67
From St. Eriks Eye Hospital, Karolinska Institutet, Stockholm, Sweden Cataract from Ultraviolet Radiation Stefan Lfgren Stockholm 2001
From St. Eriks Eye Hospital, Karolinska Institutet, Stockholm, Sweden
Cataract from Ultraviolet Radiation
Stefan Löfgren
Stockholm 2001
Dissertation abstract From St. Eriks Eye Hospital, Karolinska Institutet, Stockholm, Sweden Cataract from Ultraviolet Radiation
Stefan Löfgren, M.D. Cataract is the major cause of low vision and blindness in the world. Epidemi-ological and experimental studies link cataract to (solar) ultraviolet radiation (UVR) exposure. Current safety limits of UVR exposure are based on animal experiments, but many factors are less well known in UVR cataractogenesis. This thesis aims at strengthening the foundation for experimental UVR cataract research and to aid in future revisions of UVR safety limits. Using lactate as a marker of glycolytic activity, it was shown that in vivo UVR exposure, but not blue light, inhibits lens glycolysis. With optimised his-tochemistry of the glycolytic enzyme lactate dehydrogenase (LDH), it was de-termined that lens epithelium and nucleus have high LDH activity and cortex lower activity. LDH in the anterior parts of the lens was inhibited by in vivo UVR exposure, probably by direct photochemical action. A short penetration of UVR-B in the lens was shown using LDH activity as end-point. After in vivo exposure, young rats develop more severe cataract than old rats, with no differ-ence between sexes, and the time for maximal cataract to develop is dependent on age. The degree of cataract was quantified by measurement of in vitro lens forward light scattering. Iris pigmentation is highly UVR protective, with little importance of pupil size in pigmented eyes, while the opposite holds for albino eyes where a large pupil is more protective than a small pupil. In vitro UVR ex-posed lenses from albino rats are more sensitive than lenses from pigmented rats. The cataract type is similar in the two strains after in vitro UVR exposure, opposite of the in vivo exposure situation, where cataract type differs. Key words: Cataract, ultraviolet radiation, lens, rat, transmittance, pigment, age, pupil size.
List of papers
List of papers This thesis is based on the following papers, which will be referred to in the text by their Roman numerals: I. Löfgren S, Söderberg PG
The effect of in vivo exposure with narrow band blue light and ultraviolet radiation on rat lens glycolysis. J Photochem Photobiol B-Biol. 1995;30:145-151. (Elsevier Science).
II. Löfgren S, Söderberg PG Histochemical determination of lactate dehydrogenase activity in rat lens; influence of different parameters. Acta Ophthalmol. 1998;76:555-560. (Blackwell Publishing).
III. Löfgren S, Söderberg PG Lens lactate dehydrogenase inactivation after UV-B irradiation, an in vivo measure of UVR-B penetration. Invest Ophthalmol Vis Sci. 2001;42:1833-1836. (Association for Research in Vision and Ophthalmology).
IV. Löfgren S, Michael R, Söderberg PG Impact of age and sex in ultraviolet radiation cataract in rat. Submitted.
V. Löfgren S, Michael R, Söderberg PG Impact of iris, pupil size and eye pigment in ultraviolet radiation cataract in rat. Manuscript.
VI. Löfgren S Development of ultraviolet radiation cataract in cultured lenses from pig-mented and albino rats. Manuscript.
The published papers are reproduced with permission from the copyright hold-ers, given in brackets.
Twelve principles that characterizes a good oculist (ophthalmologist), according to Bartisch 1583:1 The oculist should:
1. Descend from religious honourable parents. 2. Worship God. 3. Have studied, speak Latin and know anatomy and pharmacology. 4. Be proficient in surgery and have started practicing it in youth. Persons
without any appropriate preparatory training or who start their ophthal-mological studies at an advanced age are incapable.
5. Have been taught by experienced oculists. 6. Have healthy eyes and normal vision. 7. Be skillful with both of his hands and able to react instantly. 8. Know how to draw in order to illustrate the instruments he needs. 9. Lead a respectable life. 10. Not be avaricious and haughty. 11. Not drink, lie and be indolent. 12. Nor promise more than he can do, and he should not praise himself.
Those were tough days
Published and printed by Karolinska University Press Box 200, SE-171 77 Stockholm, Sweden © Stefan Löfgren, 2001 ISBN 91-7349-065-2
To Ylva, Emilie and Timothy with love
Dosis Solis Facit Veneni (The dose of sun light determines the toxicity)
Paracelsus 1541
Acknowledgements
1
Acknowledgements Many people have supported me during my thesis training. I would like to spe-cially thank the following persons, institutions and foundations: • Professor Per G. Söderberg, my main tutor and mentor, for inviting me to the
world of science and for his unfailing support. • Professor emeritus Bo Lindström, my co-tutor, for giving me a glimpse of
what statistics is about and for crisp, constructive criticism of the manu-scripts.
• Assoc. Professor Enping Chen for helpful comments and general support, both in research and teaching.
• Professor emeritus Björn Tengroth, father of and former head of St. Eriks Eye Hospital, for his encouraging comments about my research.
• Professor Rudolf Rigler, Head of Department of Medical Biophysics, for of-fering lab space during my first paper.
• Drs Ralph Michael and Marcelo Ayala, my main co-workers in the team. Ralphs German sense for organizing files and protocols balanced well with Marcelos emotional and spontaneous Argentinean style. I learned and joyed, friends!
• Drs Xiuqin Dong, Manoj Kakar, John Merriam, Vino Mody Jr, Wen Qian, Jiangmei Wu, Fengju Zhang and all research colleagues and staff at St. Eriks Eye Hospital for collaboration, scientific discussions, linguistic help, and maybe even more important, interesting and fun discussions about cul-tural differences.
• Secretaries Claire Johansson, Britt-Marie Karlheden, Ulla-Britt Schützler-Pettersson, and Charlotte Ovesen for help with requisition of accepted grants, and less fun, my infinite supply of invoices.
• Research engineers Peter Goldman and Lennart Wallerman for their excel-lent craftsmanship.
• Photographer Maud Leindahl for speedy film developing and copying. • Lab technicians Margareta Oskarsson, Berit Spångberg, Anne Winter-
Vernersson, and Ylva Lagerqvist for their assistance in histology, tissue cul-ture and computer graphics.
Acknowledgements
2
• Animal care technician Monica Aronsson for keeping the animals in both good mood and condition.
• Librarian Birgitta Groundström for her Sisyphus work of keeping up with my heavy demand of articles.
• My family and friends who, at times, may have wondered if Id never leave my lab bench or the study chamber (and yes, Timothy, daddys room will be yours now).
• Myself, for putting so much effort in something so incomprehensible for my family and most of my friends. But - fascinating issues, travelling, meeting interesting people, practicing English Yes, its worth it!
Generous financial support was received from: Familjen Janne Elgqvists stiftelse Carmen och Bertil Regnérs fond Gun och Bertil Stohnes stiftelse Karolinska Institutets resebidragsstiftelser S:t Eriks Ögonforskningsstiftelse Hildur Pettersons stiftelse Anders Otto Swärds stiftelse Stiftelsen Claes Groschinskys minnesfond Sigvard & Marianne Bernadottes Forskningsstiftelse för barnögonvård Stiftelsen Sigurd och Elsa Goljes minne Eva och Oscar Ahréns stiftelse Eirs 50-årsstiftelse Svenska Sällskapet för Medicinsk Forskning Svenska Läkaresällskapet Karin Sandqvists stiftelse Rasmussens stiftelse Einar och Anna Keys resebidragsstiftelse Socialstyrelsen
Contents
1
Contents 1. Preface 2
1.1. Karolinska Institutet ......................................................................................2 1.2. The doctoral system.......................................................................................2 1.3. St. Eriks Eye Hospital ..................................................................................2
2. Introduction 3 2.1. Definition of cataract.....................................................................................3 2.2. History of lens and cataract ...........................................................................3 2.3. Lens anatomy and physiology .......................................................................5 2.4. Causes of cataract..........................................................................................8 2.5. Electromagnetic radiation and photoprotection ............................................8 2.6. UVR-B cataract ...........................................................................................10 2.7. Photophysics................................................................................................14 2.8. Photochemistry and photobiology...............................................................17 2.9. Cataract diagnose and development ............................................................19 2.10. Cataract costs and treatments ..................................................................20 2.11. Aims of the study ........................................................................................22
3. Materials and Methods 23 3.1. Animals .......................................................................................................23 3.2. Anaesthesia..................................................................................................24 3.3. Irradiation ....................................................................................................24 3.4. Spectrometry ...............................................................................................25 3.5. Eye and lens dissection................................................................................25 3.6. Lactate analysis (paper I).............................................................................26 3.7. Determination of LDH activity (papers II-III).............................................26 3.8. Lens UVR-B absorption (paper III).............................................................27 3.9. Forward light scattering (papers IV-VI) ......................................................29 3.10. Microphotography (papers IV-VI) ..............................................................31 3.11. Statistics ......................................................................................................31
4. Results and Discussion 33 4.1. Lactate analysis (paper I).............................................................................33 4.2. LDH activity in non-exposed lenses (paper II)............................................34 4.3. LDH activity after UVR-B exposure (paper III)..........................................35 4.4. UVR-B absorption in the lens (paper III) ....................................................36 4.5. Age, sex, and postexposure time (paper IV) ...............................................36 4.6. Albino and pigmented rats, in vivo (paper V).............................................39 4.7. Albino and pigmented rats, in vitro (paper VI) ...........................................40
5. Conclusions 43
6. What can be improved and what might follow? 44
7. References 45
8. Appendices 59
Preface
2
1. Preface This thesis work was performed at the Department of Medical Biophys-ics (paper I) at the Karolinska Insti-tutet and at St. Eriks Eye Hospital (remaining papers).
1.1. Karolinska Institutet The foundation of the Karolinska Institutet was preceded by over 100 years of dispute between the barber-surgeons guild and the physicians. Finally, in 1810, as a direct conse-quence of the war between Russia and Sweden in 1808-09, the Institute was born. The idea was initially to train barber-surgeons for future wars. Thus, the original name was Institutet för fältskärers danande (the Barber-Surgeon Institute). By 1811, the Institute was licensed to train also (medical) physicians, and the name was changed to Medico-Chirurgiska Institutet (the Medical-Surgical Institute). Just a few years later it was again renamed to Kongliga Carolinska Medico-Chirurgiska Institutet (the Royal Carolinska Medical-Surgical Insti-tute) to honour the king, Karl XIII. The major controversy after the foundation was the right to confer doctoral degrees. This controversy between the institute and the two older medical faculties in Uppsala and Lund lasted until 1906!
1.2. The doctoral system Initially monographs dominated, but from the fifties compound theses with several articles became more common. Today, a typical thesis consists of a thesis text and an ap-pendix of 5 to 8 articles published in peer-reviewed international journals. The time for the whole thesis-training period is on average 5 to 8 years, part- and full-time. A recent change of statutes will lead to a de-creased number of required publica-tions and a shortening of the training period to 4 years full-time. The Insti-tute emphasizes the training to result in self-contained scientists, both sci-entifically and financially. It is a pe-riod for learning experimental de-sign, statistics, method development, publishing, fund raising, and teach-ing. The thesis can thus be seen as a drivers license.
1.3. St. Eriks Eye Hospital The hospital was founded, as the only eye hospital in Scandinavia, by professor Björn Tengroth in 1990. All types of eye diseases are treated, with anterior segment and vitreo-retinal surgery as the principal ac-tivities in the nine operation rooms. St. Eriks Eye Hospital serves also as national clinic for ocular oncol-ogy.
Introduction
3
2. Introduction
2.1. Definition of cataract Cataract is defined as opacity in the lens that disturbs vision. Cataract might occur everywhere in the lens but the three common types are cor-tical, nuclear, and subcapsular cata-ract (mostly posterior). Two separate types found mainly in experimental cataract research are the anterior po-lar and the equatorial cataracts (Figure 1).
Figure 1. Cataract localisations in a lens facing up. ASC = anterior subcapsular cataract; Eq = equatorial cataract; PSC = posterior subcapsular cataract.
The origin of the name is disputed but clearly one meaning is water-fall or down rushing as the Greeks used the term καταρακτον for the Nile rapids.2 It was thought that fluid, or humour, came be-tween the lens and iris and thus dis-turbed vision. The Greeks used the name glaucosis3 for the clouding of the pupil opening. Later the term hypochyma (dense liquid behind the iris), or suffusion, overtook the meaning of glaucosis. The term cataract, or cataracta, was used by the Roman Celsus3 (c. 25 BC-AD 50).
2.2. History of lens and cataract
The ancient Greeks and Romans lo-cated the lens in the centre of the eye (Figure 2).
Figure 2. Eye by Celsus. Reprinted from History of Ophthalmology (Albert & Edwards 1996) with permission from Blackwell Publishing.
They thought the lens was the prin-cipal organ of vision. Liquid spirits of vision came from the brain, col-lected the visual information, and transported it back to the brain. The anterior position of the lens in the eye (Figure 3) was described by Galen4 (130-200), and much later rediscovered in Europe during the renaissance by Bartisch (1583) and Aquapendente (1601). The two latter located the lens directly behind the iris, without any cataract space in-between.5
Introduction
4
Figure 3. Eye by Galen. Reprinted from History of Ophthalmology (Albert & Edwards 1996) with permission from Blackwell Publishing.
Cataract surgery has been performed at least two millennia, first as couch-ing (pushing of the lens downward inside the eye) described in Indian ancient Sanskrit manuscripts, and later on as extraction. Cataract ex-traction by suction was performed by Antyllus4, a contemporary of Galen, and extraction was estab-lished in China6 during the 10th cen-tury. Even if early cases of in situ lens extraction occurred in Europe,7 extraction technique was not estab-lished until 1753 when Daviel8 in-troduced the extracapsular cataract extraction.
Introduction
5
2.3. Lens anatomy and physiology
Figure 4. Human lens anatomy Reprinted from Tissues and organs: A text-atlas of scanning electron microscopy (Kessel & Kardon 1979) and Histology of the human eye (Hogan, Alvarado & Weddell 1971) with per-mission from Dr. Richard G. Kessel and W.B. Saunders Company, respectively.
A
B C
Introduction
6
The lens function is to transmit and focus light from both near and dis-tance onto retina. The lens is devel-oped from surface ectoderm, invagi-nated during the embryonic period. The collagenous lens capsule that encloses the lens, is formed by ecto-dermic basement membrane, and continues to function as basement membrane for the epithelial cells (Figure 4).
Under the capsule, the ante-rior surface is covered by a monolayer of cuboidal epithelial stem cells, which retain the potential for cell division. However, in the central zone, the cells are quiescent unless damaged by chemical9 or me-chanical injury.10 The epithelial cells in the pre-equatorial region, also called the germinative zone, divide and form elongating fiber cells (Figure 4). The anterior end of the elongating fiber cell grows between the epithelium and the underlying fiber cells. The posterior end grows between the capsule and the underlying fiber cells. The fiber cell ends terminate in suture lines at the anterior and posterior poles where they interface with other cells differentiated simultaneously (Figure 4). During elongation cell organ-elles are necessary; but when the dif-ferentiating fiber cells reach their final length, the cell organelles are broken down by enzymatic systems. This probably enhances the trans-parency in the central part of the
lens. The disadvantage of not having organelles is that accumulated dam-age to these cells over a lifetime cannot always be repaired.
The lens cortex consists of differentiating fíber cells while the terminally differentiated postmitotic cells become a part of the lens nu-cleus. The lens does not shed any cells, and the continuous cell divi-sion throughout life means that new fiber cells are elongated superficially to the older. The older fiber cells are compressed and dehydrated,11 mak-ing the nuclear proteins more con-densed than in the cortex.
The high content of intra-cellular protein, up to 40% in the human lens nucleus12 and 50% in the rat lens nucleus,13 are responsible for the high refractive index of the lens. It was thought that the nucleus con-sists of amorphous protein, but it is now known that the embryonic nu-cleus consists of intact cells. The cross-sectional dimensions of the flattened hexagonal fiber cells (Figure 5) are about 3 x 15 µm, i.e. of the same order of diameter as a hu-man hair.
Introduction
7
Figure 5. Lens fiber cells in cross-section. Courtesy by Dr. Ralph Michael.
In the human, the length of outer cortical fiber cells is about 10 mm. The fiber cells are laterally intercon-nected with ball-and-socket junc-tions14 (Figure 6), resulting in a very short inter- cellular distance.
Figure 6. Longitudinal view of lens fiber cells with interconnecting protrusions and cavities. Courtesy by Dr. Ralph Michael.
The regular ordering and dense packing of the cells and the intracel-lular protein forms the basis of the
lens transparency.15-20 In humans, there are three main families of lens proteins, based on column chroma-tography elution: α-, β-, and γ-crystallins. About 85% of the light scattering, and consequently the re-fractive index, is due to α-crystallin,20 the most abundant pro-tein in the lens. It also functions as a chaperone, protecting other proteins from oxidative damage.21 β-crystallin is the stabilizing lens pro-tein while the γ-crystallin is more hydrated and thus induces a more easily deformed tissue, allowing for accommodation.
The low hydration of the lens deserves special attention. There is a constant inward diffusion of sodium and water, and unless the ion pumps counteract the inward current, accu-mulation of extracellular water will lead to fluctuations in refractive in-dex and ultimately opacities. An abundance of gap junctions connect the cells to a degree that the lens may be described as a syncytium for certain solutes. The equator, poste-rior pole and the epithelium have the highest concentration of Na-K-ATPase. The net current of sodium and water is hypothesized to circu-late outward from the equator and inward at both poles.22 Intracellular calcium levels must be kept low since calcium induces proteinase cascades23 leading to proteolysis and opacification24 and aggregation of crystallins.25
Introduction
8
The avascular lens relies on the aqueous and vitreous for delivery of nutrients and removal of breakdown products. There is a low partial pres-sure of dissolved oxygen in the lens surroundings. Oxygen is utilized by the aerobic mitochondrial enzyme systems in the epithelium and pe-ripheral cortex, especially at the equatorial and polar regions. The deeper parts of the lens lack mito-chondria, which is logical since oxygen diffusion into the lens inte-rior is limited. The presence of mito-chondria and other organelles in the lens interior might also induce light scattering. To maintain the neces-sary energy production despite the low concentration of mitochondria, the lens relies mainly on the (an-aerobic) cytoplasmic glycolysis.26,27 It produces only 2 ATP per glucose, while a net of 30 ATP per glucose are produced by glycolysis, (aero-bic) mitochondrial Krebs cycle and oxidative phosphorylation.28 The lens derives most of its energy (> 70%) from anaerobic glycolysis.29 ATP production in glycolysis de-pends on a supply of nicotinamide dinucleotide (NAD+). The end en-zyme in glycolysis is lactate dehy-drogenase (LDH), which reduces py-ruvate to lactate and, at the same time, regenerates NAD+ from NADH. Lactate thus produced in the lens is transported or diffuses out to the aqueous humour. The two other main metabolic pathways for glu-
cose are the hexose monophosphate shunt (HMPS), which utilizes about 14% of the glucose30 and the sorbitol pathway. The HMPS is activated when there is a demand for NADPH, for example, from aldose reductase or glutathione regenerating enzymes. During hyperglycemia, glucose is reduced in the sorbitol pathway by aldose reductase and NADPH to sorbitol, which may accumulate within the cells unless converted to fructose by polyol dehydrogenase.31
2.4. Causes of cataract The primary mechanism of cataract is still unknown. There are an abun-dance of known factors, each ac-counting for a small part of the inci-dence. What is known is basically that age leads to cataract develop-ment. Genetic factors have been es-tablished by the use of twin regis-tries.32,33 Diabetes, steroid medica-tion, trauma, smoking, ionising ra-diation and solar ultraviolet radia-tion (UVR) are all shown to corre-late significantly with cataracto-genesis.34 Unfortunately, the patho-physiology is insufficiently known for most of these factors.
2.5. Electromagnetic radiation and photo-protection
The basis of life is light, i.e. the Sun. Besides the beneficial effects of so-lar radiation, there are also harmful effects to the eye as well as the skin.
Introduction
9
All parts of the eye may be dam-aged, and for the lens it is known that ultraviolet radiation and infrared radiation (IRR) are of special con-cern. Light is by definition radiation that is perceived by the human eye. The term ultraviolet light is there-fore contradictory since UVR is in-visible for most humans. The excep-tions are young or aphakic individu-als seeing long-wavelength UVR-A.36 Many photobiologists prefer the term ultraviolet light while physi-cists commonly use radiation. IRR, light, and UVR are often com-bined into optical radiation:
Table 1. Electromagnetic spectrum Optical
radiation
Waveband
Photon en-
ergy (eV)
IRR-C 1000− 3 µm 0.001− 0.4
IRR-B 3− 1.4 µm 0.4− 0.9
IRR-A 1.4− 0.76 µm 0.9− 1.6
Light 760− 400 nm 1.6− 3.1
UVR-A 400− 315 nm 3.1− 3.9
UVR-B 315− 280 nm 3.9− 4.4
UVR-C 280− 100 nm 4.4− 12.4 Wavelength intervals as defined by the CIE.
There is a disagreement of the upper boundary of UVR-B, which by many photobiologists is taken as 320 nm. Physicists on the other hand, often use the original CIE (Commission Internationale de lÉclairage) defini-tion of 315 nm, introduced in the 1930s, and still the golden stan-
dard.35 Some authors combine UVR-A and UVR-B into the single term near-UVR.
All extraterrestrial UVR-C and the bulk of UVR-B are filtered in the atmosphere and do not reach the Earth. The irradiance on Earth can be modulated by several factors. Altitude and solar elevation angle determine the distance through the atmosphere. The varying concentra-tions of smoke and particles from large forest fires and volcano erup-tions alter the absorption properties of the atmosphere. It is little known among the public that the relative absorption of optical radiation by clouds decreases with decreasing wavelength. This means that IRR (heat) is absorbed well while a sub-stantial part of UVR-B still is trans-mitted. Ozone (O3) has received a lot of attention recently because of thinning of the stratospheric ozone layer. Most affected are the Antarc-tic and parts of the southern hemi-sphere.37
Ozone is formed when mo-lecular oxygen (O2) in the strato-sphere is split by UVR-C, and the atomic oxygen (O) blend with O2 (Figure 7).
Introduction
10
SunUVR-C
UVR-BO2
O OO3O2
O2O
UVR-C
Figure 7. Formation and breakdown of ozone.
Ozone, in turn, is an effective ab-sorber of both UVR-C and UVR-B, absorbing most radiation below 290 nm. Ozone will only affect the radiation distribution up to about 330 nm as the ozone absorption co-efficient approaches zero at this point. The ozone level fluctuates with the season, decreasing during early fall in the southern hemisphere and during the late winter in the northern hemisphere.
The destruction of strato-spheric ozone by chlorofluorocar-bons and nitrous oxide has lead to international treaties and bans in or-der to reduce production of these and similar pollutants. The decrease in ozone has lead to an increase in UVR-B on earth. A continuing trend of increased UVR-B would lead to increased incidence of cataract in the world.38 Ozone is, on Earth, a toxic and extremely reactive gas with
characteristic smell, produced by, amongst other, ordinary office equipment like copy machines and laser printers. It is used in the indus-try for disinfection of water.
2.6. UVR-B cataract Early history The support for UVR-B as etiologic factor is based on experiments, epi-demiological studies, and human cases. A connection between harm-ful rays and eye damage was pointed out 1858 by Charcot39 where he found that chemical rays from electric arcs caused inflammatory changes in the skin and external parts of the eye. This was corrobo-rated 1868 by Hess40 who placed electrical discharges close to the eyes of rabbits. In 1870 a special lens was devised in order to protect the eye from the damaging rays.41 In 1889 Widmark42 used glass filters to identify these rays as UVR. He also found in experiments that the crys-tallin lens absorbed UVR43 (1891) and that lens damage occurred after exposure to UVR (1901).44
Case studies A varying relationship between UVR-B exposure and cataract has been demonstrated in human case reports.45-47
Introduction
11
Epidemiological correlations The earliest known attempt to estab-lish epidemiology of cataract was in the 11th century by the Arabian oph-thalmologist Ammār Ali: The coun-tries in which the cataract is most frequent are the humid countries. Among them are Tunisia and Dami-ette and similar places which lie at the seashore and where fresh fish is eaten.48 In many cases, early mod-ern epidemiological studies did not include information on both the type of cataract and quantitative individ-ual UVR-B exposure. Instead the fo-cus was on geographical or physical parameters such as latitude or ambi-ent sunlight levels,49-65 altitude,57, 59,
66 or qualitative estimates of sunlight exposure.67-69 The statistical power of the above studies would have im-proved if both cataract type and in-dividual UVR-B exposure had been included in the models. Nutritional and socio-economic factors were frequently absent.
Several authors have esti-mated both individual UVR-B expo-sure and cataract type to determine their correlation.70-78 The two studies also taking into account individual ocular UVR-B exposure; the Beaver Dam76 and Chesapeake Bay73 stud-ies, both found a positive association between risk of cortical opacity and UVR-B exposure. A majority of the epidemiological studies using more sophisticated protocols for estima-tion of individual UVR-B exposure
and classification of cataract sub-types has found a correlation be-tween solar UVR-B exposure and cataract, mainly cortical. Curiously enough, high altitude was in two studies negatively correlated to cata-ract prevalence. Instead, temperature has hypothesised to be a prominent factor.79 Later on, the contradictory results were, at least partly, resolved by taking differences in ground re-flection in account.80,81 The inten-sity of diffuse skylight might then actually be smaller than the ground reflection. In order to better correlate sunlight and UVR-B exposure with cataract, it is important to estimate the ocular UVR-B dose, not only the ambient levels. The use of contact-lens dosimeters is so far the best way to individually estimate ocular UVR-B exposure.82
A compelling finding, strengthening the UVR-B cataract hypothesis, is that cataract occurs more often in the inferonasal quad-rant of the lens.83-87 This is under-standable when the facial anatomy is taken into consideration (Figure 8).
Introduction
12
Figure 8. Oblique rays of direct sunlight entering the eye. Reprinted from AIHA Journal (Sliney 1972) with permission.
It was hypothesised that oblique so-lar UVR-B might reach the mitotic region of the lens.88,89 Ray tracing, taking into consideration facial anat-omy, confirms this hypothesis.90 Experimental evidence During the past 30 years, an abun-dance of studies with controlled ex-posures have confirmed the early UVR-B cataract findings, both in vivo and in vitro. Most studies on UVR-B cataract are acute short-term but long-term UVR-B exposures or long-term experiments with repeated exposures also occur.91-95 A mile-stone in UVR-B cataract research was the thorough demonstration of the UVR-B action spectrum in the pigmented rabbit, presented by Pitts in 1977 (Figure 9).96 He showed that 300 nm UVR is the most harmful
wavelength and that the threshold dose for cataract is 1.5 kJ/m2 (in the corneal plane).
0
20
40
60
80
100
120
290 300 310 320
Wavelength (nm)
Dos
e (k
J/m
2 )
Figure 9. Action spectra for UVR-B cata-ract in rat97 (line) and rabbit96 (dotted line).
Similar action spectra for UVR-B have been presented for rats97 in vivo (Figure 9), rabbit in vivo,96 and lens epithelial cells in vitro.99 The cornea is the dose-limiting tis-sue for acute radiation damage. However, while the corneal damage from UVR-B is reversible, the lens damage is, in most cases, not. Cumulative long term solar UVR-B may thus ultimately result in cata-ract. Current safety standards for avoidance of lens UVR damage are based on experiments on adult ani-mals.96 The cornea of the young animal is thinner and therefore at-tenuates less radiation. The prolif-eration of cells in the young lens is more active than in the older, which renders the young lens potentially more sensitive to UVR-B. Repair mechanisms may vary with age. Fur-
Introduction
13
ther, structural and biochemical dif-ferences in lenses of different ages may vary the response to UVR-B. Since all proteins might be targets for UVR-B, enzymes are vulnerable. Indeed, glycolytic95,100,101 are shown to be inhibited by UVR-B. The cell membranes are damaged in dual ways since both the transmembrane proteins, like Na-K-ATPase,102,103 and the lipids can be oxidized. A disturbance of membrane integrity may lead to uncontrolled flux of ions and water, leading to opacities. The same phenomenon occurs when the ion pumps are inhibited. Water and ion disturbances are shown to occur in UVR-B cataract.104-107 There are antioxidative systems, but they might be overloaded, at least with acute exposures. Localisation of UVR-B cataract Cases with unintentional UVR-B exposure and epidemiological stud-ies have both linked anterior and posterior cataracts to human UVR-B exposure. Experimental UVR-B cataract in animals is cortical, spar-ing the nucleus. Anterior polar cata-ract seems to be more common for pigmented animals, while for albi-nos, anterior and equatorial cataract dominates.
Experimental parameters It is clear that experimental acute UVR-B cataract, as well as other oxidative stress cataracts,108 is caused by disturbances in osmotic balance. The initiating steps in the chain of events leading to opacity are insufficiently described. Damage to cell membrane lipid bilayer and membrane proteins, DNA, mito-chondria, and cytoplasmic enzyme systems are all described in UVR cataract. Ionic and water transport is disturbed, leading to short and long-range fluctuations in the refractive index, and consequently light scat-tering. Sodium and water influx, in-tracellular calcium release, de-creased ATP production, necrosis and apoptosis of epithelial and dif-ferentiating fiber cells all contribute to the opacification. The patho-physiology in the development in long-term UVR-B exposure is less well known. DNA damage and membrane damage both have their advocates.
One common problem occurs when animal research studies are compared. Different research groups use various species and strains, and animals of both sexes and of differ-ent ages. Age may play a role since antioxidative enzyme activity de-cline with age.109 Some enzyme ac-tivities in the lens differ between sexes.110 Other parameters that ren-der comparisons difficult are dose,111,112 exposure time,113 frac-
Introduction
14
tionation of dose,114,115 and time be-tween UVR-B exposure and cataract assessment.116,117 After in vivo UVR-B exposure of young rats, the time for maximum intensity of for-ward light scattering is dependent on the dose.116 However, this has not been investigated with old or very young rats.
The use of pigmented and nonpigmented animals raises diffi-culties when data are interpreted. The yellow lens pigments in some animals resemble that in humans (see 3.1. Animals). With in vivo UVR-B exposures, investigators fre-quently use pupil-dilating eye-drops in order to reduce the variation in pupil size during exposure. Little is however known about the effect of eye pigment and pupil size in the development of experimental UVR-B cataract. Concern has been raised about the use of sunglasses since the pupil should dilate, theoretically al-lowing more sunlight to enter the lens from oblique angles not passing through the sunglass.
With in vivo UVR-B expo-sure, the individual tissues interact with each other. However, some-times the in vivo dynamics may cloud the search for specific patho-physiological mechanisms. In these cases, the in vitro experiment can be a powerful tool, allowing for exclu-sion of interaction factors. Factors playing a role between in vitro and in vivo exposures are the concentra-
tion of ascorbate in the cornea and the aqueous.118,119 Ascorbate absorbs UVR-B and functions as an antioxi-dative agent. The ascorbate concen-tration in nocturnal animals like rats,120 although low, may serve a function. Some UVR-B is absorbed in the cornea and aqueous and re-emitted at longer wavelengths as fluorescence or heat.119,121,122 This factor is however of a very small magnitude. The oxygen partial pres-sure in the lens differs between in vitro and in vivo123-126 exposure, in-troducing different loads of reactive oxygen species.
While UVR-B causes epithe-lial and anterior cortical changes due to the shallow penetration in the lens, UVR-A reaches also the poste-rior parts of the lens and, depending on suitable endogenous or exoge-nous sensitisers, could initiate lens nuclear or posterior cataract. The UVR-A doses required for cataract induction are much higher than for UVR-B.97,127,128
2.7. Photophysics Radiation can be modelled in two ways, either as a wave motion or a quantum/photon (energy packet) phenomenon. Different properties of radiation are better described by ei-ther of the two models. Photon en-ergy (E) is inversely proportional to the wavelength (λ), meaning a higher photon energy for shorter wavelengths:
Introduction
15
λ
ν chhE == (1)
where h is Plancks constant, ν is frequency, and c is speed of light in the medium.
When radiation is quantified, certain parameters are used, such as irradiance (W/m2) and dose (irradi-ance times exposure time, Ws/m2 = J/m2). Radiant exposure is some-times used instead of dose. The law of reciprocity states that dou-bling the irradiance and halving the exposure time induces the same bio-logical response. This has almost been an axiom in UVR cataract re-search until recently when it was in-dicated that different exposure times at constant dose induced differences in severity of UVR-B cataract.113
Also, repeated exposures with the same total dose but different expo-sure intervals resulted in varying de-gree of cataract.115
Radiation impinging on the eye, and the lens, will be reflected, refracted, and diffracted. Reflection and refraction are subsumed by the term scattering, which is defined as an immediate secondary emission of light in all directions, with or without any loss of energy. Unfor-tunately, there are several definitions of scattering in the literature. Dif-fraction describes the bending of a wavefront hitting a disturbance, leading to a change of amplitude or phase. Parts of the radiation will also be absorbed by molecules, or chro-
mophores, in the tissue, and may be dissipated as fluorescence (momen-tanous emission of radiation of a longer wavelength), phosphores-cence (emission even after excitation stopped), or as heat. Finally, ab-sorbed radiation energy might be dissipated by chemical reactions in the chromophore or neighbouring molecules (see Ch. 2.8.).
To quantify the degree of transparency in the lens, or other tis-sues or materials, the parameters transmittance (τ) and absorbance (A), or optical density, are used:
τ1log
EElogA 10
010 == (2)
where E0 is the intensity of the inci-dent beam, E is the intensity of the transmitted beam, and τ is transmit-tance.
Transmittance numbers are multiplied to reach the total trans-mittance, while absorbances are added together. Because of a range in radiation intensity over several magnitudes, transmittance is some-times impractical, while the loga-rithmic absorbance is often more feasible. A transmittance of 0.000001 percent is the same as an absorbance of 8.0.
During evolution the eye has conserved the ability to transmit light to the retina. On the other hand, potentially dangerous radiation is blocked in the anterior parts of the eye. This was found experimentally
Introduction
16
already in 1845 by Brucke129 and re-fined in 1916 by Verhoeff and Bell.130 In 1962 Boettner & Woll-ter131 measured transmittance of the human eye. This was later extended to rabbit,132 monkey,133 and rat.134 In general, the transmittance is directly related to the thickness of the cornea and not to other characteristics of the corneal tissue.
Intense short-term UVR-B exposure induces cataract, while long-term ambient sun exposure is only hypothesized to induce cata-ract. Cornea absorbs most UVR-B but small parts still reach the lens. The amount of 300 nm UVR reach-ing the anterior lens surface ranges from 0% to 10 %, depending on spe-cies, and age of the animal.131-134 The techniques have been measure-ment of transmittance in whole lens or lens sections. Increasing propor-tions of UVR-A pass through the lens and reach retina.
The total irradiance of the so-lar radiation on earth is 0.5-1.1 kW/m2, with a large varia-tion depending on geographic, physical and meteorological fac-tors.135 The UVR-B part thereof is only about 3 W/m2, depending mainly on ground reflectance.137-139 The ratio between ocular and ambi-ent UVR-B irradiance ranges from 2% to 20%,69,140,141 resulting in an ocular UVR-B irradiance of less than 1 W/m2, in comparison with 10 W/m2 UVR-A.142
The total irradiance from the sun can be split up into a diffuse (skylight) and a direct component (Figure 10). For UVR-B, the diffuse component is on a clear day about as large as the direct component, but for over-cast days the diffuse component is proportionally larger.
Direct
EarthDiffuse
Atmosphere
Figure 10. The total irradiance is made up of diffuse and direct components (modified from Urbach143).
The diffuse component is made up of two types of radiation scattering; Mie and Rayleigh scattering. Mie, or large particle, scattering occurs when the scattering particles size is same or slightly larger than the wavelength of light, and is normally the greatest contributor. Rayleigh, or small particle, scattering (Φ) occurs when particle size is less than the wavelength, and is inversely propor-tional to the fourth power of the wavelength (λ):144
4λk
scattered =Φ (3)
where k is a composite constant, changing with the angle from the beam axis. This means that shorter wavelengths scatter more than
Introduction
17
longer. Rayleigh scattering explains the red sun at sunset because the shorter wavelengths are scattered out of the long beam path. The blue sky is also explained with the same model. Sunlight scattering may re-sult in a false sense of security be-cause with shielding from direct sunlight the bright light and the heat sensation from IRR will decrease considerably but the total UVR lev-els will be less reduced.135
An individual does not easily influence the sunlight intensity reaching Earth. Luckily enough, the personal precautions made by an in-dividual are the most powerful. The use of brimmed hats and sunglasses decrease the ocular irradiance of UVR-A and B with more than 90%.68 The fear that sunglasses paradoxically lead to increased UVR irradiance reaching the eye lens and interior because of dilation of the pupil due to blocking of light is no longer an issue as modern sun-glasses have excellent UVR-B ab-sorptive properties, and in most cases also efficient UVR-A absorp-tion. The only problem might be that most sunglasses do not protect against oblique solar radiation. The major behavioural factors are avoid-ance of mid-day sun, positioning in low ground reflection environments, and reduction of exposure time. Grass reflects about 1% UVR-B, while dry beach sand and fresh snow reflects 17% and 88%, respec-
tively.135,145 The general belief of water as a good reflector of UVR-B is valid for diffuse skylight but not direct sunlight. Calm water has a low reflectance, of course depending on solar angle, while foaming water has a very high reflectance. The physical (eyebrow configuration, depth of orbits) and physiological (squinting, pupil size) properties of the face have a varying impact on ocular irradiance.
Several countries have im-plemented a UVR index system to facilitate for the public to know when the UVR levels are high.146
Schools in Australia and New Zea-land have introduced caps and solar protective textiles in the school uni-form in order to protect the children to UVR-B.
2.8. Photochemistry and photobiology
The First law of photochemistry, postulated by Grotthus and Draper in 1818, states that only absorbed photons can induce chemical effects. When the absorbing chromophore is chemically changed during the reac-tion, the reaction is called a direct photochemical reaction. When the absorbing chromophore does not change chemically but causes a chemical change in another, neighbouring, molecule, the reaction is called a photosensitised reaction. A photosensitiser is a molecule, or part thereof, that absorbs photons
Introduction
18
and enters a higher, unstable, energy level, or excited state. The surplus energy is then immediately released to nearby molecules. There are two principle pathways for the sensitiser to act (Figure 11).
UVR Target
Sens Radical
O2 ROS
D
I
II
S
Figure 11. Simplified scheme of photo-induced energy transfer. D = direct path-way; S = sensitiser.
1) Type I or radical pathway the sensitiser interacts directly or indi-rectly (via an intermediate molecule) with the target molecule; 2) Type II or singlet oxygen pathway the sen-sitiser interacts with oxygen in the tissues with formation of reactive oxygen species (ROS), such as singlet oxygen, which in turn induce chemical changes in the target mole-cule. The resulting photochemical reactions may lead to conforma-tional changes of enzymes, thereby inactivating them, aggregation of structural proteins, or membrane damage.
The small part of UVR-B that reaches the Earth has high enough photon energy to react with cells and induce photochemical changes. The energy per photon is increasing with
decreasing wavelength meaning that UVR-B generally has a higher reac-tivity than longer wavelengths. The presence of molecules with preferen-tial absorption in a distinct wave-band modulates the molecular reac-tivity. Amino acid residues in en-zymes, nucleic acids and crystallins absorb UVR-B strongly, resulting in oxidation and potential loss of func-tion of the molecules. Of the absorb-ing amino acid residues in the lens, tryptophan absorbs more than 95% of the UVR-B, with tyrosine, phenylalanine, cystein, and cystine contributing to some extent.147 Some of these amino acids have an absorp-tion tail reaching into the UVR-A spectrum.
Three main anti-oxidative molecules in the eye are the tri-peptide glutathione148 (GSH), ascor-bic acid149 (vit C), and α-toco-pherol150 (vit E). Sulfhydryl groups in glutathione (GSH) are susceptible to oxidation, but the GSH-regenerating enzyme system enables GSH to act as a quencher. Ascorbic acid is a water-soluble quencher pre-sent in the aqueous and lens cell cy-toplasm while α-tocopherol is lipid soluble and present in the cell membrane lipid bilayer, mainly protecting against lipid peroxidation. If the protective anti-oxidative systems are photobleached, i.e. saturated by exposure with radiation, damage occurs. The key lenticular antioxidative enzymes are catalase,
Introduction
19
are catalase, glutathione peroxidase, and superoxide dismutase.151,152 They are all highly active in the lens epithelium,153 the first region to en-counter ROS from the anterior chamber and UVR.
UVR-A and light is absorbed less efficiently and requires photo-sensitisers for transfer of energy to initiate chemical changes in critical molecules. Such sensitisers in the lens are some of the kynurenine me-tabolites derived from trypto-phan.154,155 They have an absorption tail extending from UVR-B into the UVR-A and are shown to generate ROS after UVR-A and UVR-B ab-sorption.156,157
Enzyme inhibition is proposed to, at least partly, be initiated by conformational changes due to breakage of disulfide bridges by en-ergy transfer from adjacent aromatic amino acids.158,159 One pre-requisite of inactivation is that the affected amino acid residues are essential, i.e. participate in the substrate-binding or a critical tertiary structure.
When molecular damage has occurred and repair is not possible, the scavenging systems are acti-vated. Several proteinases degrade the faulty proteins and thus recycle the amino acids.
2.9. Cataract diagnose and development
The cataract diagnose is usually set when the patient complains of de-
creased visual acuity or glare. The opacities disturb vision by absorp-tion and light scattering. According to WHO, cataract is the leading cause of blindness in the world, counting for more than 50% of the blind. The prevalence of cataract is about 50% at the age of 65 and then increasing.
Classification of human cata-racts is based on location and ap-pearance of the opacity, or according to the colour of the nucleus. There are three common types; cortical, nuclear, and posterior subcapsular cataract (PSC). All types are present as age-related cataracts, and the prevalence numbers in a white re-tired American population are 30-70% nuclear, 4-15% cortical, and 5-7% PSC.77
There are several techniques available to detect and quantify cata-ract. Among the non-invasive tech-niques, the slit-lamp microscope is the main tool. Scheimpflug photog-raphy160 (Figure 12) allows focus of the whole lens and is thus preferable in quantitative studies.
Introduction
20
Figure 12. Scheimpflug image of a lens with nuclear and signs of anterior cortical cataract. Courtesy by Dr. Wen Qian.
This technique is especially suitable for early and moderately advanced nuclear cataract. Cortical cataract is usually asymmetric and cannot eas-ily be represented by a single slit image. Instead, cortical cataract is often quantified by retroillumination imaging. Several slit-lamp based grading systems to be used in the clinical setting has evolved around the world, such as LOCS (Lens Opacities Classification System), the Wilmer System, the Oxford System, and the CCESG System (Coopera-tive Cataract Epidemiology Study Group).161 The main difficulty for in vivo quantification is to find an easy and objective method that works for all types of cataract. None of the current methods fulfil all these re-quirements. There are also a number of in vitro or invasive techniques, such as measurement of forward light scattering,162 transmit-
tance,163,164 wavefront aberration,92 and fluorescence.124
The different types of cataract have separate molecular and cellular characteristics. The nuclear cataract is commonly viewed as an aggrega-tion and water-insolubilisation of crystallins, while the cortical cata-ract resembles the experimental os-motic cataract. Oxidative stress seems to be an early event in all cataracts.
2.10. Cataract costs and treatments
During the past 2000 years, the only way to treat cataract has been surgi-cal removal of the opaque lens tissue from the visual pathway. In the early days by couching and nowadays by phacoemulsification, i.e. fragmenta-tion by ultrasound and removal of the lens tissue inside the capsule. The operation ends with the inser-tion of a plastic intraocular lens (IOL) in the capsular bag. Dr. Har-old Ridley in England invented the IOL in the 1940s.165 He inserted plastic lenses into eyes in order to avoid the bulky phakic glasses the patients had to use after cataract surgery. The IOL was a huge step toward a patient-friendly solution of the cataract problem.
No proven prophylactic meas-ures are available, nor any medical treatments. Some drugs have been tried against cataract in diabetes, but with no success. Dietary antioxi-
Introduction
21
dants may be beneficiary but this has not been convincingly shown in lar-ger studies. Avoidance of known risk factors such as smoking and sunlight should decrease the inci-dence of cataract but those trials are yet to come. In the absence of any preventive measures that can be ap-plied in a public health setting, the only recourse is surgery. Cataract surgery is a cost-effective interven-tion, with sight restored after a rela-tively low-cost operation. The ob-stacle is the large number of people needing surgery, leading to a heavy burden on the health care system. In Sweden (with 9 million citizens) more than 60 000 cataract operations were performed in 1999, to a cost of 800 Euros each. Cataract surgery is a bearable burden for the health care system in Sweden and other devel-oped countries. For the developing countries, lack of doctors and func-tioning health care systems pre-cludes general access to cataract surgery.
Research on UVR cataract is of importance both for the general population and for the industry. The industry needs to know how much, or little, UVR can be tolerated in the
working environment. Based on all available scientific knowledge, the ICNIRP (International Commission on Non-Ionizing Radiation Protec-tion) has derived exposure limits for UVR exposure (Figure 13).166 The limits are valid for incident radiation onto skin or eye during an 8-hour period.
1.E+00
1.E+01
1.E+02
1.E+03
1.E+04
1.E+05
1.E+06
250 300 350 400Wavelength (nm)
Expo
sure
lim
it (J
/m2 )
Figure 13. Eight-hour exposure limits for eye and skin according to ICNIRP.
The public gain from UVR cataract research since prophylactic measures and nonsurgical treatment aiming at preventing or delaying cataract will eventually emerge. If the onset of cataract could be delayed 10 years, the cataract prevalence would de-crease by 50%.167
Introduction
22
2.11. Aims of the study Current safety limits of UVR exposure are based on animal experiments. Hu-man experiments would be unethical since cataract, in principle, is irreversible. There are numerous factors that are less well known about UVR cataractogene-sis. This thesis aims at strengthening the foundation for experimental UVR cata-ract research, concerning both chemical and biophysical effects of UVR. It is anticipated that future revisions of UVR safety limits will be facilitated. The specific aims were:
• To determine if in vivo UVR-B and blue light exposure affect lens glycolysis (I).
• To optimise LDH enzyme histochemistry for lens studies (II).
• To investigate the regional UVR-B inactivation pattern of LDH and to de-termine UVR-B penetration depth in the lens with LDH histochemistry (III).
• To determine if age and sex influence UVR-B cataractogenesis (IV).
• To determine if pupil size and iris pigment influence UVR-B cataractogene-sis (V).
• To determine if in vitro UVR-B cataractogenesis differs between pigmented and nonpigmented animals (VI).
Materials & Methods
23
3. Materials and Methods
3.1. Animals The animals were treated according to Swedish regulations and ethical approval was obtained from the Northern Stockholm Animal Ex-periments Ethics Committee.
A diversity of animal species have been used in UVR cataract re-search, including primates, other mammals, birds, and fishes. The strict regulations for utilization of human eyes and lenses for in vitro studies have favoured use of lenses from animals such as rat, pig, and cow. The most commonly used spe-cies for in vivo experiments are probably rabbit, rat, and mice. The rat eye resembles the human eye. Hence, it is reasonable to expect that pathophysiological mechanisms in rat eyes can be transferred to hu-mans.
Even if pigmented rat eyes are more similar to human eyes than al-bino rat eyes, the rat is still noctur-nal. This holds also for rabbit and mouse. One major argument against nocturnal animals is that they have much less ascorbate in the anterior segment than diurnal animals.120 The ground squirrel has been proposed as a good model since it is diurnal, melanin pigmented, and has the yel-low lenticular pigment that humans have, N-formyl kynurenine (NFK).94,168 Primates and some other animals have this yellow pigment
while the common animals used for in vivo (rabbit, rat, and mice) and in vitro (rat, pig, and cow) UVR cata-ract research all lack the pigment. NFK colours the lens yellow and functions as both UVR and blue light filter. Unfortunately, the ground squirrel is not available from commercial breeders.
In papers I-IV outbred albino rats (Sprague-Dawley) were used. Due to the heavy allergenic potency of male rats (and allergy symptoms of the author) a shift to female rats were made after paper I. During the progress of the thesis, it became clear that more information was needed about UVR cataract devel-opment in pigmented animals. The Brown-Norway rat was chosen as the pigmented strain because it is probably the most commonly used pigmented rat strain in UVR cataract research and comparative informa-tion is therefore available.93,169 The Brown-Norway rats in papers V-VI were inbred, hence an inbred albino strain, the Fischer-344, was chosen for comparison. One important dif-ference between inbred and outbred rats is the smaller size of the inbred rats, and eyes, at corresponding ages.
The youngest rats used in the studies were 3 weeks old and the oldest 1.5 year. Six-week-old rats are post-weanling and prepubertal. The mean life length of Sprague-Dawleys is over 2 years but morbid-
Materials & Methods
24
ity and mortality increases steeply after about 1.5 year.
In papers I-III, the rats were killed by cerebral dislocation. Be-cause of the larger rats in paper IV, CO2 asphyxiation was utilized be-fore the cerebral dislocation. This technique was also used in the re-maining papers.
3.2. Anaesthesia The rats were anaesthetised in order to keep them immobilized during ir-radiation. All but the older rats (pa-per III) were anaesthetised without any pre-treatment. Isoflurane gas was used in paper III to briefly se-date the rats before the intraperito-neal injection of a combination of ketamine and xylazine (papers I-V). This combination is effective and safe170 but has side effects that may interfere with experiments on the eye. The marked proptosis and lid retraction caused by xylazine might influence the osmotic components in the aqueous and eventually the lens. Transient cataract occurs almost immediately after induction of an-aesthesia and lasts about 1-2 hours.171,172 The xylazine-ketamine anaesthesia is also shown to aggra-vate UVR-induced cataract (Zhang, Löfgren, and Söderberg, unpub-lished). Rat identification was ob-tained with earmarks, or ear plus tail marks.
3.3. Irradiation Dose is here defined as the energy of UVR-B per unit area reaching the anterior surface of the cornea (I-V) or lens (VI).
In order to evaluate effects of UVR-B, the type and quantity of ra-diation must be known. There are two principal ways to achieve UVR-B for the planned experiments. One is to use a laser and the other to filter broadband radiation from a lamp with an interference filter or to make a waveband selection with a monochromator. An interference fil-ter-based high-pressure mercury lamp source was used in papers I-III, and a monochromator source for the remaining papers. The advantage with a monochromator is that the waveband is usually narrower than with a filter. The drawback with a monochromator is the large loss of energy output. Thus, a compromise between bandwidth and output must be achieved. A UVR-B laser appears as the perfect instrument but common UVR lasers emit only in a single UVR wavelength. If more wave-lengths are needed more lasers have to be purchased at significant ex-pense. There are tuneable UVR-B lasers on the market but they are pulsed, typically with nanosecond pulsewidth and very high irradiance, causing non-linear absorption.
Irradiance was measured with a thermopile system calibrated to an
Materials & Methods
25
ANSI (American National Standard Institute) traceable source by the Swedish National Bureau of Stan-dards. The advantage of a thermo-pile is the flat response over a wide waveband and the stability over time.
Even ten times the highest ir-radiances used in this thesis did not increase the temperature of water in a quartz cuvette with an immersed lens. The temperature meter was sensitive to 2 decimals.
The exposure time in the the-sis varied from 6 minutes (blue light; paper I) to 30 minutes (UVR-B; pa-per VI).
3.4. Spectrometry Spectral intensity distribution was determined with a spectrometer. The relative spectrum can be quantified if the spectrometer is calibrated to a black or grey body radiator. Abso-lute spectrum can be calculated if the relative spectrum from the spec-trometer is combined with total ir-radiance measured by the thermopile system. The interference-filtered source displays a wider spectrum with a minor peak at the mercury line at 365 nm. The true full width at half maximum (FWHM) was 6.2 nm for the monochromator source, markedly narrower than the 10 nm theoretical value of the monochro-mator. The FWHM for the interfer-ence filter source cannot be esti-mated due to the dual peak
(Figure 14). Both spectra peaked at 302.6 nm.
0.00
0.50
1.00
280 300 320 340 360 380 400
Wavelength (nm)
Rel
ativ
e sp
ectr
al ir
radi
ance
(W
/m2 /n
m)
Dual monochromator
Interference filter
Figure 14. The two spectra used in this the-sis.
The interference-filtered source dis-plays a wider spectrum with a minor peak at the mercury line at 365 nm. The true full width at half maximum (FWHM) was 6.2 nm for the mono-chromator source, markedly nar-rower than the 10 nm theoretical value of the monochromator. The FWHM for the interference filter source cannot be estimated due to the dual peak. Both spectra peaked at 302.6 nm.
3.5. Eye and lens dissection
The enucleated eye was first rinsed in buffered saline and then posi-tioned cornea facing down on a pa-per towel. Extrabulbar tissues were removed and the sclera was opened around the equator. Using curved forceps without teeth, the lens was lifted out, and immediately im-mersed in a Petri dish filled with buffered saline. All extralenticular tissue such as vitreous or ciliary
Materials & Methods
26
body was removed under a dissec-tion microscope. The time from enu-cleation to free lens in vitro was about 5 min.
3.6. Lactate analysis (paper I)
Paper I was initiated by a pilot study where lenses from 9 nonexposed rats were cultured for 24 h. Lactate pro-duction, as released to the medium, was estimated at the end of the cul-ture period in order to estimate an appropriate sample size, with feasi-ble settings of the α and β errors, and minimum detectable difference.
In the main study, lenses from 22 plus 22 rats exposed in vivo to unilateral blue light (435 nm) or UVR-B (303 nm), were incubated aerobically in culture medium. After 5, 10, and 20 h, aliquots of culture medium were withdrawn and lactate quantified. LDH and NAD+ were added and the lactate was oxidized to pyruvate under simultaneous re-duction of NAD+ to NADH. The ab-sorbance of formed NADH was measured spectrophotometrically and compared with a standard curve. The results of the main study en-couraged an extension with 21 more rats exposed in vivo to UVR-B (303 nm). This time, lactate in ali-quots taken at 2, 4, and 6 h after ex-posure, and intralenticular lactate after 6 h, was quantified.
3.7. Determination of LDH activity (papers II-III)
Enzyme histochemistry The distribution of glycolytic activ-ity in a tissue may be determined by biochemical methods where glucose metabolites or glycolytic enzyme ac-tivities are measured in subcompo-nents of tissue. Another technique is enzyme histochemistry, which al-lows direct estimation of enzyme ac-tivity in different regions of a tissue on a relative scale. Once the histo-chemical procedure has been estab-lished, the information on the distri-bution of activity can be acquired. Histochemistry is therefore prefer-able for mapping of regional enzyme activity.
The importance of tissue-specific methodology in histochemi-cal studies has been pointed out.173 It was therefore decided to optimise, for lens purposes, a method for his-tochemical determination of lactate dehydrogenase.174,175
Reagent solution was added to serial 10 µm whole-eye cryosections on a slide. The stain nitrobluetetra-zolium (NBT) was reduced in an electron-transfer multistep reaction resulting in conversion from a wa-ter-soluble non-coloured molecule to water-insoluble coloured formazan (Fig. 1, paper II).175
In the first part of paper II, an optimisation of concentration of the various contents in the enzyme solu-
Materials & Methods
27
tions was conducted on lenses from 12 rats. For each chemical, the plan was to choose the concentration that gave the highest enzyme activity. When this was done, the second part of paper II was initiated, also with 12 rats. Using the optimised reagents established in the first part, activity of lactate dehydrogenase was deter-mined in different parts of the lens.
Sample size and sources of varia-tion Data from the second part of paper II were analysed with ANOVA. The variation components (sources of variation) thus obtained were used for optimisation of the experimental design. In order to reduce the sample size in future studies one may de-crease the influence of the variance estimates in the enzyme activity measurements. The general idea is to minimize total variation by dividing each variation component estimate with a maximum feasible number of replications, such as number of ani-mals, sections, and measurement points.
With known intra-individual variation, a pre-planned sample size could be determined.
Modified histochemistry in paper III The use of NEM and pyruvate in the incubation medium differed from that in paper II. Pyruvate was in-cluded in the control medium as a product inhibitor176 in order to
minimize the staining reaction in the lens nucleus in the control sections due to endogenous lactate. Without addition of pyruvate, the endoge-nous lactate would cause a false low test-minus-control activity. Sulfhy-dryl groups are known to reduce NBT into coloured formazan, and for that reason NEM was added to both media in order to block this non-specific staining reaction, espe-cially in the nucleus.176
Microdensitometry The density of the staining was quantified with a microdensitometer equipped with an interference filter. The suitable wavelength is deter-mined by the absorption curve of the dye. NBT is reduced in two steps, first to reddish and finally to blue, and the absorption curves of the two states cross each other at the isobes-tic point, at 585 nm.177 The transmit-tance was measured in the epithe-lium, anterior cortex, nucleus, and posterior cortex, and finally trans-formed into absorbance.
3.8. Lens UVR-B absorption (paper III)
The total attenuation of UVR in the lens consists of both absorption and scattering. The transmission of UVR in whole lenses and lens sections has been measured in several species. This approach however does not dis-criminate between absorption and scattering. In order to estimate the
Materials & Methods
28
absorption, that is, the biologically interesting part of the attenuation, a biological indicator molecule was sought. It had to be present in all parts of the lens, and it should be possible to quantify the effect of ab-sorbed UVR.
Lens UVR-B penetration model The inactivation (%) of LDH at 0 hours latency (Ω), was used as a measure of the intensity of UVR-B in the tissue during exposure. Inacti-vation was calculated as 1 minus the ratio of relative activity in the ex-posed lens (absorbance units, Ae), and the relative activity in the non-exposed contralateral lens (Ac; Eq. 4).
c
e
AA−=Ω 1 (4)
According to the second law of pho-tochemistry and the law of reciproc-ity, the inactivation of LDH (Ω) is, at each position in the lens, directly proportional to the dose of UVR-B (He). The proportionality constant is k (Eq. 5).
Ω= kH e (5) According to the Lambert-Beer law, the linear absorption coefficient (α) can be estimated if the intensity of the radiation is known at two points along the path of the radiation (Eq. 6), the distance between the points being l2 to l1.
12
2:
1:ln
llHH
e
e
−
=α (6)
Substituting He in Eq. 6 according to Eq. 5, it is seen that the linear ab-sorption coefficient (α) can be calculated from the inactivation of LDH at the two points along the path of the radiation (Eq. 7).
12
2
1ln
ll −
ΩΩ
=α (7)
Penetration depth is here defined as 2/α, corresponding to a transmit-tance of 1/e2 (~14%) at the penetra-tion depth.
Materials & Methods
29
3.9. Forward light scattering (papers IV-VI)
Figure 15A. Schematic of the Light Dissemina-
tion Meter117 Figure 15B. Ray tracing of the Light Dis-
semination Meter178 Fig. 15A was reprinted with permission by Blackwell publishing. Fig. 15B was reprinted with permission from Karger Publishing. The degree of cataract was quanti-fied with an objective technique de-veloped by Söderberg 1990.117,162 A probing white light from a cold light source was directed from underneath the lens. The forward scattered light from the lens was collected by the optics of a camera equipped with a photodiode in the film plane (Figure 15). The light-induced current from the photodiode was measured with an ampere meter. The readings were calibrated against the readings from
light scattering from a lipid emul-sion of the drug diazepam. This al-lows conversion of relative current readings of forward light scattering to absolute Equivalent Diazepam Concentration (EDC). The light scattering data were normalized by a log10(EDC+1) transformation, as suggested by Söderberg 1990.117,162 The transformed unit is the tEDC.
The absolute readings have the advantage to be comparable over time. The standard curve demon-
Materials & Methods
30
strates that if the concentration of scatterers becomes too high, the in-tensity of forward light scattering decreases. This decrease is secon-dary to back scattering and absorp-tion. The absorption is an effect of increased path length of the scat-tered light due to multiple light scat-tering. If the light scattering in the lens is high enough to decrease the intensity of forward scattered light, the complete lens appears white in incident illumination, or dark in the centre with a peripheral brighter ring in dark-field illumination.
The technique allows a detec-tion of less than 1% change in light scattering, the size determined by setting of probing light intensity.
Age, sex, and postexposure time (paper IV)
Age sensitivity experiments In the first age sensitivity experi-ment, four groups of 20 Sprague-Dawley rats aged 3, 6, 17, and 52 weeks were included. The rats re-ceived 8 kJ/m2 UVR-B unilaterally. The time interval between UVR ex-posure and cataract measurement was one week.
In the second age sensitivity experiment, three groups of 22 rats aged 6, 17, and 52 weeks were in-cluded. Again, the rats received 8 kJ/m2 unilaterally. The time inter-val between exposure and cataract measurement was this time eight weeks.
Sex sensitivity experiment A total of 22 male and 22 female 6-week-old Sprague-Dawley rats were included. The rats received 5 kJ/m2 unilaterally. The time inter-val between UVR-B exposure and cataract measurement was one week.
Albino and pigmented rats, in vivo (paper V) Forty-two each of Brown-Norway and Fischer-344 rats (age 6 weeks) were divided into two eye-drop treatment groups. Half of the rats from each strain received the mydri-atic tropicamide in both eyes while the other half received the miotic pi-locarpine in both eyes. A UVR-B dose of 5 kJ/m2 was delivered uni-laterally. A 5 kJ/m2 corneal in vivo dose is slightly above the dose shown to induce irreversible cataract in rabbits and rats.96,111 The time in-terval between UVR-B exposure and cataract measurement was one week. One week after exposure the eyes were inspected for pathologic changes (eyelid inflammation and corneal damage including erosions and edema), graded from 0 to 3. Hy-phema was graded during eye dis-section.
Albino and pigmented rats, in vitro (paper VI) A total of 23 Brown-Norway and 22 Fischer rats were included. From each rat one lens was exposed to UVR-B while the other served as a
Materials & Methods
31
control. The lenses were positioned anterior side down in buffered saline solution in a Petri dish and the radia-tion was directed from beneath using an aluminium mirror. A 1.8 kJ/m2 UVR-B dose was delivered during 30 minutes. Using corneal and aque-ous transmittance values from mon-key and rat, the actual dose was cal-culated to conform to the 5 kJ/m2 in vivo dose given in paper V. Immedi-ately after the UVR-B exposure, both lenses from each rat were trans-ferred to culture medium in new dishes with vented lids. The dishes were prefilled with 4 ml Medium 199, with antibiotics and serum. In-cubation environment was set to 37 °C and humidified air with 5% CO2 and the medium was changed every other day. Each lens was measured for light scattering daily during 7 days. At each occasion, light scattering was measured in triplicate for each lens.
3.10. Microphotography (papers IV-VI)
All lenses in papers IV-VI were pho-tographed via a dissection micro-scope, both for logging and for find-ing correlations between type of cataract and degree of light scatter-ing. The lenses were imaged in inci-dent light from a paraxial cold-light ring illumination above the lens, or in dark-field illumination from an angle under the lens. The illuminat-
ing light came from opposite direc-tions (Figure 16).
Darkfield illumination
Brightfield illumination
Figure 16. Two types of photography illu-mination.
3.11. Statistics Confidence intervals (CI) were pre-ferred to t-tests, since the CI more intuitively gives the variation. It is easy to grasp the result of mean paired-sample differences between exposed and nonexposed lenses, since zero will be excluded from the interval if there is a significant dif-ference. Sample size calculations were carried out for each type of methodology, using feasible settings of α and β errors: Table 2 Decision Reality Accept H0 Reject H0
H0 true
Confid. level 1-α
Type I error Sign. level
α
H0 false
Type II error β
Power (strength)
of the test 1-β
H0 (null hypothesis): µ = µ0 (the two means are equal)
Materials & Methods
32
ANOVA was frequently used be-cause most studies had more than two groups to compare. If an ANOVA was significant, subsequent comparison tests were performed. When it was obvious, or analyses indicated considerable inhomogene-ity of variance or departure from normality, non-parametric ANOVA and comparison tests were used.
Less utilized methods were Pearson χ2 test of contingency tables, Fischer exact test, arcsine transfor-mation, linear regression, estimation of variance components, and poly-nomial regression. Throughout the thesis the significance level was set at 0.05 and the confidence coeffi-cient to 0.95.
Results & Discussion
33
4. Results and Discussion
4.1. Lactate analysis (paper I)
The absolute accumulated levels of lactate produced in nonexposed lenses after 20 h incubation were similar to data from other studies on young rats.179,180
Blue light exposure did not induce any significant effects on lens lactate production (Fig. 2, paper I), and the trend of stimulated lactate production was not significant. UVR-B exposure, on the other hand, induced a significant and steep inhi-bition of lactate production at 2 to 6 h after exposure (Fig. 1, paper I). To exclude UVR-B inactiva-tion of lactate transport mechanisms as confounding factor, the intralen-ticular lactate levels were quantified six hours after exposure. Lactate concentration in the exposed lenses was higher than in the nonexposed but total lactate production in UVR-B-exposed lenses was still in-hibited.
A significant recovery and re-turn to base-line occurred between 5 and 20 h (Fig. 1, paper I). The av-erage lactate production, from 5 to 20 h, in lenses from UVR-B-treated rats was significantly lower than that of blue light-treated rats, as revealed by ANOVA.
A similar solar UVR-B dose as the one received by the rats could be achieved staring into the Swedish sun during 95 minutes, with peak irradiance during the whole expo-sure.181 Although the spectrum is not the same, the solar peak irradiance is not present for 95 minutes, and the aversion reflex caused by the (visi-ble) light intensity render it impossi-ble to stare at the sun any longer than a few seconds, the reasoning still gives an idea of the dose level.
Precalculated statistical power was 0.9. Blue light transmission through cornea and aqueous is higher than for UVR-B, leading to higher blue light irradiance than UVR-B irradiance at the lens sur-face. Wavelength-dependent differ-ences in transmission within the lens might result in an attenuation of the same order for the two wave-bands.132,133,182 However, the propor-tions of the attenuation factors ab-sorption and scattering are unknown. The conclusion must be that there is no, or very low, blue light absorp-tion by glycolytic enzymes in the lens. An inhibition of the glycolysis is consistent with studies on inactiva-tion of glycolytic enzymes after ex-posure with UVR-B.95,100,101 The re-covery may be due to several fac-tors. Reversible inhibition of en-zymes has been shown for cyto-chrome oxidase 183 and lactate dehy-
Results & Discussion
34
drogenase.95 The postulated inhibi-tion of the Krebs cycle and the oxi-dative phosphorylation together with increased energy demands due to re-pair mechanisms and increased de novo synthesis of proteins leads to a local decrease in the energy level, i.e. ATP, in the epithelium. This in turn stimulates a compensating in-creased glycolysis in the epithe-lium27 and in the cortical lens fibers. The early UVR-B inhibition of the glycolysis reported here sup-ports the theory that shortage of en-ergy induces processes that increase the light scattering in the lens.
4.2. LDH activity in non-exposed lenses (paper II)
Optimisation of LDH histochemistry The final incubation medium, or re-agent mixture, was obtained by choosing the highest concentration or incubation time not associated with the formation of large dye ag-gregates. This type of aggregates would lead to suboptimal densitome-try. It has been stressed that a tis-sue stabilizer should be used be-cause the loss of enzyme, substrates and dye from the sections will de-crease.173 However, it was not possi-ble to use a tissue stabilizer such as polyvinyl alcohol because the lens nuclei often fell off the slides during
the procedural steps, despite many efforts to attach the sections. The high protein concentration, more than 65%, in the rat lens nucleus13 probably plays a role in the attach-ment.
LDH activity The LDH activity varies considera-bly within the lens, and the highest activities were found in the epithe-lium and nucleus (Fig. 4, paper II).
The distribution pattern of LDH activity in young rat lens is in accordance with a biochemical study95 −the LDH which in young rats, as is well known, shows higher activities in the nucleus than in the cortex. Two studies on LDH isoenzymes have shown an increase in LDH-5 activity towards the lens nucleus.184,185 Thus, the LDH isoen-zyme pattern may explain the high LDH activity in the lens nucleus. The staining may also be caused by non-specific reaction between NBT and sulfhydryl groups in the com-pacted proteineous nucleus.
Sample size and sources of variation With a minimum detectable differ-ence between treated and control lenses of 20% of the control value, i.e. effect size, and a power of 0.80, the estimated minimum sample sizes are for the epithelium 10 (5), the an-terior cortex 10 (7), the nucleus 20 (14), and for the posterior cortex
Results & Discussion
35
40 (30). Numbers in parenthesis are the calculated sample sizes, which will be underestimations since the estimated variance for mean paired-sample difference is based on un-treated animals. In a treatment study a new source of variation will be added. In an investigation including all lens regions, with the same set-up as in the present study, the sample should comprise at least 40 animals because the sample size for the pos-terior cortex will be the restricting factor.
The statistical optimisation of the method suggests that a reason-able set-up is the same as the present with 3 slides, 3 sections and 3 measurements.
4.3. LDH activity after UVR-B exposure (paper III)
For each time point (0, 2, and 6 hours), the enzyme activity in non-exposed eyes was significantly higher in the corneal epithelium and in the lens nucleus than in the re-maining lens regions. The UVR-B exposed eyes showed the same pat-tern.
In (Fig. 3, paper III), the ra-diation effect is shown for each time point and eye region. The LDH ac-tivity in the cortex of nonexposed lenses was similar to that in paper II. The nuclear levels were, however, lower. This is explained by the addi-tion of N-ethylmaleimide, which
partly blocked sulfhydryl groups, thus reducing non-specific formazan staining.
The LDH activity in the cor-nea and the outer anterior part of the lens decreased immediately after UVR-B exposure, which suggests a photochemical mechanism. There was no significant difference be-tween the three time points. There-fore the effect of UVR-B-induced ocular inflammation on the enzyme activity was small.
The decrease in lens LDH ac-tivity in the outer anterior cortex during the 6 hours of the study was approximately 20%, which conforms with the small effect on total lens lactate production seen after UVR-B exposure (paper I). This is also in accordance with the short penetra-tion of UVR-B in the lens. Evidence has been presented that the anterior, and not the posterior, parts of the lens are responsible for the devel-opment of UVR-B cataract. Only an-terior and not posterior UVR-B ex-posure of lenses had an effect on the ion pump activity in the lens epithe-lium and the development of cata-ract.163
The apparent UVR-B effect in the lens nucleus at 0 hours (Fig. 4, paper II) is most likely an artifact caused by sub-optimal morphology in this region and the technical measurement problems associated with it. There is always a statistical 5% risk that the confidence interval
Results & Discussion
36
excludes the true mean, in this case, hypothetically 0. Nuclear recovery from 0 to 6 hours after exposure is also unlikely. Recovery was not pre-sent in any other region and because there are no cell organelles in the nucleus, there can be no de novo production of new enzyme.
4.4. UVR-B absorption in the lens (paper III)
The penetration depth (T= 1/e2) in the anterior cortex was estimated to 0.45 mm, corresponding to ap-proximately 14 % residual UVR-B intensity. The attenuation of 300 nm UVR-B as expected from in vitro measurements of the linear absorp-tion coefficient in whole monkey133 lenses and sectioned anterior cortex from a 70-year-old human134 lens is plotted in (Fig. 5, paper III). The ab-sorption coefficient was in both lat-ter studies based on transmittance measurements and then transformed to absorbance. The monkey data suggest a considerably shallower penetration than our rat data, whereas the human lens data had a penetration depth of 0.36 mm, slightly shallower than our 0.45 mm.
The radiation was delivered by an interference filter source, hence the penetration would be ex-pected to go deeper than a 300 nm centred waveband. The deeper pene-tration of the absorption-derived curve compared with the other
transmittance-derived curves further support this. The other two curves were obtained with a spectropho-tometer, which provide a narrower bandwidth. In the present study, the UVR-B spectrum peaked at 302.6 nm and had a skew distribu-tion towards UVR-A.
The relatively shallow pene-tration depth of UVR-B in the lens necessitates a focus on lens epithe-lium and anterior cortex and indi-cates that whole-lens measurements might be less efficient.
4.5. Age, sex, and postex-posure time (paper IV)
The control lenses were all subjec-tively clear, with an age-dependent increase in forward light scattering.
Age sensitivity 1 week after UVR-B exposure The UVR-B−exposed lenses exhib-ited anterior subcapsular cataract and equatorial cataract, sometimes with spokes extending towards the posterior suture. In the 3-week-old group, 12 of 20 irradiated lenses were severely damaged.
Nine of the twelve severely damaged lenses (Fig. 5A, 5B, pa-per IV) and three of the lenses with quantifiable intensity of forward light scattering had nuclear cataract in addition to cortical cataract.
The proportions of severely damaged lenses after 8 kJ/m2 300 nm UVR-B was significantly
Results & Discussion
37
higher for rats aged 3 weeks in com-parison with rats aged 6 weeks. The same UVR-B exposure created variation of light scattering among 6, 17, and 52 week-old rats. Further, the same UVR-B dose induced more light scattering in 6-week-old rats than in 17 and 52 week-old rats, with no difference between the two latter groups.
Age sensitivity 8 weeks after UVR-B exposure For all three age groups the expo-sure to UVR-B increased the light scattering in exposed lenses relative to controls (Fig. 4, paper IV). Ex-posed lenses exhibited more exten-sive cataract compared to same age groups one week after exposure (Fig. 5, paper IV).
There were severely damaged lenses in the two younger groups in-cluding almost all exposed lenses in the 6-week-old group. The propor-tion of such was lower for the 17 week group and zero for the 52 week group. All groups differed significantly from each other.
Sex sensitivity For both sexes, 5 kJ/m2 300 nm UVR-B induced light scattering in the exposed lenses. There was no difference in light scattering be-tween sexes.
A pilot study with old rats in-dicated that a UVR-B dose below 8 kJ/m2 might not induce cataract.
The chosen dose of 8 kJ/m2 is well above the threshold dose for perma-nent UVR cataract in rabbits96 and young rats.97,111 The strong reaction in the 3-week-old rats in the first age experiment was not anticipated. Since the aim of the second age ex-periment was to compare postexpo-sure time, the dose was kept at 8 kJ/m2. The 3-week-old group was omitted because of the occurrence of severely damaged lenses already af-ter one week. For the 6-week-old rats in the sex experiment, 5 kJ/m2 was known from earlier experiments to be appropriate. The technique of forward light scattering is not designed for quanti-fication of cataract in lenses with severe damage. Consequently, the analyses of these lenses will have relatively less statistical power than the planned parametric statistics. This is important in future planning of experiments. Such planning would be facilitated by the data pre-sented in this study.
The suspicion was raised that the two-to-three-fold difference in cataract severity between the two younger and the two older groups at one-week follow-up was due to dif-ferences in eye dimension. Lenses from young rats are smaller and the anterior chamber shallower than in older rats. This means that with the same corneal dose, more radiation would reach the lens of a younger rat. Measurements on cryosectioned
Results & Discussion
38
rat lenses revealed a 25% difference in corneal thickness between the youngest and oldest rats (Fig. 6, pa-per IV).
Combining the actual corneal thickness and anterior chamber depths with the corneal and aqueous absorption coefficients for 300 nm from Maher133 gives a range of doses at the lens anterior surface from 3.8 to 2.9 kJ/m2 (from youngest to oldest rats), with a 8 kJ/m2 corneal dose. Linear regression analysis on esti-mated dose vs. age shows a slope significantly different from zero, in-dicating a true difference between the four doses. Since the dose-response function for UVR-B cata-ract is approximately linear in this dose range,111 the age-dependent dif-ferences in intra-ocular dimensions do not change the lenticular UVR-B dose in a magnitude that can explain the differences in cataract develop-ment among the age groups.
Lenses from young humans transmit more UVR than lenses of old humans.131 If the same holds for 300 nm UVR and rat lenses, it might be of importance since mitochondria are targets for UVR-B and lenses from young rats have a distribution of mitochondria reaching deeper into the lens than older rats.186
The cell division rate, and the lens growth rate, is higher in young rats. The mitotic region is located in the pre-equatorial zone behind the iris, which, in albino animals, trans-
mits a substantial part of UVR-B. When combining the lesser UVR-B absorption in the anterior segment, possibly lesser intralenticular lens UVR-B absorption, deeper mito-chondrial distribution and higher mi-totic activity in young animals, it is clear that they have a higher risk po-tential of developing UVR-B cata-ract.
The type of cataract for all but the 3-week-old rats was equatorial with anterior haze and cortical spokes extending to the posterior su-ture. We did not expect to find the nuclear cataract seen in the 3-week-old rats. It has, until now, almost been an axiom that experimental UVR-B cataract causes cortical cata-ract.
The increase in cataract sever-ity between 1 and 8 weeks after irra-diation is consistent with the cataract development occurring after 20 kJ/m2 300 nm UVR but in dis-agreement with that after 5 kJ/m2 300 nm UVR.116 There it was shown that with the higher UVR-B dose, cataract development progressed af-ter one week, while for the lower dose no further increase in light scattering occurred at time points up to 32 weeks. The reason for this dis-agreement with the 5 kJ/m2 dose is most probably the difference in ra-diation spectrum. They used an in-terference filter based source, which produced a wider UVR-B waveband, including more radiation in the up-
Results & Discussion
39
per UVR-B wavelengths, while in the current study a monochromator-based source was used (Figure 14).
The current safety limits for optical radiation (UVR, light and in-frared radiation) exposure do not consider age. Regardless of the mechanisms for the age dependency, the fact remains that young rats are more sensitive to UVR-B than older rats. Thus age should be considered as a factor during future revisions of UVR-B safety limits.
The example of UVR skin carcinogenesis may serve as a warn-ing as we know that sunburn epi-sodes in childhood predispose the child to skin cancer in adulthood.
4.6. Albino and pigmented rats, in vivo (paper V)
The average pupil size in the two strains after instillation of eye-drops was 1.7 mm with pilocarpine and 3.5 mm with tropicamide.
UVR-B-exposed eyes Fischer cataracts were mainly lo-cated equatorially with extension to the posterior cortex (Fig. 1B, paper V). Several lenses had anterior haze and equatorial vacuoles (Fig. 1C, paper V). In a few cases a ring-shaped anterior cataract was appar-ent (Fig. 1D, paper V). The Fischer groups differed significantly from each other and they also had more cataract than the Brown-Norway rats (Fig. 2, paper V).
Almost half of the exposed Brown-Norway lenses, in both groups, exhibited a small anterior polar subcapsular spot-like cataract (Fig. 1E, 1F; paper V). All groups except the miotic-treated Brown-Norway rats developed sig-nificantly more light scattering in exposed lenses compared to control lenses (Fig. 2, paper V). On the other hand, there was no significant difference in light scattering be-tween the two Brown-Norway groups (Fig. 2, paper V).
In general, the eye-lid margins were edematous and conjunctival injection was present. The corneas exhibited stromal infiltrates, ero-sions, haze, and marginal vasculari-zation. Hyphema was often present. Fischer rats regularly showed more ocular inflammation. The more prominent ocular inflammation in the Fischer rats, also having more severe cataract, indicates at least a co-variation between cataract and ocular inflammation.
There was also slightly, but consistently, more corneal damage and eyelid inflammation in the my-driatic-treated UVR-B-exposed eyes compared to the miotic-treated. Hy-phema and lid inflammation was al-most nonexistent in exposed Brown-Norway eyes, which most likely is due to the dark melanin in the pig-mented rat skin and iris.
The increased light scattering in the miotic-treated Fischer group,
Results & Discussion
40
compared to the mydriatic-treated, is most probably caused by the thinner, stretched-out iris transmitting more UVR-B to the lenticular germinative zone, the cell dividing area in the lens epithelium close to the equator.
Anterior polar cataract is not associated with albino rats. It has earlier been described in Brown Norway rats exposed to similar UVR-B doses.93,187
It is not surprising that albino rats are more sensitive to UVR-B than pigmented rats. Melanins (in the iris) have antioxidant proper-ties188 and also function as UVR heat sink, converting small parts of the UVR to heat. The cooperation of iris melanin and ascorbate in the aqueous results in UVR-induced production of hydrogen peroxide and superoxide anions,189,190 which is neutralized by the normal antioxi-dative pathways.151
The small difference in UVR-B cataract development with small or large pupil in pigmented eyes does not support the fear of sunglasses as harmful for the lens. However, if the sunglasses are not tightly fit, oblique sunrays might reach sensitive parts of the lens.
4.7. Albino and pigmented rats, in vitro (paper VI)
The changing of medium every other day disturbed the lenses. Within a few minutes of changing the me-dium, the lenses developed a tran-
sient anterior haze. The haze disap-peared after about 15 minutes. Due to the development of haze at me-dium change, the light scattering in the lenses was measured immedi-ately before change of medium. In several dishes, a slight clouding of the medium appeared after about 5 days. It reappeared even after change of medium.
UVR-exposed lenses
Cataract appearance One day after exposure, some ex-posed lenses developed a faint hazy-granular subcapsular cataract, con-fined to the anterior surface (Figure 17A-B). On day 2, it had progressed and was evident in all exposed lenses. The granules were best viewed from the posterior aspect. By day 2-3 shallow small equatorial vacuoles and deeper cortical opaci-ties posterior to the equator appeared (Figure 17C). By day 4-5, the poste-rior opacities progressed to the pos-terior suture region, creating a trian-gular clear zone at the pole (Figure 17D-E).
All three types of cataract progressed, resulting in: 1) anterior subcapsular cataract with a floccular appearance; 2) very large shallow equatorial vacuoles; 3) shell-like opacification of the whole mid-to-deep posterior cortex, not crossing over the equator (Figure 17F-G). No nuclear cataract was observed.
Results & Discussion
41
UVR-exposed lenses
Day 1 Day 3 Day 4 Day 7
Figure 17. UVR-exposed lenses after 1-7 days in organ culture. A, D, F) Incident illumination. B, C, E, G). Dark-field illumination. All lenses are viewed posterior side up except Fig. 3G, which is viewed from an oblique angle. Grid unit length is 0.79 mm. B is m magnified 30% vs the others.
Forward light scattering After a lag phase of one day, the light scattering in the exposed lenses increased steadily with time after exposure (Fig. 4, paper VI). By day 2, the light scattering was signifi-cantly stronger in exposed lenses than in nonexposed lenses in both strains, as indicated by the confi-dence interval for the mean paired-sample difference between exposed and nonexposed lenses (Fig. 4, paper VI). Lenses from Fischer rats regis-tered the highest readings, following the trend of increasing difference over the course of the week. The in-crease in light scattering was best modelled with a 2nd order polyno-mial, and the 2nd order regression coefficient for Fischer rats was sig-
nificantly higher than for Brown-Norway rats.
The clouding of medium might be a sign of contamination, despite antibiotics. Because of bulky equipment in different locations (UVR-B source, light dissemination meter and microscope with camera), the lenses could not be handled in laminar flow environment at all times outside the incubator. The daily measurements introduced sev-eral opportunities for contamination since the culture dish lids had to be removed during scattering measure-ments and photography.
The anterior granular-like subcapsular cataract is most likely caused by apoptosis and necrosis of the epithelial cells. Both the time
A D F
B E G C
no image
Results & Discussion
42
frame and physical appearance cor-relate well with in vivo studies using approximately the same lenticular UVR-B doses.111,191
The appearance of the equato-rial opacities is similar after expo-sure to other oxidative or osmotic agents.192,193
The cortical opacity surround-ing, but initially not involving, the posterior suture lines is also caused by disturbed ion and water transport mechanisms. Because of the short penetration of UVR-B in the lens,133,134,paper III no UVR-B reaches the posterior lens region. The fiber cells are however elongated, reach-ing from the UVR-B-exposed ante-rior cortex to the posterior. It cannot be ruled out that anterior UVR-B damage is signalled intracellularly to the posterior fiber cell endings. Ul-timately the pump load will be too strong for the ion pumps, and indi-rectly the mitochondria, concen-trated around the posterior su-tures.194 As a result, the posterior su-ture region per se becomes opaci-fied.
Equatorial vacuoles were pre-sent in both strains, in contrast with the earlier in vivo experiment where only the albino rats developed vacu-oles. These vacuoles, described in a chloride channel inhibitor model,193 also appear due to malfunctioning ion transport mechanisms. There are regional differences in transport di-
rection in the lens, with the equator having the highest concentration and activity of ion pumps and gap junc-tions, resulting in an net outward current.22 When UVR-B hits the equatorial region, the ion pump function deteriorates, resulting in an accumulation of extracellular water.
The lens light scattering (in tEDC units) as measured in the Petri dish is 12% lower than for the cu-vette-based measurements in earlier non-culture experiments. Correcting for this, the light scattering pre-sented is still 3-to-10-fold higher than in the in vivo experiment (paper V). Given all differences between in vitro and in vivo exposure, with a majority giving protection in the in vivo situation, it is not surprising to find that UVR-B cataract was more severe in the present in vitro ex-periment compared to the preceding in vivo experiment with similar len-ticular dose (paper V).
The results suggest that there is a small but real difference in inherent UVR-B sensitivity between pig-mented and albino rat lenses. The UVR-B cataract type induced in vi-tro differs from in vivo cataract in pigmented rats, but not in albino rats. The degree of in vitro-induced cataract in both albino and pig-mented rats is higher than can be expected from in vivo UVR-B lenticular dose calculations.
Conclusions
43
5. Conclusions
• In vivo UVR-B exposure, but not blue light, inhibits lens glycolysis.
• With optimised LDH histochemistry it was determined that lens epithelium and nucleus have high LDH activity and cortex low activity.
• LDH is inactivated in the anterior parts of the lens and remaining UVR-B in-tensity at 0.45 mm depth is about 15%.
• Young rats are more sensitive to UVR-B than old rats, with no difference be-tween sexes, and the time for maximal cataract to develop is dependent on age.
• Iris pigmentation is highly UVR-B protective, with little importance of pupil size in pigmented eyes, while the opposite holds for albino eyes where a large pupil is more protective than a small pupil.
• In vitro UVR-B-exposed lenses from albino rats are more sensitive than lenses from pigmented rats, and the cataract type differs between the two strains.
Improvements and future
44
6. What can be improved and what might follow?
Paper I Biochemical or nuclear magnetic resonance (NMR) analysis of lens sections, as a complement to media aliquots, would increase the prob-ability of detecting effects on the glycolysis.
Paper II The attachment of the sections to the slides has to be improved. It might be worthwhile to try fibrin glue. Also, thicker sections could be tried, but this can lead to nonlinearity in the staining reaction. Stabilisation with an enzyme-inert substance might improve the quality of the sec-tions. The densitometry was time-consuming and should preferably be automated in the form of a pro-grammable motorized x-y table. A modern CCD equipped scanning densitometer would be a solution.
Paper III The combination of appropriate UVR-B laser(s) and histochemical or biochemical determination of several end-points would give a powerful compound knowledge about the biological penetration depth. Discrimination between UVR-B absorption and other UVR-B attenuating factors in lens section would be possible with the use of integrating spheres.
Papers IV-VI The proportions of absorption and scattering in cataractous lenses can be determined with an integrating sphere. This might improve the for-ward light scattering measurements for lenses with moderate to severe cataract. At least this would facili-tate establishment of a light scatter-ing limit, above which forward light scattering is less suitable.
In vitro exposure of lenses of different ages might lead to a better understanding of the age dependent sensitivity. It is necessary to find out if young individuals need to avoid sunlight more than older ones.
The effect of UVR entering the eye in an oblique angle should be further studied. The clinical ob-servation of infero-nasal cataracts should be investigated with a suit-able model system. Also, the protec-tive properties of the iris ought to be addressed.
The chain of events ultimately leading to UVR cataract is not known. The search is still out for the earliest detectable changes. It is rea-sonable to expect cellular studies to be the tool for this search. On the other hand, in vivo research is needed in order to understand the effect of different UVR exposure patterns on the lens.
References
45
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2. Hirschberg J. The history of ophthalmology. The monographs, Vol.1. The eye and man
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3. Albert DM. Greek, Roman, and Arabian Ophthalmology. In: Albert DM, Edwards DD,
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4. Lascaratos J, Marketos S. A historical outline of Greek ophthalmology from the Helle-
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Appendices
59
8. Appendices
