The presence of areas of hypoxia is a prominent feature of various inflamed, diseased tissues, including malignant tumors, atherosclerotic plaques, myocardial infarcts, the synovia of joints with rheumatoid arthritis, healing wounds, and sites of bacterial infection. These areas form when the blood supply is occluded and/or unable to keep pace with the growth and/or infiltration of inflammatory cells in a given area. Macrophages are present in all tissues of the body where they normally assist in guarding against invading pathogens and regulate normal cell turnover and tissue remodeling. However, they are also known to accumulate in large numbers in such ischemic/hypoxic sites. Recent studies show that macrophages then respond rapidly to the hypoxia present by altering their expression of a wide array of genes. In the present study, we outline and compare the phenotypic responses of macrophages to hypoxia in different diseased states and the implications of these for their progression and treatment.

Hypoxia Regulates Macrophage Functions in Inflammation

Radiation therapy is an established modality in the treatment of head and neck cancer patients. Compromised wound healing in irradiated tissues is a common and challenging clinical problem. The pathophysiology and underlying cellular mechanisms including the complex interaction of cytokines and growth factors are still not understood completely. In this review, the current state of research regarding the pathomechanisms of compromised wound healing in irradiated tissues is presented. Current and possible future treatment strategies are critically reviewed.

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Wound healing after radiation therapy: Review of the literature

Rats have been widely used in the study of skin wound healing and the efficacy of different treatment modalities. This particular animal species is often selected for its availability, low cost, and small size. To define the current use of rat skin wound healing models, this manuscript provides a review of articles published between 2000 and 2003 that chose rats as their research animals. Of the 55 articles reviewed, it was found that 38.2% of the studies used incisional models and 38.2% used excisional models, with some studies using combinations. The majority of the studies (78.2%) used the rat's dorsum as the wound location. Male Sprague Dawley in the 250-300 gram weight range were the most preferred rats. Sodium pentobarbital/pentobarbitone was the most commonly used anesthetic choice. Similarities and differences in the selected experimental conditions are noted and questions are raised with regard to comparability between studies and the ability to transfer the data from the animal model to the human clinical situation. Attempts to compare studies for the advancement of wound healing knowledge are being hampered by the differences found between the studies. Standardization in reporting could facilitate comparisons and may instigate additional research that favors the inevitable comparisons between the studies. Thus, universal reporting requirements need to be developed for animal wound healing studies.

Rat models of skin wound healing: A review

Radiotherapy is an invaluable weapon when treating cancer. However, the deleterious effects of radiation, both immediate and long-term, may have a significant effect on local tissues. Problematic wound healing in radiation-damaged tissue constitutes a major problem that is frequently overlooked during the management of patients who require radiotherapy, or have had radiotherapy in the past. Poor wound healing may lead to chronic ulceration, pain, secondary infection and psychological distress and compromise the outcome of general or reconstructive surgery. We discuss the pathophysiology of poor wound healing following radiotherapy, specific problems for radiation-damaged tissue and potential treatments to improve wound healing of irradiated tissues.

Radiotherapy and wound healing.

Chronic wounds are a major clinical problem that lead to considerable morbidity and mortality. We hypothesized that an important factor in the failure of chronic wounds to heal was the presence of microbial biofilm resistant to antibiotics and protected from host defenses. A major difficulty in studying chronic wounds is the absence of suitable animal models. The goal of this study was to create a reproducible chronic wound model in diabetic mice by the application of bacterial biofilm. Six-millimeter punch biopsy wounds were created on the dorsal surface of diabetic (db/db) mice, subsequently challenged with Pseudomonas aeruginosa (PAO1) biofilms 2 days postwounding, and covered with semiocclusive dressings for 2 weeks. Most of the control wounds were epithelialized by 28 days postwounding. In contrast, none of biofilm-challenged wounds were closed. Histological analysis showed extensive inflammatory cell infiltration, tissue necrosis, and epidermal hyperplasia adjacent to challenged wounds-all indicators of an inflammatory nonhealing wound. Quantitative cultures and transmission electron microscopy demonstrated that the majority of bacteria were in the scab above the wound bed rather than in the wound tissue. The model was reproducible, allowed localized cutaneous wound infections without high mortality, and demonstrated delayed wound healing following a biofilm challenge. This model may provide an approach to study the role of microbial biofilms in chronic wounds as well as the effect of specific biofilm therapy on wound healing.

Delayed wound healing in diabetic (db/db) mice with Pseudomonas aeruginosa biofilm challenge: a model for the study of chronic wounds

Radiation-impaired wound is characterized by delayed healing, nonhealing, and carcinogenesis. The mechanism remains unclear. Matrix metalloproteinases (MMPs) are one family of key regulators of the process of wound healing. Their abnormal expression plays important roles in the formation of some chronic skin ulcers. The objective of this project was to study the expression of MMP1 in surgical and radiation-impaired wound healing and its effects on the healing process and tissue remodeling. A rat model of radiation-impaired wound healing was used. Routine light microscopy, electron microscopy, immunohistochemistry, and in situ hybridization, all of which enabled the detection of MMP1 expression during the healing process, were performed. The wound healing process was impaired and delayed. In rats receiving 25Gy gamma-ray locally, the irradiated wounds healed 6 days later than the nonirradiated controls. The following changes in MMP1 expression were found: (1) In the early inflammatory phase and in the period of granulation tissue formation, MMP1 expression was only slightly if at all affected in the newly formed epidermis of irradiated wounds compared with controls. Later, the epidermal expression of MMP1 in radiation wounds was comparatively increased following the delay of the healing process. (2) MMP1 expression in irradiated wounds was markedly decreased in fibroblasts, endothelial cells, and macrophages compared with controls. The expression phase was prolonged because of the delay of the healing process. The reduced expression of MMP1 in granulation tissue retards such important processes as cell migration, angiogenesis, and tissue remodeling, thus slowing the healing process. The expression ofMMP1 in the proliferating keratinocytes may help re-epithelialization. However, in the late healing period, overexpression of MMP1 in the epidermis may hinder the establishment of basal membrane and the formation of granulation tissue, and affect the tissue remodeling process.

Expression of MMP1 in Surgical and Radiation-Impaired Wound Healing and Its Effects on the Healing Process

We studied the apoptosis of mouse circulating lymphocytes and its mechanism induced by 2, 4, 6, and 8 Gy of whole-body γ-irradiation and the expression of bax and bcl-2 gene products as related to apoptosis. We found that, in the early stage after irradiation (4th-7th day), the percentage of lymphocyte apoptosis increased rapidly. Four hours after 2, 4, 6, and 8 Gy of γ-irradiation, the apoptotic lymphocytes were 2.6, 3.8, 5.5, and 10.4 times those in the controls, respectively. A good correlation was found between the intensity of apoptosis and the radiation dose. As the radiation dose increased, the absolute counts of peripheral lymphocytes decreased sharply; 4 hours after 2, 4, 6, and 8 Gy of γ-irradiation, the lymphocyte counts were 82, 63, 47, and 22% of the controls, respectively. The peak value of lymphocyte apoptosis was observed on the 7th day after irradiation using the in situ terminal labeling method, and the apoptotic lymphocytes were found to be 16 times the number in the controls. These results were in accordance with those obtained by the May-Grunwald-Giemsa staining method. The absolute counts of peripheral lymphocytes dropped to their lowest value on the 7th day after irradiation, suggesting that lymphocyte apoptosis might be the major cause of lymphocytopenia in the early stage after irradiation. The abnormal expression of bax and bcl-2 gene products in irradiated lymphocytes was closely related to apoptosis in peripheral lymphocytes.

Apoptosis of circulating lymphocytes induced by whole body gamma-irradiation and its mechanism.

Objective To study the relationship between the expression of Bax, Bcl-2 proteins, and apoptosis in radiation compound wound healing of rats. Methods Apoptosis, Bax and Bcl-2 proteins were estimated by in situ terminal labeling (TUNEL) and immunohistochemical methods. Results (1) Changes of the apoptosis in wound healing showed three typical characteristics: early occurrence, high frequency and delayed disappearance after radiation to rats when compared with those of simple wound group, which might be an important reason for radiation-induced delayed wound healing. (2) The expression of Bax protein increased evidently with the increment of apoptosis and showed a good corresponding relationship with the apoptotic frequency in the process of wound healing. While the expression of Bcl-2 protein decreased obviously as the apoptosis reached a maximum and showed increasing tendency up to normal level when the apoptosis decreased distinctively. Conclusions Bax and Bcl-2 proteins play an important role in the apoptotic regulation of radiation compound wound healing in rats.

Relationship between expression of Bax and Bcl-2 proteins and apoptosis in radiation compound wound healing of rats.

The expression of endogenous interleukin-3 (IL-3) gene in mouse bone marrow after whole body irradiation was assessed by immunocytochemistry, in situ hybridization (ISH), and in situ reverse transcription PCR (IS RT-PCR). The results showed obvious injury to the bone marrow followed by recovery 4 weeks after radiation. IL-3 protein was increased in the cytoplasm of recovering hematopoietic cells, especially at 21 days after radiation, while the mRNA was negative, except at 15 days when the mRNA was weakly positive by ISH. IS RT-PCR showed that IL-3 mRNA was strongly positive in the cytoplasm of recovering hematopoietic cells, especially at 10 to 15 days after radiation. We postulate that endogenous IL-3 gene may accelerate hematopoietic recovery in irradiated bone marrow and that IS RT-PCR may objectively report the ragulation of IL-3 gene expression.

Interleukin-3 gene expression in irradiated mouse bone marrow.

Apoptosis of hemopoietic cells in bone marrow following radiation has been rarely reported, let alone studied quantitatively and pathologically. LACA mice were irradiated with 2.5, 4.0, 5.5, and 7.0 Gy gamma-rays, and the bone marrow was examined at 6 hours, 1 day, and 3 days after radiation. Semi-thin and thin sections were examined by light and electron microscopy. The number and area density of the apoptotic cells were assessed by means of a Cambridge Quantimet 970 Image Analyzer. We found that apoptosis occurred in only a few hemopoietic cells in the control mouse bone marrow, whereas 6 hours after radiation there were many apoptotic hemopoietic cells in each sample of irradiated bone marrow. Compared with controls, both the number and area density of the apoptotic cells markedly increased in the bone marrow of animals in every radiation dose group, and the difference was statistically significant (p < 0.01). Furthermore, the number and area density increased as the radiation dose increased. Our findings suggest that apoptosis is the main mode of radiation-induced hemopoietic cell death.

Hong Yang

Papers

Expression of MMP1 in Surgical and Radiation-Impaired Wound Healing and Its Effects on the Healing Process

Apoptosis of circulating lymphocytes induced by whole body gamma-irradiation and its mechanism.

Relationship between expression of Bax and Bcl-2 proteins and apoptosis in radiation compound wound healing of rats.

Interleukin-3 gene expression in irradiated mouse bone marrow.

Apoptosis of hemopoietic cells in irradiated mouse bone marrow.