Maxfacts

Some general considerations

High-energy photon irradiation is a well-established, and often successful, treatment modality in the management of many different malignancies and some other conditions. Unfortunately, this success comes at the price of a wide range of common short- and long-term adverse effects. In this regard, radiation therapy applied to the head & neck region is not different to radiation therapy applied to other regions of the body. There are common underlying mechanisms of the body’s attempts at healing radiation injuries and the resulting damage, irrespective of the irradiated body region. What differs for different irradiated regions of the body are the respective adverse effects on function(s), these are specific to specific regions of the body. For example, after irradiation in the breast region, lung and heart tissues are at risk of fibrosis formation, with corresponding ill effects. Adverse effects afflicting healthy structures following high-energy photon irradiation in the head and neck region are a consequence of the very dense structure of different vital functions in this relatively small region of the body. It may be useful to subdivide these adverse effects not just into short- and long-term effects, but to think about effects related to direct tissue damage (including dermatitis and mucositis, soft- and hard tissue necroses, damage to salivary glands leading to xerostomia, dysphagia, dental caries and difficulties with speech, late secondary malignancies) and adverse effects related to the body’s attempts to heal the radiation injuries (mainly fibrosis, for example leading to trismus, dysphagia or a painful, stiff neck (‘woody neck’)). Below we take a look specifically at fibrosis and the pathology of healing of radiation injury.

Challenges with researching the pathology of wound healing after radiation injury

Given that fibrosis, of variable severity, is a common long-term effect following high-energy photon irradiation and has been known for a long time, one might expect that the pathology of healing of radiation injury should be a well-researched and well-understood process. Unfortunately, this is not the case and there are several conflicting hypotheses about the pathology of this process (some older hypotheses have been shown to be wrong, or too simplistic, or based on a confusion between causes and correlations). When researching the pathology of healing of radiation injury, it is not really possible to design meaningful laboratory investigations on cell cultures and monitoring single parameters. Neither are investigations using animal models particularly meaningful as the physiology & pathology of, for example, rodents will be different from that of humans. Part of the challenges to investigate this healing process relates to the fact that radiation injury typically results from a series of injuries to healthy tissue over a period of time, the period of a radiation-therapy course. This is difficult to model in a laboratory experiment using cell cultures. Probably more importantly, however, the real difficulty for such research stems from the fact that wound healing following radiation injury is a complex process taking place in a complex tissue environment that keeps changing and adapting over time. Accordingly, any attempts to associate overall effects with single causes are bound to be wrong; healing of wounds in general, and in particular of radiation injuries, are multi-factorial processes. Thus, observation of the course of events and outcomes for patients, combined with some laboratory experiments, after careful interpretation appear to be the best current route to improved understanding of these healing processes and the reasons for things to go wrong, leading to fibrosis, the long-term result of the body’s attempt to repair tissue damage after radiation injury. Still, many questions remain. For example, there is a correlation between the occurrence of initial dermatosis/mucositis and late-onset fibrosis. However, there is no clear correlation between the severity of the initial reaction/inflammation (dermatosis and/or mucositis) and long-term development of severe fibrosis, adding to the difficulty to identify people at risk of developing severe fibrosis later on. The remaining gaps in the understanding of the pathology following radiation injury also account for the failure to date of a number of mitigation / treatment attempts.

Course of events after radiation injury

The wound healing processes following radiation injury are different from the processes involved in the healing of mechanical (surgical), chemical or biological (infection) injuries in several respects. It is, therefore, instructive to compare the processes, identifying similarities as well as differences.

Healing response to normal traumatic injury.

The body’s response to normal traumatic tissue injury consists of a series of finely balanced events, all taking place in an organised way involving many tissue components. It is an immediate reaction to a singular event, and it consists of a series of overlapping orchestrated steps, stirred through and controlled by the consecutive release and removal of a large number of different cyto- and chemokines and growth factors (all types of messenger molecules) at different stages of the process. First, trauma triggers activation of the coagulation system (clotting is the natural mechanism to stop bleeding). This is followed by an inflammatory response (to attract a variety of messenger molecules to the site of injury) to initiate epithelial regeneration (re-growth of the thin outer layer of body structures by replenishing lost cells), formation of granulation tissue (to protect the injury site during further healing), and finally deposition of new connective tissue components (for example, different types of collagen alongside formation of new blood vessels) in the extracellular matrix (the spaces surrounding cells) and ongoing remodelling to provide a stable repaired tissue structure. This process typically reaches the stage of a mature scar over a period of 18 months, but further remodelling can continue for years.

Healing response to radiation injury.

An important difference is the repeating nature of tissue injury over a course of radiotherapy. ‘Normal/healthy’ tissue surrounding a tumour will change considerably over a course of delivery of irradiation fractions. The initially normal tissue will become increasingly inflamed wherever it is repeatedly exposed to high-energy irradiation, so that the initially normal tissue will be a significantly different form of ‘normal’ tissue after delivery of a course of irradiation.

Also following radiation injury, initially the coagulation system is triggered, even if no bleeding occurs from disrupted blood vessels. It is thought that reactive oxygen species formed by irradiation cause the immediate inactivation of thrombomodulin (a membrane protein that regulates and in particular reduces coagulation) on the endothelial cell layer (the thin lining) of the affected blood vessels which is further enhanced by inflammation. Subsequently, the expression and function of all tissue (and other) factors and proteins are altered, and in this way help to establish a permanently altered structure and behaviour of the endothelial cell layer. In essence, these changes amount to a permanently established pro-coagulant endothelial cell layer in irradiated blood vessels and are likely responsible for the initiation and continuation of a delayed radiation injury. Some altered factors, in particular thrombin (an enzyme that regulates coagulation as well as cell proliferation, inflammation, collagen deposition and endothelial permeability) have additional lasting effects on the healing and remodelling behaviour.

A second aspect where the healing processes of traumatic and radiation injuries differ from each other concerns the inflammatory response to injury. Typically for a traumatic wound, the blood levels of inflammation-driving factors increase rapidly in order to initiate further healing at the site of injury. The inflammatory response then reduces quickly because all of these pro-inflammatory factors and molecular messengers have a short life span once released; in addition, the onset of the production of anti-inflammatory factors at this stage further curbs inflammation. Overall, after a traumatic injury the inflammatory response is a short episode (unless wound infection occurs). In contrast, the inflammatory response to radiation injury does not decrease in such a controlled and rapid manner. The inflammatory response to radiation exposure starts quickly, most probably caused by direct cell damage and the endothelial layer of exposed blood vessels, leading to vasodilation and redness and inflammation of the skin. The release of thrombin (see above), histamine several prostaglandins and other factors drive this reaction. This inflammatory reaction does not resolve rapidly and is believed to lead to the long-term development of a perpetuated inflammatory environment, which in turn is believed to underpin the uncontrolled and ongoing deposition of ‘wrongly constructed’ connective tissue – the cause of fibrosis. There is little chance of these inflammatory response(s) to resolve over a course of radiation therapy: each irradiation fraction adds to the inflammatory response which, consequently, accumulates over time (‘fractionated inflammatory insult’). In fact, repeated injury and ongoing inflammatory responses enhance each other. The exact profiles of the inflammatory reactions as a function of timing and dose patterns of irradiation schemes are not well investigated. Similarly, there are no definitive investigations into the corresponding effects of larger areas of lower-dosage exposure from some IMRT schemes (intensity modulated radiation protocols), compared with ‘traditional’, non-modulated radiotherapy schemes involving smaller areas with high-dosage exposure.

In the healing process of both traumatic and radiation injuries, the inflammatory response overlaps with, and triggers, the beginning of tissue rebuilding. In the healing of a traumatic wound, this process of ramping up tissue repair runs in parallel to a controlled and orderly ramping down of the inflammatory response. In contrast, the unresolved and dysregulated inflammatory response following radiation injury leads on to a similarly dysregulated process of tissue rebuilding over long periods of time. Some of the visible reactions to radiation injury (skin and mucosa) tend to resolve relatively quickly after a course of radiation therapy and are thought to be related to the activation of epithelial stem cells.

Nevertheless, there are ongoing transformation processes which carry on compromising healing and contribute to the delayed healing and development of long-term effects such as fibrosis. The derailed and disorganised tissue rebuilding starts early in the process with the creation of so-called inflammatory infiltrates, driving the differentiation of fibroblasts into fibrocytes which, together with changes in the vascular endothelial tissue over time lead to the excessive and ongoing production and deposition of extracellular matrix proteins and collagen (the connective tissue surrounding cells). Of the multiple factors driving this perpetuated process, the transforming growth factor beta 1 (TGF-β1) is the main factor driving the conversion of fibroblasts (needed for tissue rebuilding) into myofibroblasts (responsible for the excessive deposition of connective tissue). Once myofibroblasts have been activated, the production of excessive connective tissue can continue permanently by activation of another factor, connective tissue growth factor. Despite the central role of TGF-β1 in the process, a large number of further cytokines and growth factors are involved in the complex network of interacting factors. Some key stages of the pathological healing process can be identified. Ahead of the establishment of fibrosis, chronic inflammation and its effects on endothelial cells with related vascular effects play a key role. In the next stage, fibrosis forms in patchy active areas with high levels of active myofibroblasts in an already poorly organised extracellular matrix. The development converges to a long-term late phase which is characterised by the presence of atrophic tissue, retractile fibrosis and increasing loss of parenchymal cells (loss of functional tissue). A simplified view / summary of this ongoing dysregulated process may be to consider the development of fibrosis a failure of the controlled down-regulation of normal fibrogenesis as occurs in the healing of traumatic wounds.

The finer details of the physiological and biochemical mechanisms that lead from initial radiation injury to vascular dysfunction and eventually to tissue sclerosis, fibrosis and contraction are not yet completely understood. In particular, the underlying causes for the persistence of toxic effects on endothelial cell lines and fibroblasts, long after the regression of normal tissue reactions (even after decades), remain unclear. It has been speculated that lasting mutations in these cell lines, caused by high-energy radiation may be responsible. Radiotherapy leads to lasting damage of the vasculature, at different scales. For a long time, this damage was considered to be responsible for the long-term consequences of radiation injury. However, it is still not entirely clear if tissue ischaemia (restricted blood flow and oxygenation) is the/a cause or a result of radiation injury.

There are also lasting effects on the healing of traumatic wounds in previously irradiated tissue as damage to fibroblasts and their function impairs the healing of traumatic wounds (despite the overactive deposition of connective tissue material; the most likely explanation for this conundrum being that while (damaged) fibroblasts in irradiated tissue may be able to deposit an abundance of collagen, they can no longer proliferate in response to a traumatic wound). Such considerations are important in the design of combined radiotherapy and surgery treatment schemes. From a clinical point of view, it is also relevant to note that other chronic conditions involving endothelial-dysfunction related defects (including diabetes, hypertension, vascular diseases, obesity, lupus erythematosus, scleroderma, rheumatoid arthritis) have been found to be correlated with increased late effects of radiation injury.