Uncontrolled blood sugar is a major cause of vascular complications and delayed wound healing in diabetes mellitus. During wound healing process, normally, apoptosis is responsible for events such as removal of inflammatory cells and evolution of granulation tissue into scar which occur during the late phase of wound healing. Early apoptosis can lead to abnormal wound healing by removing granulation tissue including fibroblast, endothelial cell, and small vessels. To determine the role of apoptosis in association with hyperglycemia in diabetic wound healing, apoptosis-related intracellular marker such as expression of Bcl-2 protein by immunohistochemistry and normal histology has been studied. Histological findings show higher level of apoptosis and diminished granulation tissue formation in diabetic rats wounds along with minimal expression of Bcl-2 in diabetic rats wounds when compared with nondiabetic rats wounds. It can be concluded from this study that elevated blood sugar level may be associated with increased apoptosis and the least expression of Bcl-2 protein which might cause deregulation of the wound healing processes in streptozotocin-induced diabetic rats.
Apoptosis is a physiological cell death process, possesses very distinct features, and is almost different from necrosis which is characterized by morphological changes, including DNA fragmentation, membrane alteration, and formation of apoptotic bodies. It is one of the physiological cell death processes, which governs developmental biology and cellular homeostasis in an organism. There is enormous kind of evidence that the sequence of cellular events that characterize healing of cutaneous wound and other tissue repair processes is tightly regulated and controlled by a distinct temporal pattern of cellular apoptosis [
Immunohistochemical expression of Bcl-2 has been reported in several lymphatic, neurogenic, and epithelial neoplasms [
All animal procedures were performed with the approval of the Animal Ethical Use and Care Committee at the North Eastern Hill University, School of Life Sciences, Shillong, India. They were housed under ideal laboratory conditions, maintained on standard pellet diet, and watered
Four days after streptozotocin injection, the animals were tested for glucose in blood by using commercially available glucose estimation kit to confirm the diabetes. Animals having blood sugar level greater than 250 mg/dL were included in the study.
After confirmation of diabetes, rats were anesthetized with ketamine (80 mg/kg body weight) intraperitoneally, and full-thickness excisional wounds were made on the back of the eight-week-old rats in 8 mm diameter. Biopsy specimens were obtained at the 5th, 10th, 20th, and 30th days after preparation of wounds and kept in 10% neutral buffered formalin for histology and immunohistochemistry.
Wound tissue was excised, ringed in cold phosphate buffer saline (PBS), and fixed in 10% neutral buffered formalin. After washing with distilled water, tissue was placed in dehydration process through graded ethanol. After two changes with xylene a block was prepared and cut into 5 mm thin sections by microtome machine. Sections were stained with hematoxylline and eosin, mounted, and finally observed under light microscope.
DNA fragmentation was studied by the methods described elsewhere [
Five µm thick sections were cut from the specimens and placed on poly-L-lysine-coated slides. They were then dewaxed in xylene and rehydrated in graded alcohol. The endogenous peroxidase activity was consumed by immersing the sections in 0.1% hydrogen peroxide in absolute methanol for 20 minutes. Nonspecific binding was blocked by incubating the slides in 20% fetal calf serum in PBS for 20 minutes. A monoclonal antibody directed against Bcl-2 oncoprotein was obtained from Santa Cruz Biotechnology, USA. Before application of the primary antibody, the sections were heated in a microwave oven in 10 mM citric acid monohydrate, pH 6.0 for 3 minutes. After one hour of incubation with the primary antibody (dilution 1 : 50) a biotinylated secondary anti-mouse antibody was applied followed by the HRP-biotin-peroxidase complex. The color was developed by adding diaminobenzidine and mounted. Negative control was prepared by omitting the primary antibody. For positive control follicular lymphoma was used.
The mean blood sugar level in control group rats on the 5th, 10th, 20th, and 30th days was 75.62
Mean blood glucose level, apoptotic index, and DNA fragmentation in control rats.
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Mean blood glucose level, apoptotic index, and DNA fragmentation in rats with diabetes.
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At the earliest time point provisional matrix with inflammatory cells and dilated blood vessels could be observed underneath a newly formed epithelial layer. Reepithelialization was completed after wounding. Maturation of granulation tissue was detected and characterized by the presence of new blood vessels, inflammatory cells, and collagen fibers organized into a dense connective tissue in control group (Figure
Histological findings in control rats and rats with diabetes. (a) The migration of inflammatory cells, formation of new blood vessels, and tissues architecture (indicated by arrow) in control rats and (b) diminished formation of granulation tissue, provisional matrix, and delayed healing in wounds of rats with diabetes (indicated by arrow) (H & E staining).
Control rats
Rats with diabetes
Apoptotic cells in control group and in diabetic rats wounds group were identified on the basis of morphological features that induced central cell apoptotic bodies, uniformly condensed chromatin, and densely stained nucleolus or membrane bound apoptotic bodies containing one or more nuclear fragments. The apoptotic cells were determined by counting more than 300 cells in at least three separate regions. Apoptotic cell death was monitored by morphological analysis that showed increased number of apoptotic cells in diabetic wound group on the 30th day (Figure
Distinct number of apoptotic cells were found and observed in both groups, but it was significantly higher in wounds of rats with diabetes (b) when compared with control group (a) (H & E staining) (arrow clearly showed the apoptotic cells characterized by nuclear condensation and fragmentation in both groups).
Control rats
Rats with diabetes
The Bcl-2 staining and its expression as protooncogene were observed in granulation tissue of control and diabetic rats wounds on the 30th day. Bcl-2 staining and expression were positive and more intense in control rats wounds. In the case of diabetic rats wound, no Bcl-2 positive staining or Bcl-2 expression was observed as the time advances (Figure
Immunohistochemical localization and expression of the bcl-2 (antiapoptotic protein) in (a) control rats and (b) wounds of rats with diabetes.
Control rats
Rats with diabetes
Wound healing processes can be divided mainly in three distinct stages: (i) fibroblast proliferation and secretion of mucopolysaccharides, (ii) replacement of mucopolysaccharides by collagen molecule and maturation of these molecules by cross-linking, and (iii) remodeling and scar formation. Cell proliferation, protein synthesis, and protein release are also evaluated in wound healing process. Healing of wounds in diabetes is characterized by delays in the repair process as well as the decrease in the tensile strength of healing wounds. Deficiencies in fibroblast numbers have been reported to represent an important aspect of delayed wound healing in diabetes. It has been suggested that aberrant growth factor expression, altered inflammatory responses, or enhanced glycosylation of proteins may be involved alternatively; enhanced apoptosis may decrease fibroblast numbers, which could contribute to the impaired diabetic healing. In this study we found that inflammatory cells and dilated blood vessels could be observed underneath a newly formed epithelial layer in nondiabetic rats. Maturation of granulation tissue was detected and characterized by the presence of new blood vessels, inflammatory cells, and collagen fibers organized into a dense connective tissue in control group (Figure
Apoptosis has been identified as the mechanism of cell death in a number of degenerate diseases. Apoptosis is an active process that has described well biochemical and morphological characteristics including lack of inflammatory response and DNA fragmentation caused by endonuclease activity. It is identified in histological sections by pyknotic nuclei, cytoplasmic condensation, and DNA fragmentation which can be stained by in situ end labeling (TUNEL) of single or double strands break in DNA. The study reports that both experimental diabetes in rats and diabetes mellitus in humans are accompanied by increased apoptosis and biochemical changes in wound healing processes. It was found that inflammatory cells undergo apoptosis [
Apoptosis has also been implicated in dermal reconstruction. In a study by Rossio-Pasquier et al., athymic nude mice were grafted with split-thickness human skin biopsies [
Kane and Greenhalgh further the role of apoptosis-related markers during inflammation at the leading edge of the epithelium was explored [
The authors hereby certify that there is no conflict of interests with any financial organization regarding the material discussed in this paper.
The authors would like to thank the Council of Scientific and Industrial Research (CSIR) for providing the financial help.