More than 100,000 transplantation worldwide makes corneal transplant one of the most common procedure performed annually. The immunologically privileged status of the eye, within the first year after transplantation, the successful rate of the eye transplant is very high. However, the corneal transplant’s prognosis is similar to that of the liver, corneal and renal transplantation. The immunologic rejection has been the top reason for allograft failure. Currently topical application of corticosteroid is used as a treatment in corneal transplantation for patient. For the patients with high risk, this treatment is not very effective. Therefore it is urgent to improve the process of the prognosis after corneal transplantation for continued therapeutic and preventive efforts.
Mesenchymal Stem Cells play an important role in regenerative medicine. Their role in delayed senescence, secretion of pro-angiogenic growth factors, and potential of immunomodulation make them very much suitable option for the wound treatment but the harsh conditions like hyperglycemia induced oxidative stress reduces their therapeutic abilities. However, to combat this problem, strategy to precondition stem cells with different chemicals has been used by researchers to enhance their therapeutic potential and functionality. The high frequency of oxidative stress in diseased and damaged tissues and adult stem cells demote functionality of adult stem cells in diabetic individuals resulting in transplant rejection. Diabetes is amongst the major risk factors involved in development of chronic wounds and may result in amputation of the body parts. The burden of transplant failure increases day by day due to accelerating rate of diabetes and aging. Organ failure results due to decreased growth factors’ secretion, waited epithelialization, collagen deposition and reduced angiogenesis in diabetic people. For preconditioning, traditionally, different medicinal approaches are used which include compounds derived from herbs (aloe vera), compounds derived from animal (propoils, honey), traditional dressings and silver (cotton wool, gauze) and other living organisms (maggots). The pharmacological agents such as N-Acetyl-L-Cysteine (NAC), Curcmuin and Cafiec acids have been used for the reduction of reactive oxygen species (ROS) in the non-obese diabetic or severe combined immunodeficient mice and showed significant improvement in engraftment of the human cells.
It is expected that the current study will help in the development of fruitful schemes for the treatment of corneal transplant in the diabetic patients.
Diabetic mice model preparation
In case of mice, the level of glucose in the blood is considered as normal if it is 80-100 mg/dl. For the induction of diabetes mellitus, mice are injected intraperitonally with a single dose of 6mg/kg streptozotocin. After 48 hours the blood sample is obtained from the tails of the mice. If the glucose level in the blood sample is more than 250 mg/dl then it is considered as diabetic.
MSC culture and expansion
Mesenchymal Stem Cells can be derived from bone marrow cells and adipose tissue of mice. For this purpose the bone marrow cells are isolated from tibia and femur and cultured at 37oC and 5% CO2. Alternatively, adipose tissues are collected from the peritoneal cavity of the mice, cleaned and meshed and cells are filtered and cultured in an incubator.
For this purpose, the exhausted medium is removed from the cell culture and it is replaced with medium having antioxidants dissolved in it.
Prior to transplantation, the mice are given the pre-conditioned MSCs through the vein of the tail, intravenously. The transplantation of orthotopiccorneal is performed for all the mice except the control group.
Flow cytometry is carried out for the characterization of MSCs. For this purpose, monoclonal antibodies are used.
Histology and histochemistry
Histological study is conducted. It includes enucleation of eyes, their fixation, dehydration, sectioning and rehydration followed by staining and observation.
Serum is obtained from the mice models after transplantation and antibody levels are detected.
RNA isolation and real-time polymerase chain reaction
Total is extracted from the cornea through Trizol reagent medium and cDNA is synthesized via reverse transcription. To determine the mRNA expression level quantitative polymerase chain reaction is performed.
Delayed-type hypersensitivity assay
For the delayed-type-hypersensitivity assay, footpad swelling is measured with engineer’s micrometer and specific footpad swelling is calculated.
For statistical analysis, GraphPad Prism software can be used.