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Evaluating Post-irradiation Tissue Alterations

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Towards evaluating post-irradiation tissue alterations

Abstract

We seek to call to attention the paucity of data concerning irradiation effects on the extracellular matrix and of organised tissues. Examples of such research are cited as are some of the limiting factors towards obtaining meaningful results. We further seek to engender a range of research towards further improving the quality of life, most pointedly of those receiving radiotherapy.

Keywords: Irradiation, Extra Cellular Matrix, Tissues

Introduction

Not long after the discovery of X-rays it was recognised that tissue effects can occur as a result of irradiation. Most notable of the early as opposed to late changes in tissue properties was the observation of skin erythema (the reddening of skin), first recognised at relatively high doses received from soft x-rays, the severity of reddening increasing with dose. Indeed, the observation of the occurrence of skin erythema was soon to be followed by if not the first, then at least one of the earliest means of measuring dose, with the degree of reddening equated with dose through the so-called skin-erythema dose (SED). By the very early 1920s it had become more than apparent that x-ray exposures were producing a range of effects, including opacifiation of the lens of the eye (cataract formation) and of even greater alarm, malignancy; see for instance (1) concerning the widely recorded developments leading to the X-Ray Martyrs and to the first controls on radiation exposures. It would of course be unacceptable nowadays and indeed proscribed within any modern radiation controls legislation to knowingly produce such deterministic effects in an individual, other than for the purpose of producing net benefit for the individual, as for instance would typically be determined in medical practice in which the net benefit far outweighs the risk.

The safe, optimal use of radiation for the benefit of humankind has advanced considerably since those first very tentative steps, and with widening exploitation of radiation methods there now exist a wide range of fundamental studies aimed at providing strong underpinning knowledge of radiation effects in living tissues. These include both studies at the intracellular level (with which this article will not concern itself, not least because of the massive range of studies already devoted to the areas of endeavour) as well as of extracellular effects, the latter including the extracellular matrix (ECM). As an example of the latter, interest has been shown in changes in protein structure, starting with investigations using high doses of radiation up to about 1 MGy, such as the work of Cassel in 1959, reported by Bailey (2). While at such high doses it is of no surprise that one could detect these extracellular changes even given the instrumentation capabilities available at that time, the sensitivity of instrumentation for ECM and organised tissue changes occurring at doses of clinical interest is rather more challenging, confronting this challenge is one of the prime themes of present review. This interest relates to the continuing desire to provide the most effective outcome for the patient while suppressing untoward side-effects. Here note is made that while the aim of radiotherapy is to deliver the maximum radiation damage to tumour cells, damage can occur to organs at risk (OAR), such as for instance the heart in breast radiotherapy. Epidemiological studies of patients receiving radiotherapy for the left breast have shown a significant increase in cardiovascular death (3), albeit there being well-established practices for limiting dose to the tissues of the heart. In what is to follow, we review efforts aimed at studying changes in the pericardium, the intention being that such efforts be regarded as a model system, on the basis of which other organised tissue effects can also be studied. We similarly review radiation induced effects in hyaluronic acid, important throughout the body, not least in terms of lubractive role.

Pericardium is a part of the heart, mainly made of collagen fibres and can show changes in its structure at doses as low as a few Gy (4). In our own studies, we have used hyaluronic acid (HA) and collagen as models for effects of radiation on tissues. HA is prevalent between the two layers of the pericardium, offering smooth motion between the serous and visceral layers. It is also of course prevalent in the skin and in the space between the articulating bones of skeletal joints, offering similar lubricating roles. The HA and the framework offered by the collagenous systems provide for support and stability of motion and changes in the mechanical properties of these can result from insults that include irradiation as well as disease processes. Using well controlled radiation doses allows us to study both of these tissue components as models for investigation of biomechanical changes in a variety of tissues, not only directly as a result of irradiation but also in examining associated functionality changes from disease-based deformations. It should be mentioned that most of the work cited herein concerns doses at radiotherapy levels, an exception being food studies wherein the work also concerns viscosity changes, albeit at much higher doses (up to 10 kGy). Our work has attracted a favourable degree of citation, Table 1 indicating the prime drivers of those referencing present studies.

Rheological studies

Given irradiation to a sufficient dose, changes can be observed in the viscosity of the HA that forms the basis of synovial fluid (being one example of ECM). As an aside it can be noted that HA is a non-Newtonian fluid, in particular showing dependency upon shear rate. Changes in the viscosity of HA can be explained in terms of depolymerisation and polymerisation, as investigated by our group (15), use being made of rotating viscometers to alter shear rate. The rotating viscometer as well as the falling sphere method represent particular forms of tool whose use enable measurement of the effects of irradiation on the structure of HA and of other proteins. The importance of this work is also clearly seen in measuring the side effects of irradiation of sensitive structures such as the rectum in prostate radiotherapy, pericardium effects in regard to left breast radiotherapy or indeed even in studies of the viscosity of food sauces, as seen in Table 1. HA is found to be in all body fluids and organs, as in for instance, in the vitreous humour, synovial fluid of the joints, umbilical cord, and skin (16). Table 2 shows the concentration of HA in some tissues and tissue fluids. One of the distinctive properties of HA is its high-molecular weight and therefore high viscosity, making it a crucial element of the extracellular matrix (2).

Effect of radiation on HA

Out own work on HA has concerned the synovial fluid of articulating joints, with HA forming a major component of this fluid, playing the dominant role in joint lubrication. In particular, studies have shown HA to be the major determinant of viscoelastic behaviour in synovial fluid (18, 19). It has been established that after typically the third decade of life the body will begin to lose the ability to produce HA, making it all the more important that irradiation changes in HA attract attention, particularly in regard to radiotherapy of the joints, with loss in viscosity and hence of wear resistance being expected to impact upon the quality of life.

In more detail, irradiation of HA will result in ionization and excitation of the atoms of HA and surrounding ECM, to the extent that this may lead to changes in the physical and chemical nature of the polymeric HA. Alterations can be a result of several effects, including chain scissions and cross-linking (2, 20) and bond deformation (21). Chain scissions will result in reduction of the molecular weight and the associated viscosity (2). Conversely, cross-linking will result in increasing viscosity, a reflection of increasing molecular weight. Since viscosity is an important property of the HA polymer, giving rise to its viscoelastic behaviour in the synovial joint, it is important to investigate this in regard to any concomitant radiation cosmesis effects i.e. effects impacting on the quality of life of the individual following radiotherapy. While chemical degradation of HA induced by reactive oxygen-derived species (ROS) has attracted considerable attention in regard to HA depolymerisation, irradiation induced depolymerisation or bond deformations at low levels of dose has been largely neglected. Thus, it has been the intention of the studies previously reported by this group to make measurements of viscosity and shear stresses on HA solutions, conducted at different shear rates, use being made of various types of viscometer for different concentrations (0.01% – 1%w/v) of HA.

Collagen studies

In regard to irradiation effects upon collagen, it first needs to be mentioned that collagen forms an essential framework that not only provides biomechanical support to tissues but also moderates nutrient flow within the body, to be altered by both physical insult such as irradiation and also from the presence of disease, as in infiltrating ductal carcinoma. Collagen fibres are fundamentally fibrous proteins, made up of long filaments arranged side by side, sometimes forming networks and also sometimes forming annuli. Indeed, there exists a rather extensive range of types of collagen, approaching 30 variations, such that between them they can accommodate a range of biophysical needs. Thus it is of no surprise to learn that among the particular challenges in irradiation studies is the wide variation in collagen fibre types and widths, not least given that collagen fibre width represents one of the most important physical properties of collagen fibres, potentially altering as result of irradiation. It is clear that studies of for instance biomechanical stress-strain are particularly prone to misinterpretation.

Effect of irradiation on pericardium (fibrous ECM)

Before discussing the radiation effects on pericardium it is first worth briefly discussing the effects on both proteins and collagen. Irradiation is one of the mechanisms (physical or chemical mechanisms) that can provide for modification of the structure of proteins. In general, irradiation of proteins can cause ionization and excitation of the atomic constituents, leading to changes in the physical and chemical nature of peptide chain. This can take place as a result of three different actions, bond deformation, chain scissions and cross-linking of the hydrogen bonds (2). One or more of these effects can take place in both dry and aqueous states of proteins. It should be noticed that in dry state, the radiation effects are predominantly direct action on the amino acids as a result of disruption of the secondary structure of proteins. Conversely, irradiation in aqueous state also involves action of free radicals, where and radicals interact with the surrounding molecules in the solution, mainly hydrogen atoms.

In the early 1960s a number of studies investigated the effects of irradiation on collagen, a major component of the extracellular matrix (2, 22-24). The studies were conducted at particularly elevated doses, from a few tens of kGy, through to extremely high doses of ~ 1 MGy, as in the work of Cassel in 1959, as reported by Bailey (2). Unsurprisingly, at such doses detectable changes were observed. Changes on collagen structure as a result of irradiation are similar to those occurring for other fibrous proteins. Irradiation can be connected to changes to hydrogen bonds that make up the backbone of the triple helix and might lead to chain scission or/and cross linking within the basic structure which can affect its mechanical strength.

In regard to pericardial alterations, with potential mortality from cardiovascular disease, a particular concern is that of ultrastructural changes of the heart following therapeutic thoracic irradiation. This was first documented by Burch and his group in work published in 1968 (25). The changes of the heart tissues were found to differ from those resulting from ischemia or infarction (25). Further such early work was published on the effect of radiation on the heart, changes being investigated through variations in electrocardiography (ECG) readings (26). Studies of tissue morphology started in the late 1960s, the heart being considered at the time to be of low sensitivity to radiation at radiotherapeutic doses (27) until in rabbits Fajardo and Stewart demonstrated radiation induced damage at low single doses (up to 40 Gy) (28).

Radiation induced changes to the heart, resulting from radiotherapy of cancers of organs close to the heart have been reported in a number of studies (see below). These changes have also been observed in different animal species, including rats (29), rabbits (27, 28) and monkeys (30). Effects in different organs in the chest, such as breast, oesophagus and lymphoma were subsequently documented for humans (31-33). In regard to the incidence of heart disease following breast radiotherapy, this has been estimated to be 3.4 per cent, compared to 5.8 per cent for lymphomas cancer patients (34). Mechanisms for radiation-induced changes in the heart can be broadly summarised to be a result of direct interactions with cell nuclei, with subsequent degradation of DNA, together with compromised vascular, extracellular matrix, neural damage and affects upon the heart mediated by the viscous and mechanical alterations. Radiation-induced damage on the heart can be observed in the pericardium, myocardium, valves and coronary arteries or interstitial cellular medium (ECM) (3, 26, 31, 35). Although the aforementioned structures of the heart can be potentially damaged by irradiation, pericardium has been found to be more frequently affected than other structures, in particular the parietal part (36). It has also been noticed in the myocardium that damage is more frequent in the anterior wall of the left ventricle than the right ventricle. This may explain the reported reduction in heart output after irradiation (37, 38). It should also be mentioned her that the incidence of damage is dose related, depending on the volume of the heart irradiated (32).

Issues in AFM studies of pericardium with fat

In study of bovine pericardium samples by the present group, use was made of the tapping mode and a hard cantilever. In doing so, although some images were initially found to be of acceptable quality, there was observed to be a progressive reduction in spatial resolution. Guidance showed this to be a result f a contaminated tip, the hard cantilever applying excessive force on the sample surface. Remnant fat on the sample added to such difficulties. Since it would be impractical to change the tip for each new scan, technical advice lead to use of reduced hardness cantilevers for soft samples.Although as an alternative, use could be made of chemical treatment to remove fat deposits, described in the literature as a means of stabilizing pericardium, it has nevertheless been reported that the fibre width and d-spacing will change as result of use of those chemicals (39-42). Such chemical treatment might also affect the mechanical properties of samples, possibly increasing stiffness. Glutaraldehyde (GA) and dimethyl suberimidate (DMS) are examples of such chemical agents that could be applied in pericardium processing in order to produce prostheses. It should be mentioned that these chemicals affect pericardium, mainly through a cross-linking mechanisms (39-42). In considering making use of chemical treatment, specifically as applied in treating food products, no evidence has been found to negate change in the properties of such samples. Therefore, in our own work we have consistently avoided such treatment. A summary table showing some of these chemicals is shown in table 3, with their possible effects on collagen fibres, observed using AFM scanning.

Table 3 Summary of chemicals and effect on topography/other properties of collagen fibres.

Concluding Remarks

In this commentary piece, we have sought to stimulate further research in this important area of endeavour. It would seem to bea matter of curiosity that while remarkable efforts have been made in regard to intracellular research and quite deservedly so, very limited efforts have been made in regard to the ECM and at the organised tissue level. We have pointed out a number of drivers of such research, also indicating some of the challenges towards obtaining meaningful results when confronted by multifactorial dependencies, failure to take these into careful account limiting the quality of results. An important fundamental limitation of such irradiation effect studies is that these are typically conducted in silico and with the lack of vitality of life, one is unaware of the possible effect of recovery of tissues post-irradiation.

Table 1 Prime drivers of research citing work reviewed herein.

Field of interest

Study purpose

Breast radiotherapy

Pericardial stress-strain

Prostate radiotherapy

HA degradation

Rectal dose as a result of prostate radiotherapy

Dose reduction through collagen injection

Rectal dose as a result of prostate radiotherapy

HA sensitivity

Rectal dose as a result of prostate radiotherapy

HA degradation

Pericardium dose

HA degradation

Effect of radiation on HA expression

HA degradation

Biomedical applications reviewed: hot topics areas cited twice

HA and collagen changes

Biotechnology/Effect of radiation on different sauces

Viscosity measurements

Aortic valve

Viscoelastic properties

Prostate radiotherapy

HA degradation

Table 2 Concentration of hyaluronan in some tissues and tissue fluids.

Tissue or fluid

Concentration, mg/l

Human umbilical cord

4100

Human synovial fluid

1420-3600

Bovine nasal cartilage

1200

Human vitreous body

140-338

Human dermis

200

Human thoracic lymph

8.5-18

Human urine

0.1-0.5

Human serum

0.01-0.1

Table 3 Summary of chemicals and effect on topography/other properties of collagen fibres.

Chemical used in prep

Effect seen using AFM

Tannic acid

Considerable change in surface topography & thickness of collagen fibrils, also in fibril arrangement on tissue surface

Glutaraldehyde

1. Creates inter- and intra-molecular cross-links.

2. Tissue calcification and mechanical damage which could have an impact on the collagen D-banding pattern.

Dimethyl suberimidate (DMS)

Increases Young modulus although this might be connected with natural degradation of the biological samples

Different cross-linking reagents: glutaraldehyde (GA), dimethyl suberimidate (DMS) and tannic acid (TA)

1. Considerable changes in the surface topography of collagen fibrils and in the spatial organization of the fibrils within the tissue.

2. changes in the D-spacing pattern


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