale et al., 2014). Another procedure that will be implemented in this experiment will be through light microscopy which is used to magnify small samples with the use of visible light and lenses (Tomer et al., 2014). And finally, western blot analysis will also be done to successfully evaluate the experimental study. In western blot proteins are detected and analyzed. The proteins are separated from the gel into a membrane through the process of electrophoresis and are specifically visualized (Eaton et al., 2013).

In Vivo Scar Healing Experiment

2-month-old male and female C57BL/6 mice will be taken. The body weight should be around 25–40 grams. The male and female mice should be housed individually. The animals should have access to free drinking water and standard chow. They should be maintained on a cycle of 12 hours light and darkness. The study will be carried out in strict accordance with the recommendations by the Australian Code of Practice for the Housing and Care of Laboratory Mice, Rats, Guinea Pigs and Rabbits. The protocol will be approved by Australian and New Zealand Society for Laboratory Animal Science (Whittaker, 2014). A single surgery will be performed under Xylasine or Ketamine anesthesia, and all efforts will be maintained to reduce the animal suffering.

Even burn wounds will be ensured by shaving off the hair on the dorsum. The dorsum is considered as an ideal choice because it becomes difficult for the mice to reach the wound area and create further injuries in that region. The mouse will be placed on its back in a plastic frame template, and a window will expose a predetermined skin surface area. The exposed area from the template will be immersed in a water bath at 100°C for 8 seconds. This will inflict a thickness burn (Domergue, Jorgensen & Noël, 2015). The mice will be observed for any pain or discomfort signs and will be treated with buprenorphine if required. The temperature of water bath and the exposure time can vary.

The healing process will be monitored and evaluated for many days during the first week under a slit lamp and then once every week. Unwounded skin from identical locations of WT and FMOD^−/− animals were collected as controls. Tissue samples for histology will be bisected between two 4−0 nylon sutures and they will be fixed in 10% formalin (Wosgrau et al., 2015). After fixation, the samples will be dehydrated. They will be embedded in paraffin followed by and 5-μm section cuts for H&E staining.

For frozen sections, the tissues will be frozen in OCT, 5μm sections will be cut.Indirect immunofluorescence (IF) will be performed. The primary antibodies will be used on the mounts incubated at 4°C overnight: αMβ2, αLβ2 and αEβ7. Secondary that is conjugated to either rhodamine or fluorescein will be used with blocking conditions. The incubation period of the sections will be 1 hour in room temperature. Donkey antihuman IgG or donkey antirat IgG will be used (Stelzer, 2015). Sections will be cover slipped DAPI mounting media followed by examination under a confocal microscope or fluorescence microscope  with a digital camera. We will run negative controls where there was omission of primary antibodies. We will observe at least 3 tissues per antibody.  The captured images on the confocal microscope will be evaluated with software for image analysis like Media Cybernetics, Bethesda, MD or Image Pro Plus v.7.

Indirect Immunofluorescence Microscopy

The tissues will be fixed either in 4% paraformaldehyde for H&E (hematoxylin-eosin) or Karnovsky's with ½ strength for TEM (transmission electron microscopy). For TEM, the scarred tissues, 60–90 ? thick will be cut on ultramicrotome and examined under an electron microscope. For H&E, we will cut and stain 6-μm sections. The sections will be examined under a light microscope that has a digital camera fitted with it.

Epithelial tissue will be scraped from the mice after scarring it in by using a water bath following euthanization management. The tissues will be subjected to flash freezing in liquid nitrogen. Furthermore, they will be processed for Western blot analysis. Eight sections of tissues will be used for each experiment. Tissues will be homogenized and lysed. Protein will be subjected to purification and assay using Biorad; Hercules, CA. Total proteins will be loaded in equal amounts on a nonreducing Tris-glycine gel (4%–20%). The proteins will be transferred to an Immobilon-P or Millipore membrane which will be stained with Sigma or Ponceau S. This will check transfer efficiency (Taylor & Posch, 2014). Membranes will be blocked with TBS containing 5% milk and probed with the primary antibodies αMβ2, αLβ2 and αEβ7and will be incubated at 4°C overnight. These membranes will be incubated with a secondary antibody that is conjugated with HRP at room temperature for 1 hour: donkey antihuman IgG (αMβ2) or donkey antirabbit IgG (αEβ7). Chemiluminiscence with Millipore will be used to visualize immunoreactive bands. The same procedure will be repeated thrice. ImageJ v.1.5 software will be used to measure the intensity of the bands. Prism 5.0 will be used to plot the fold enhancement values.

Conclusion

Thus from the following discussion, an experimental design can thus be constructed in order to study the underlying mechanism of some specific integrins involved in improving the healing and thereby reduce the scar formed in burns. From this experiment it can be detected whether the specific integrins have been upregulated inside the epithelium during the healing of burn scars in mice. This will provide evidence for understanding the mechanisms of excessive scarring and wounds. It will help to design novel therapeutic approaches and interventions that will utilize integrins as targets for scar reduction.

References

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Domergue, S., Jorgensen, C., & Noël, D. (2015). Advances in research in animal models of burn-related hypertrophic scarring. Journal of Burn Care & Research, 36(5), e259-e266.

Eaton, S. L., Roche, S. L., Hurtado, M. L., Oldknow, K. J., Farquharson, C., Gillingwater, T. H., & Wishart, T. M. (2013). Total protein analysis as a reliable loading control for quantitative fluorescent Western blotting. PloS one, 8(8), e72457.

Koivisto, L., Heino, J., Häkkinen, L., & Larjava, H. (2014). Integrins in wound healing. Advances in wound care, 3(12), 762-783.

Olczyk, P., Mencner, ?., & Komosinska-Vassev, K. (2014). The role of the extracellular matrix components in cutaneous wound healing. BioMed research international, 2014.

Stelzer, E. H. (2015). Light-sheet fluorescence microscopy for quantitative biology. Nature methods, 12(1), 23-26.

Taylor, S. C., & Posch, A. (2014). The design of a quantitative western blot experiment. BioMed research international, 2014.

Tomer, R., Ye, L., Hsueh, B., & Deisseroth, K. (2014). Advanced CLARITY for rapid and high-resolution imaging of intact tissues. Nature protocols, 9(7), 1682.

Weber, C. E., Li, N. Y., Wai, P. Y., & Kuo, P. C. (2012). Epithelial-mesenchymal transition, TGF-β, and osteopontin in wound healing and tissue remodeling after injury. Journal of burn care & research: official publication of the American Burn Association, 33(3), 311.

Whittaker, A. (2014). Animal research regulation in Australia-does it pass the test of robustness?.

Wosgrau, A. C. C., da Silva Jeremias, T., Leonardi, D. F., Pereima, M. J., Di Giunta, G., & Trentin, A. G. (2015). Comparative experimental study of wound healing in mice: pelnac versus integra. PloS one, 10(3), e0120322.