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Morphological and histological changes in eye lens: Possible application for estimating postmortem interval
Legal Medicine 17 (2015) 437–442
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Legal Medicine journal homepage: www.elsevier.com/locate/legalmed
Morphological and histological changes in eye lens: Possible application for estimating postmortem interval Gemma Prieto-Bonete, Maria D. Perez-Carceles ⇑, Aurelio Luna Department of Legal and Forensic Medicine, Biomedical Research Institute (IMIB-Arrixaca), Regional Campus of International Excellence ‘‘Campus Mare Nostrum”, University of Murcia, Spain
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Article history: Received 26 February 2015 Received in revised form 3 September 2015 Accepted 14 September 2015 Available online 14 September 2015 Keywords: Postmortem interval Eye lens Corneal transparency Proteins Histology
a b s t r a c t Establishing the postmortem interval is a very complex problem in Forensic Science despite the existence of several macro- and microscopic methods. In the case of ocular methods, most are based on an evaluation of the biochemical components of the vitreous humour 24–36 h after death, but, to our knowledge, there are no studies on the relationship between lens and the postmortem interval. Since the lens is protected between the vitreous humour and the aqueous humour inside the eyeball, postmortem changes are assumed to start later in the lens. To evaluate the usefulness of using the lens to establish the postmortem interval, we examined 80 rabbit lens enucleated 24, 48, 72 and 96 h after death, assessing changes in sphericity and absorbance at different wavelengths and any histological alterations. Both sphericity and absorbance were seen to decrease to a statistically significant extent, and there was a gradual loss of structure and organisation of the lens components as a function of the postmortem interval. Modifications in the lens were seen to be useful for determining the postmortem interval between 24 and 96 h. Ó 2015 Elsevier Ireland Ltd. All rights reserved.
1. Introduction Establishing the postmortem interval continues to be one of the most complex problems in Forensic Science. Studies based on ocular data to establish PMI are few and tend to be based on an evaluation of the biochemical components of the vitreous humour, such as sodium, potassium, chloride, lactate and hypoxanthine 24–36 h after death [1–4]. The lens is positioned in the eye behind the iris. The anterior lens surface is bathed by aqueous humor, while the posterior lens surface is in contact with the vitreous body [5]. Contains 1000–3000 layers of fiber cells [6]. The adult lens contains two kinds of fiber cells: (i) those located in the cortex (the outermost layers of the lens), which are not yet mature and still contain organelles (including mitochondria), all of which are degraded through protease- and nuclease-regulated processes, leaving behind membrane-enclosed bags of crystallines, and (ii) those located in the nucleus (the core of the lens), which are mature and do not contain organelles [6]. Into subcellular organelle evacuation during maturation is necessary to ensure the transparency of the lens, as ⇑ Corresponding author at: Department of Legal and Forensic Medicine, Biomedical Research Institute (IMIB-Arrixaca), Regional Campus of International Excellence ‘‘Campus Mare Nostrum”, School of Medicine, University of Murcia, E-30100, Spain. E-mail address: [email protected] (M.D. Perez-Carceles). http://dx.doi.org/10.1016/j.legalmed.2015.09.002 1344-6223/Ó 2015 Elsevier Ireland Ltd. All rights reserved.
organelles scatter light, whereas ordered proteins (crystallins) do not. Protein synthesis and protein degradation are minimal or non-existent, and crystallins and perhaps other proteins that were synthesized at the birth of the cell persist throughout the life of the organism [7]. The transparency of the eye lens thus depends on the regular alignment of elongated fiber cells, which perform the difficult task of stacking together neatly to fill a spheroidal volume, filled with cytoplasmic crystallins and cytoskeletal intermediate filaments encased in membranes made from a few integral membrane proteins. The lens proteins belong to common protein families, but the lens tends to have its own unusual version [8]. Crystallines, which are expressed as three different isoforms (a-crystallin, b-crystallin and c-crystallin), are major cytoplasmic components of the vertebrate eye lens that constitute >90% of the total protein content in eye lens fiber cells and >35% of their wet weight [7]. The chaperone action of a-crystallin is vital for maintaining eye lens transparency. The reasons for using rabbit as an experimental model to study the lens are the following: First, both the rabbit and human lens have branched sutures, although the former is of the ‘‘line” type and the latter of the ‘‘star” type. For this reason, the rabbit lens can be considered as a simplification of the more complex organisation of the fibers of the human eye [9–13]. Secondly, the rabbit lens is closer in size and sphericity to the human lens than other
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Fig. 1. Sphericity of the lenses and statistically significant differences between right and left eyes for postmortem intervals.
lens commonly used in experiments, such as mouse or rat. The first study to use rabbit eye as an experimental model measured the thickness of the cornea by ultrasounds [2]. Lastly, in this study what we have achieved of the functional parameters of rabbit lens provides a basis for comparison with the human lens [10,14–16]. While there are many studies on the morphological and histological changes related to certain pathologies, we have found no reference to the use of the lens to establish the PMI. Determining the changes in lens transparency postmortem would be a useful starting point for testing the possibility of determining visual impairment conditions’ in a cadaver [17]. The postmortem evaluation of the lens would provide the added benefit of associating different forms and stages of cataracts with accidents, thus affording new information relevant to the development of preventive measures [18]. Apart from estimating the PMI, the age of the subject could be ascertained between 24 and 48 h after death by applying radiocarbon techniques to the lens [17]. In conclusion, the postmortem determination of lens opacity would provide helpful information that could be used during legal proceedings and it would be also a good complement to clinical data, and fundamental in cases where there is no medical documentation. The aim of the present study was to assess whether the postmortem morphological and histological modifications that take place in the lens may be related to the postmortem interval itself.
2. Materials and methods 2.1. Sample collection The lens (n = 80) were taken from 40 rabbits with an average age of 84.02 days old (75–95 days old) sacrificed in a local meatprocessing company [19]. All the animals used were treated in the normal way and were not killed for the sake of the experiment described. In the laboratory of the Forensic Medicine Department of the University of Murcia (Spain), the lens were left exposed to
the air in a room with a mean temperature of 21.3 °C at 24 hpm, 21.4 °C at 48 hpm, 22.4 °C at 72 hpm and 22.7 °C at 96 hpm. The animals were cared for following Spanish law (RD 1201/05) according to the principles of EU directive EU 86/609. This study was approved by the Ethics Committee of the University of Murcia (Spain). 2.2. Enucleation and measurements of sphericity and absorbance The 80 lens were classified into four groups of 20 samples each. Every 24 h all the lens from a given group were enucleated (first group 24 h postmortem, second group 48 hpm, third group 72 hpm and the last group 96 hpm) by making a lateral incision and cutting the orbital muscles [20]. Once extracted, the 20 lens were placed in physiological saline, the their absorbance was measured at 365, 370, 375, 415 and 420 nm using a Shimadzu UV-160 spectrophotometer [21]. The absorbance of each eye lens was measured directly in a 1-cm plastic cuvette designed and developed in our laboratory, and an empty cuvette was used as blank. The special design of the cuvette allowed the lens to be held in place vertically, so that its own weight and the effect of gravity did not affect its structure since it was supported by the edges, leaving the central part in its correct anatomical position when light rays were directed at it. After measuring, the lenses were photographed using a Nikon DX digital camera (AF-S DX NIKKOR), at 20 cm distance and with a resolution of 10.2 megapixels. The image analysis program image tool UTHSCSA was used to measure the greatest and smallest diameter, and to calculate the sphericity of the lens. In this way, we could assess any modification in transparency or translucence as a function of the PMI. 2.3. Histological study After visual examination by image tool, the eyes lens were fixed in 10% buffered formalin for 22–30 days and embedded in paraffin. Sections (4 nm thick) were obtained from each paraffin block and placed on SuperFrost (Menzel-Gläser, Braunschweig, Spain) plus
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Fig. 2. Macroscopic image of the lens: (a) 24 h postmortem; (b) 48 h postmortem; (c) 72 h postmortem; (d) 96 h postmortem.
glass slides. After deparaffinization and rehydration in graded alcohols, haematoxylin and eosin and was applied. 2.4. Statistical analysis The spss 20.0 package (SPSS Inc., Chicago, IL, USA) was used for statistical analysis of the data, calculating the mean, standard deviation (SD) and 95% confidence levels. The Kruskal–Wallis test, a non-parametric test for more than two independent samples, was used to compare groups. Also, specific contrasts for each variable grouped according to diagnostic category were carried out using the non-parametric Mann–Whitney test for two independent samples. p values less than 0.05 were considered statistically significant.
absorbance values between the lenses at any wavelength within the same PMI. The absorbance values for each wavelength are indicated in the table. As can be seen, absorbance decreases as the postmortem interval increases (Table 1). The data were compared and analysed by non-parametric tests. The Kruskal–Wallis test for the IPM variable revealed a significant difference between the groups at 365 nm, 370 nm, 375 nm, 415 nm and 420 nm. Mann–Whitney tests were used to investigate any significant results obtained from the initial Kruskal–Wallis analysis (Table 1). When the lens absorbance values were compared for different PMIs statistically significant differences were found between the lenses in all cases. 3.2. Lens sphericity: at different PMIs
3. Results 3.1. Measurement of absorbance in right and left lenses at different wavelengths and for different postmortem intervals In all the right and left lenses for every sampling time postmortem there were no statistically significant differences in the
Mean and 95% confidence interval of lens sphericity were: 0.976 ± 0.005 (0.965–0.988) at 24 h postmortem, 0.967 ± 0.007 (0.956–0.981) at 48 h postmortem, 0.950 ± 0.007 (0.929–0.971) at 72 h postmortem and 0.913 ± 0.015 (0.881–0.947) at 96 h postmortem. For examination of the sphericity variable, the Kruskal–Wallis test produced a significant difference with IPM
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Fig. 3. (A) Lens 24 h post-mortem: (a) The cell nucleus (black arrow) (HE, 4); (b) the lens capsule and epithelium and, below these, adhering lens fibres (black arrows) (HE, 10); (c) detail of the image observed in Figure a. The transparent capsule (black arrow) and epithelial cells joined to the uniformly distributed lens fibres (HE, 40). (B) Lens at 48 h post-mortem: (a) posterior face: well preserved epithelium in the peripheral zone (black arrow). (HE, 10); (b) nuclei of epithelial cells (HE, 40); (c) lens fibres (black arrow) uniformly distributed and joined, with no space between them (HE, 40). (C) Lens 72 h post-mortem: (a) the lens fibres are separated (black arrow) (HE, 10); (b) young disaggregated fibres (HE, 10); (c) detail of Figure b. The space between the young fibres is increasing (black arrow) (HE, 40). (D) Lens 96 h post-mortem: (a) eosinophil structures (black arrow) (HE, 10); (b) agglomeration of randomly disposed elongated eosinophil structures (black arrows) (HE, 40).
Table 1 Mean, confidence interval and p-values of the absorbance values at different wavelengths and for each post-mortem interval.
*
Postmortem interval (h)
W 365 nm Mean (95% CI)
W 370 nm Mean (95% CI)
W 375 nm Mean (95% CI)
W 415 nm Mean (95% CI)
W 420 nm Mean (95% CI)
24 48 72 96
468.8 418.1 388.5 334.9
537.1 464.5 409.2 325.3
509.3 410.2 393.5 320.3
462.8 399.9 366.6 313.3
464.8 (426.5–503.0) 411. 9 (397.5–426.3) 381.8 (359.2–404.4) 321.7 (279–364.4)
p-value Kruskal–Wallis 24 vs 48* 24 vs 72* 24 vs 96* 48vs 72* 48 vs 96* 72 vs 96*
<0.0001 0.036 0.001 <0.0001 0.031 0.001 0.026
(427.6–509.9) (400.1–436) (365.2–411) (293–376.7)
(488.7–585.4) (431.1–532.1) (379.5–438.9) (281.6–369.1)
<0.0001 0.006 <0.0001 <0.0001 0.002 <0.0001 0.008
(463.5–555.1) (424.8–455.7) (367–420.1) (279–361.7)
<0.0001 0.003 <0.0001 <0.0001 0.003 <0.0001 0.007
(419.3–506.27) (384.4–415.3) (344–389.2) (270.2–354.5)
<0.0001 0.002 <0.0001 <0.0001 0.012 <0.0001 0.041
0.001 0.023 0.001 <0.0001 0.041 <0.0001 0.041
p-Value Mann–Whitney.
(p < 0.0001). Statistically significant differences in sphericity were found between the different PMIs in all cases. Moreover there was a tendency for the sphericity to decrease as the PMI increased (Fig. 1). 3.3. Macroscopic evaluation of transparency A macroscopic study of the lenses at each PMI pointed to the tendency for transparency to decrease with time. After 24 and 48 h, the transparency was still apparent, while at 72 it had begun to diminish and at 96 h the lenses were totally opaque and had lost their sphericity (Fig. 2).
3.4. Postmortem histological analysis of eye lens At 24 and 48 h postmortem the lens structure are similar. Histologically, all the cell layers could be observed - the external capsule, cell epithelium, cortex and nucleus (Figs. 3A and B). Adhering to and as a continuation of the epithelium, note the uniformly distributed fibres of lens both in the cortex and nucleus (Figs. 3Ab, Ac, Bb, Bc). Nucleated fibres corresponding to young fibres can be observed in the equatorial zone (Fig. 3Aa). However, at 72 h, alterations in the structure and order of the lens layers began to appear (Fig. 3Ca). Neither the capsule nor the layer of epithelial cells could be distinguished, although the fibers of the
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cortex and nucleus had lost their uniformity, being disaggregated and separated probably due to the loss of the mechanisms that held them together (Figs. 3Cb and Cc). Similarly, at 96 h neither the capsule nor epithelial cells could be distinguished (Fig. 3Da) and, instead of individual fibers, aggregated eosinophils were evident (Figs. 3Da and Db).
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the cell membranes is not related with the loss of transparency, but is separate phenomenon [30]. There are many studies concerning cornea turbidity to determine the postmortem interval using image analysis and it has been shown that cornea opacity increases with time after death [29,31]. 4.3. Loss of lens internal structure and sphericity
4. Discussion As bibliographical search revealed no studies on the estimation of the PMI by reference to the lens, which led us to analyse morphological (macroscopic and histological) and biochemical modifications related with the same to see whether such information could provide information on the PMI between 24 and 96 h. Our study shows how the lens becomes opaque with time, losing transparency, sphericity of the anterior and posterior face and absorbance at the long wavelengths tested. All these morphological changes in the macroscopic structure are accompanied by a loss of the internal structure of the cell fibers and cells. 4.1. Loss of transparency with time The loss of transparency with time is due to the metabolic processes that take place between the lens and the aqueous and vitreous humor and the aggregation of crystallins proteins in the fibers of the lens nucleus. The lens epithelium is fundamental in the opaque process since it contains a region with a high metabolic level, which is responsible for the vital transport of different electrolytes, amino acids and metabolic product. The reactions pump introduces potassium into the lens as sodium is expelled. The transparency of the lens is partly due to the correct functioning of this pump, and when it stops with the death of the subject potassium levels fall in the lens, favouring the entry of chlorine, accompanied by water and calcium. Together with the cell necrosis that occurs after death, in which lysosomal enzymes play a part, this leads to an increase in opacity, i.e. loss of transparency [21,22]. The in vivo process of opacification, known as cataracts, is produced by different mechanisms to those which act postmortem. The oxidising agents alter the protein structure and affect the mechanisms of aggregation, cross-linking, peptide cleavage and the introduction of active carbons in the protein chains [7,23–25]. Besides these processes, crystallins appear in the fibers a protein fraction of high molecular weight [26]. Protein–protein disulphides are responsible for the increase in protein of high molecular weight, found in only in cataractous lenses [27]. It has been hypothesized that lens proteins aggregate to large particles that scatter light, which causes lens opacity [28]. Autopsies performed within 10–30 h of death can confidently use this method for the accurate estimation of postmortem a time. However, with shorter and longer times after death, the accuracy may diminish. Corneal opacity can also occur due to the transpiration of fluid and the degeneration of endothelial cells, and may therefore be accelerated in high temperatures [29]. Upon death or removal of an eye the cornea absorbs the aqueous humour, thickens, and becomes hazy. A hazy cornea presents edema, paleness, obvious thickening and tiny frills on the surface, and becomes concave due to dehydration and consequent shrinkage; finally, its endodermis adheres to the crystalline lens. 4.2. Decrease in absorbance with PMI Absorbance was seen to decrease with the length of the PMI, reaching a minimum at 96 h. This decrease was due to the loss of orientation of the fibers in the lens [7]. The damage suffered by
In the anatomopathological study the PMI-dependent destructuralization was also clear in the epithelium central zone or nucleus and the capsule. At 24 and 48 h after death, the lens structure was conserved as if it had been enucleated immediately after death just as it was after enucleation, preserving all its cell layers and fibers intact. However, after 48 h the anterior capsule was looser than the posterior since the latter was more hydrated through contact with the vitreous humour and so was less affected by postmortem phenomena. The integrity of the crystallin proteins of the fibers depends on vital metabolic processes that involve their aggregation, oxidation, proteolysis, deamination, glycosylation and transpeptidation [32,33]. The cessation of metabolic exchange processes increases the quantity of free glucose within the lens, provoking excessive glycosylation of the crystallin proteins. Consequently the lens attracts water, changing the permeability of the sodium and so altering the distribution of ions [34]. This is accompanied by an increase in the concentration of sodium and a loss of potassium and amino acids. Moreover, the high sorbitol concentration attracts water, provoking the breakage, liquefaction and separation of the fibers, and asymmetry in the lens sutures, creating vacuoles in the cortex and affecting the lens structure. As a result of the loss of the intracytoplasmic content during the cell degeneration process, a dense material, known as multilamellar bodies, appears, producing changes in the density of the cytoplasmic membranes and separating the fibers [35,36]. Something similar occurs with cornea. The change in corneal opacity is believed to be secondary to the change in hydration, while the increased water content of the stroma is the main cause of corneal swelling. Other causes of loss of structure include architectural destruction of the collagen fiber network, functional alteration of corneal endothelia, dysregulation of proteoglycan hydration and the ion concentration of the corneal stroma. The part of the cornea covered by the eyelid is more likely to become hazy sooner. When the ambient temperature is relatively high, opacity tends to occur early [31]. One limitation of the study is that it only looks at the lens of presumably healthy eyes. It has been seen that temperature has a significant influence on protein degradation and the survival of endothelial cells [37,38], and so the ambient temperature should be taken into account. Light per se does not affect lens transparency, unlike the cornea in which changes in opacity are believed to be secondary to the change in hydration, while the increased water content of the stroma is the main cause of corneal swelling [31], both of which are closely related with the eye being open [29,39]. Lens and cornea opacity are two independent processes. Age is another factor to consider, since cataracts tend to be formed with age, while it is generally accepted that corneal opacity increases with age due to a decrease in the number of corneal endothelial cells, which results in an increase in the thickness of the cornea owing to inflow of aqueous humor to the cornea [29]. Other influential factors include the individual variation and presence of cataracts, in which case measurements of transparency cannot be used, and only sphericity can. Lastly, it should be mentioned that the procedures described can be applied in human autopsies so that the eyeball does not have to be removed, making ophthalmological endoscopy a valuable tool in forensic autopsy [40]. Another technique, postmortem
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monocular indirect ophthalmoscopy, consists of a light source attached to a headband along with a hand-held lens. This permits a wide view of the fundus after death. To facilitate skill acquisition in monocular indirect ophthalmoscopy, a simple and inexpensive teaching model can be constructed from hinged, cylindrical plastic containers [41]. When the Miyake-Apple posterior video/photographic technique is used (the most widely used technique), the eyes are bisected through the equator and glued to a glass slide. Posterior images can be obtained using an extra microscope connected to a camera recorder below the eye. Such procedures are usually carried out with an open sky preparation. This technique is the easiest and provides a wide spectrum of information [42–44].
[14]
[15]
[16]
[17]
[18]
[19]
5. Conclusion The results obtained in this study suggest that measurements of lens sphericity and absorbance and a histological analysis of the lens may be regarded as complementary tools that will provide useful information for estimating the PMI between 24 and 96 h. Further studies in human will provide more information on the proposed method. It is possible to apply radiocarbon dating of the human eye lens crystallines to reveal proteins [15] by means of a histopathological study of postmortem eyes. Lastly, complementary immunohistochemistry can be used for skill acquisition in monocular indirect ophthalmoscopy to detect lens protein and structural changes.
[20] [21]
[22] [23] [24]
[25]
[26] [27]
Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.legalmed.2015. 09.002.
[28]
[29]
[30]
References [1] H.V. Chandrakanth, T. Kanchan, B.M. Balaraj, H.S. Virupaksha, T.N. Chandrashekar, Postmortem vitreous chemistry – an evaluation of sodium, potassium and chloride levels in estimation of time since death (during the first 36 h after death), Int. J. Leg. Med 20 (2013) 211–216. [2] G.L. Lü, F.X. Jiang, X.S. Xu, Y.J. Jiang, Z.G. Li, X. Wang, H. Shi, L.C. Yu, C.C. Xu, Estimation of postmortem interval by detecting thickness of cornea using ultrasonic method, Fa Yi Xue Za Zhi 28 (2012) 89–91. [3] R.T. Matias, X.L. Rae, L. Ebiham, The localization of transport properties in the frog lens, Biophys. J. 48 (1985) 423–434. [4] Z. Mihailovic, T. Atanasijevic, V. Popovic, M.B. Milosevic, Could lactates in vitreous humour be used to estimate the time since death?, Med Sci. Law 51 (2011) 156–160. [5] S. Bassnett, Y. Shi, G.F. Vrensen, Biological glass: structural determinants of eye lens transparency, Philos. Trans. R Soc. Lond. B Biol. Sci. 366 (2011) 1250–1264. [6] W.K. Subczynski, M. Raguz, J. Widomska, L. Mainali, A. Konovalov, Functions of cholesterol and the cholesterol bilayer domain specific to the fiber-cell plasma membrane of the eye lens, J. Membr. Biol. 245 (2012) 51–68. [7] B.H. Toyama, M.W. Hetzer, Protein homeostasis: live long, won’t prosper, Nat. Rev. Mol. Cell Biol. 14 (2013) 55–61. [8] A.R. Clark, N.H. Lubsen, C. Slingsby, SHSP in the eye lens: crystallin mutations, cataract and proteostasis, Int. J. Biochem. Cell B 44 (2012) 1687–1697. [9] A. Gwon, The rabbit in cataract/IOL surgery, Anim. Model Eye Res. (2008) 184– 204. [10] S. Al-khudari, S.T. Donohue, W.M. Al-Ghoul, K.J. Al-Ghoul, Age-related compaction of lens fibers affects the structure and optical properties of rabbit lenses, BMC Ophthalmol. 7 (2007) 19. [11] J.R. Kuszak, J.G. Sivak, K.L. Herbert, S. Scheib, W. Garner, G. Graff, The relationship between rabbit lens optical quality and sutural anatomy after vitrectomy, Exp. Eye Res. 71 (2000) 267–281. [12] N. Braun, M. Zacharias, J. Peschek, A. Kastenmüller, J. Zou, M. Hanzlik, S. Weinkauf, Multiple molecular architectures of the eye lens chaperone aBcrystallin elucidated by a triple hybrid approach, Proc. Natl. Acad. Sci. 108 (2011) 20491–20496. [13] A. Laganowsky, J.L. Benesch, M. Landau, L. Ding, M.R. Sawaya, D. Cascio, D. Eisenberg, Crystal structures of truncated alphaA and alphaB crystallins reveal
[31]
[32] [33] [34]
[35] [36]
[37]
[38]
[39] [40] [41] [42] [43]
[44]
structural mechanisms of polydispersity important for eye lens function, Protein Sci. 19 (2010) 1031–1043. X. Liu, M. Zhang, Y. Liu, P. Challa, P. Gonzalez, Y. Liu, Proteomic analysis of regenerated rabbit lenses reveal crystallin expression characteristic of adult rabbits, Mol. Vis. 14 (2008) 2404. J.R. Kuszak, C.A. Enneser, J. Umlas, The ultrastructure of fiber cells in primate lenses: a model for studying membrane senescence, J. Mol. Struct. Res. 100 (1988) 60–66. J.R. Kuszak, H.G. Brown, Embryology and Anatomy of the Lens. Embryology and Anatomy of the Lens. Principles and Practice of Ophthalmology: Basic Sciences, Philadelphia Press, 1994. N. Lynnerup, H. Kjeldsen, S. Heegaard, C. Jacobsen, J. Heinemeier, Radiocarbon dating of the human eye lens crystallines reveal proteins without carbon turnover throughout life, PLoS One 3 (2008) e1529. V. Stemberga, A. Petaros, D. Kovacevic, M. Coklo, N. Simicevic, A. Bosnar, The assessment of lens opacity postmortem and its implication in forensics, J. Forensic Leg. Med. 2013 (20) (2013) 1142–1144. J. DiMattio, In vivo entry of glucose analogs into lens and cornea of the rat, Invest. Ophthalmol. Vis. Sci. 25 (1984) 160–165. L. Harris, J.B. Gehrsitz, Sodium pump in human lens, Am. J. Ophthalmol. 36 (1953) 39–44. S. Bassnett, S. Stewart, O. Duncan, Efflux of chloride from rat lens: influence of membrane potential and intracellular acidification, Exp. Physiol. 73 (1988) 941–949. O. Duncan, The site of the ion restricting membranes in the toad lens, Exp. Eye Res. 8 (1969) 406. O.A. Candia, P.J. Bentley, C.D. Milis, Asymmetrical distribution of the potential difference in the toad lens, Nature 227 (1970) 852. V.A. Lucas, J.R. Zigler, Identification of the monkey lens glucose transporter by photoaffinity labeling with cytochalasin, Invest. Ophthalmol. Vis. Sci. 29 (1988) 630. C.A. Paterson, N.A. Delamerre, L. Mawhorter, Na, K-ATPase in stimulated eye bank and cryoextracted rabbit lenses, and human eye bank lenses and cataracts, Invest. Ophthalmol. Vis. Sci. 24 (1983) 1534–1538. J.J. Harding, Disulphide cross-linked protein of high molecular weight in human cataractous lens, Exp. Eye Res. 17 (1973) 377–383. A. Spector, Debdutta Roy, Disulfide-linked high molecular weight protein associated with human cataract, Proc. Natl. Acad. Sci. 75 (1978) 3244–3248. R.E. Perry, M.S. Swamy, E.C. Abraham, Progressive changes in lens crystallin glycation and high-molecular-weight aggregate formation leading to cataract development in streptozotocin-diabetic rats, Exp. Eye Res. 44 (1987) 269–282. W. Kawashima, K. Hatake, R. Kudo, M. Nakanishi, S. Tamaki, S. Kasuda, A. Ishitani, Estimating the time after death on the basis of corneal opacity, J. Forensic Res. 6 (2015) 1. F. Malacaze, P. Chollet, E. Cavrois, Role of interleukin 6 in the inflammatory response after cataract surgery: an experimental and clinical study, Arch. Ophthalmol. 109 (1991) 1681–1683. L. Zhou, Y. Liu, L. Li, L. Zhuo, M. Liang, F. Yang, S. Zhu, Image analysis on corneal opacity: a novel method to estimate postmortem interval in rabbits, J. Huazhong Univ. Sci. Technol. [Med. Sci.] 30 (2010) 235–239. G.P. Ceschi, L.G. Artraria, Clear lens extraction for correction of high grade myopia, Klin. Monbl. Augenheilkd. 212 (1998) 280–282. J.L. Rae, J.R. Kuszak, The electrical coupling of epithelium and fibers in the frog lens, Exp. Eye Res. 36 (1983) 317–326. L.W. Andrew, G.J. Jin, Prospective evaluation of early visual and refractive effects with small clear corneal incision for cataract surgery, J. Cataract Refract. Surg. 22 (1996) 1456–1460. N. Brown, Slit-image photography, Trans. Ophthalmol. Soc. UK 89 (1969) 397– 408. C.M. Cunanan, P.M. Tarbux, P.M. Knight, Surface properties of the intraocular lens material and their influence on in vitro cell adhesion, J. Cataract Refract. Surg. 17 (1991) 767–773. Y.O. Poloz, D.H. O’Day, Determining time of death: temperature-dependent postmortem changes in calcineurin A, MARCKS, CaMKII, and protein phosphatase 2A in mouse, Int. J. Leg. Med. 123 (2009) 305–314. C.M. Xu, H.S. Zhang, S.F. Qian, The influence of temperature on the feature parameter of the endothelial cell living rate from corpse cornea, Chin. J. Forensic Med. 14 (1999) 33–34. C. Moreschi, U. Da Broi, P. Lanzetta, Medico-legal implications of traumatic cataract, J. Forensic Leg. Med. 20 (2013) 69–73. R. Amberg, S. Pollak, Postmortem endoscopy of the ocular fundus. A valuable tool in forensic postmortem practice, Forensic Sci. Int. 124 (2001) 157e62. P.E. Lantz, A simple model for teaching postmortem monocular indirect ophthalmoscopy, J. Forensic Sci. 54 (2009) 676e7. B.L. Davis, C.D. Nilson, N. Mamalis, Revised Miyake-Apple technique for postmortem eye preparation, J. Cataract Refract. Surg. 30 (2004) 546–549. D.J. Apple, E.S. Lim, R.C. Morgan, J.C. Tsai, T.D. Gwin, S.J. Brown, A.N. Carlson, Preparation and study of human eyes obtained postmortem with the Miyake posterior photographic technique, Ophthalmology 97 (1990) 810–816. K. Miyake, C. Miyake, Intraoperative posterior chamber lens haptic fixation in the human cadaver eye, Ophthalmic Surg. 16 (1985) 230–236.
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Morphological and histological changes in eye lens: Possible application for estimating postmortem interval Gemma Prieto-Bonete, Maria D. Perez-Carceles ⇑, Aurelio Luna Department of Legal and Forensic Medicine, Biomedical Research Institute (IMIB-Arrixaca), Regional Campus of International Excellence ‘‘Campus Mare Nostrum”, University of Murcia, Spain
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Article history: Received 26 February 2015 Received in revised form 3 September 2015 Accepted 14 September 2015 Available online 14 September 2015 Keywords: Postmortem interval Eye lens Corneal transparency Proteins Histology
a b s t r a c t Establishing the postmortem interval is a very complex problem in Forensic Science despite the existence of several macro- and microscopic methods. In the case of ocular methods, most are based on an evaluation of the biochemical components of the vitreous humour 24–36 h after death, but, to our knowledge, there are no studies on the relationship between lens and the postmortem interval. Since the lens is protected between the vitreous humour and the aqueous humour inside the eyeball, postmortem changes are assumed to start later in the lens. To evaluate the usefulness of using the lens to establish the postmortem interval, we examined 80 rabbit lens enucleated 24, 48, 72 and 96 h after death, assessing changes in sphericity and absorbance at different wavelengths and any histological alterations. Both sphericity and absorbance were seen to decrease to a statistically significant extent, and there was a gradual loss of structure and organisation of the lens components as a function of the postmortem interval. Modifications in the lens were seen to be useful for determining the postmortem interval between 24 and 96 h. Ó 2015 Elsevier Ireland Ltd. All rights reserved.
1. Introduction Establishing the postmortem interval continues to be one of the most complex problems in Forensic Science. Studies based on ocular data to establish PMI are few and tend to be based on an evaluation of the biochemical components of the vitreous humour, such as sodium, potassium, chloride, lactate and hypoxanthine 24–36 h after death [1–4]. The lens is positioned in the eye behind the iris. The anterior lens surface is bathed by aqueous humor, while the posterior lens surface is in contact with the vitreous body [5]. Contains 1000–3000 layers of fiber cells [6]. The adult lens contains two kinds of fiber cells: (i) those located in the cortex (the outermost layers of the lens), which are not yet mature and still contain organelles (including mitochondria), all of which are degraded through protease- and nuclease-regulated processes, leaving behind membrane-enclosed bags of crystallines, and (ii) those located in the nucleus (the core of the lens), which are mature and do not contain organelles [6]. Into subcellular organelle evacuation during maturation is necessary to ensure the transparency of the lens, as ⇑ Corresponding author at: Department of Legal and Forensic Medicine, Biomedical Research Institute (IMIB-Arrixaca), Regional Campus of International Excellence ‘‘Campus Mare Nostrum”, School of Medicine, University of Murcia, E-30100, Spain. E-mail address: [email protected] (M.D. Perez-Carceles). http://dx.doi.org/10.1016/j.legalmed.2015.09.002 1344-6223/Ó 2015 Elsevier Ireland Ltd. All rights reserved.
organelles scatter light, whereas ordered proteins (crystallins) do not. Protein synthesis and protein degradation are minimal or non-existent, and crystallins and perhaps other proteins that were synthesized at the birth of the cell persist throughout the life of the organism [7]. The transparency of the eye lens thus depends on the regular alignment of elongated fiber cells, which perform the difficult task of stacking together neatly to fill a spheroidal volume, filled with cytoplasmic crystallins and cytoskeletal intermediate filaments encased in membranes made from a few integral membrane proteins. The lens proteins belong to common protein families, but the lens tends to have its own unusual version [8]. Crystallines, which are expressed as three different isoforms (a-crystallin, b-crystallin and c-crystallin), are major cytoplasmic components of the vertebrate eye lens that constitute >90% of the total protein content in eye lens fiber cells and >35% of their wet weight [7]. The chaperone action of a-crystallin is vital for maintaining eye lens transparency. The reasons for using rabbit as an experimental model to study the lens are the following: First, both the rabbit and human lens have branched sutures, although the former is of the ‘‘line” type and the latter of the ‘‘star” type. For this reason, the rabbit lens can be considered as a simplification of the more complex organisation of the fibers of the human eye [9–13]. Secondly, the rabbit lens is closer in size and sphericity to the human lens than other
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Fig. 1. Sphericity of the lenses and statistically significant differences between right and left eyes for postmortem intervals.
lens commonly used in experiments, such as mouse or rat. The first study to use rabbit eye as an experimental model measured the thickness of the cornea by ultrasounds [2]. Lastly, in this study what we have achieved of the functional parameters of rabbit lens provides a basis for comparison with the human lens [10,14–16]. While there are many studies on the morphological and histological changes related to certain pathologies, we have found no reference to the use of the lens to establish the PMI. Determining the changes in lens transparency postmortem would be a useful starting point for testing the possibility of determining visual impairment conditions’ in a cadaver [17]. The postmortem evaluation of the lens would provide the added benefit of associating different forms and stages of cataracts with accidents, thus affording new information relevant to the development of preventive measures [18]. Apart from estimating the PMI, the age of the subject could be ascertained between 24 and 48 h after death by applying radiocarbon techniques to the lens [17]. In conclusion, the postmortem determination of lens opacity would provide helpful information that could be used during legal proceedings and it would be also a good complement to clinical data, and fundamental in cases where there is no medical documentation. The aim of the present study was to assess whether the postmortem morphological and histological modifications that take place in the lens may be related to the postmortem interval itself.
2. Materials and methods 2.1. Sample collection The lens (n = 80) were taken from 40 rabbits with an average age of 84.02 days old (75–95 days old) sacrificed in a local meatprocessing company [19]. All the animals used were treated in the normal way and were not killed for the sake of the experiment described. In the laboratory of the Forensic Medicine Department of the University of Murcia (Spain), the lens were left exposed to
the air in a room with a mean temperature of 21.3 °C at 24 hpm, 21.4 °C at 48 hpm, 22.4 °C at 72 hpm and 22.7 °C at 96 hpm. The animals were cared for following Spanish law (RD 1201/05) according to the principles of EU directive EU 86/609. This study was approved by the Ethics Committee of the University of Murcia (Spain). 2.2. Enucleation and measurements of sphericity and absorbance The 80 lens were classified into four groups of 20 samples each. Every 24 h all the lens from a given group were enucleated (first group 24 h postmortem, second group 48 hpm, third group 72 hpm and the last group 96 hpm) by making a lateral incision and cutting the orbital muscles [20]. Once extracted, the 20 lens were placed in physiological saline, the their absorbance was measured at 365, 370, 375, 415 and 420 nm using a Shimadzu UV-160 spectrophotometer [21]. The absorbance of each eye lens was measured directly in a 1-cm plastic cuvette designed and developed in our laboratory, and an empty cuvette was used as blank. The special design of the cuvette allowed the lens to be held in place vertically, so that its own weight and the effect of gravity did not affect its structure since it was supported by the edges, leaving the central part in its correct anatomical position when light rays were directed at it. After measuring, the lenses were photographed using a Nikon DX digital camera (AF-S DX NIKKOR), at 20 cm distance and with a resolution of 10.2 megapixels. The image analysis program image tool UTHSCSA was used to measure the greatest and smallest diameter, and to calculate the sphericity of the lens. In this way, we could assess any modification in transparency or translucence as a function of the PMI. 2.3. Histological study After visual examination by image tool, the eyes lens were fixed in 10% buffered formalin for 22–30 days and embedded in paraffin. Sections (4 nm thick) were obtained from each paraffin block and placed on SuperFrost (Menzel-Gläser, Braunschweig, Spain) plus
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Fig. 2. Macroscopic image of the lens: (a) 24 h postmortem; (b) 48 h postmortem; (c) 72 h postmortem; (d) 96 h postmortem.
glass slides. After deparaffinization and rehydration in graded alcohols, haematoxylin and eosin and was applied. 2.4. Statistical analysis The spss 20.0 package (SPSS Inc., Chicago, IL, USA) was used for statistical analysis of the data, calculating the mean, standard deviation (SD) and 95% confidence levels. The Kruskal–Wallis test, a non-parametric test for more than two independent samples, was used to compare groups. Also, specific contrasts for each variable grouped according to diagnostic category were carried out using the non-parametric Mann–Whitney test for two independent samples. p values less than 0.05 were considered statistically significant.
absorbance values between the lenses at any wavelength within the same PMI. The absorbance values for each wavelength are indicated in the table. As can be seen, absorbance decreases as the postmortem interval increases (Table 1). The data were compared and analysed by non-parametric tests. The Kruskal–Wallis test for the IPM variable revealed a significant difference between the groups at 365 nm, 370 nm, 375 nm, 415 nm and 420 nm. Mann–Whitney tests were used to investigate any significant results obtained from the initial Kruskal–Wallis analysis (Table 1). When the lens absorbance values were compared for different PMIs statistically significant differences were found between the lenses in all cases. 3.2. Lens sphericity: at different PMIs
3. Results 3.1. Measurement of absorbance in right and left lenses at different wavelengths and for different postmortem intervals In all the right and left lenses for every sampling time postmortem there were no statistically significant differences in the
Mean and 95% confidence interval of lens sphericity were: 0.976 ± 0.005 (0.965–0.988) at 24 h postmortem, 0.967 ± 0.007 (0.956–0.981) at 48 h postmortem, 0.950 ± 0.007 (0.929–0.971) at 72 h postmortem and 0.913 ± 0.015 (0.881–0.947) at 96 h postmortem. For examination of the sphericity variable, the Kruskal–Wallis test produced a significant difference with IPM
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Fig. 3. (A) Lens 24 h post-mortem: (a) The cell nucleus (black arrow) (HE, 4); (b) the lens capsule and epithelium and, below these, adhering lens fibres (black arrows) (HE, 10); (c) detail of the image observed in Figure a. The transparent capsule (black arrow) and epithelial cells joined to the uniformly distributed lens fibres (HE, 40). (B) Lens at 48 h post-mortem: (a) posterior face: well preserved epithelium in the peripheral zone (black arrow). (HE, 10); (b) nuclei of epithelial cells (HE, 40); (c) lens fibres (black arrow) uniformly distributed and joined, with no space between them (HE, 40). (C) Lens 72 h post-mortem: (a) the lens fibres are separated (black arrow) (HE, 10); (b) young disaggregated fibres (HE, 10); (c) detail of Figure b. The space between the young fibres is increasing (black arrow) (HE, 40). (D) Lens 96 h post-mortem: (a) eosinophil structures (black arrow) (HE, 10); (b) agglomeration of randomly disposed elongated eosinophil structures (black arrows) (HE, 40).
Table 1 Mean, confidence interval and p-values of the absorbance values at different wavelengths and for each post-mortem interval.
*
Postmortem interval (h)
W 365 nm Mean (95% CI)
W 370 nm Mean (95% CI)
W 375 nm Mean (95% CI)
W 415 nm Mean (95% CI)
W 420 nm Mean (95% CI)
24 48 72 96
468.8 418.1 388.5 334.9
537.1 464.5 409.2 325.3
509.3 410.2 393.5 320.3
462.8 399.9 366.6 313.3
464.8 (426.5–503.0) 411. 9 (397.5–426.3) 381.8 (359.2–404.4) 321.7 (279–364.4)
p-value Kruskal–Wallis 24 vs 48* 24 vs 72* 24 vs 96* 48vs 72* 48 vs 96* 72 vs 96*
<0.0001 0.036 0.001 <0.0001 0.031 0.001 0.026
(427.6–509.9) (400.1–436) (365.2–411) (293–376.7)
(488.7–585.4) (431.1–532.1) (379.5–438.9) (281.6–369.1)
<0.0001 0.006 <0.0001 <0.0001 0.002 <0.0001 0.008
(463.5–555.1) (424.8–455.7) (367–420.1) (279–361.7)
<0.0001 0.003 <0.0001 <0.0001 0.003 <0.0001 0.007
(419.3–506.27) (384.4–415.3) (344–389.2) (270.2–354.5)
<0.0001 0.002 <0.0001 <0.0001 0.012 <0.0001 0.041
0.001 0.023 0.001 <0.0001 0.041 <0.0001 0.041
p-Value Mann–Whitney.
(p < 0.0001). Statistically significant differences in sphericity were found between the different PMIs in all cases. Moreover there was a tendency for the sphericity to decrease as the PMI increased (Fig. 1). 3.3. Macroscopic evaluation of transparency A macroscopic study of the lenses at each PMI pointed to the tendency for transparency to decrease with time. After 24 and 48 h, the transparency was still apparent, while at 72 it had begun to diminish and at 96 h the lenses were totally opaque and had lost their sphericity (Fig. 2).
3.4. Postmortem histological analysis of eye lens At 24 and 48 h postmortem the lens structure are similar. Histologically, all the cell layers could be observed - the external capsule, cell epithelium, cortex and nucleus (Figs. 3A and B). Adhering to and as a continuation of the epithelium, note the uniformly distributed fibres of lens both in the cortex and nucleus (Figs. 3Ab, Ac, Bb, Bc). Nucleated fibres corresponding to young fibres can be observed in the equatorial zone (Fig. 3Aa). However, at 72 h, alterations in the structure and order of the lens layers began to appear (Fig. 3Ca). Neither the capsule nor the layer of epithelial cells could be distinguished, although the fibers of the
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cortex and nucleus had lost their uniformity, being disaggregated and separated probably due to the loss of the mechanisms that held them together (Figs. 3Cb and Cc). Similarly, at 96 h neither the capsule nor epithelial cells could be distinguished (Fig. 3Da) and, instead of individual fibers, aggregated eosinophils were evident (Figs. 3Da and Db).
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the cell membranes is not related with the loss of transparency, but is separate phenomenon [30]. There are many studies concerning cornea turbidity to determine the postmortem interval using image analysis and it has been shown that cornea opacity increases with time after death [29,31]. 4.3. Loss of lens internal structure and sphericity
4. Discussion As bibliographical search revealed no studies on the estimation of the PMI by reference to the lens, which led us to analyse morphological (macroscopic and histological) and biochemical modifications related with the same to see whether such information could provide information on the PMI between 24 and 96 h. Our study shows how the lens becomes opaque with time, losing transparency, sphericity of the anterior and posterior face and absorbance at the long wavelengths tested. All these morphological changes in the macroscopic structure are accompanied by a loss of the internal structure of the cell fibers and cells. 4.1. Loss of transparency with time The loss of transparency with time is due to the metabolic processes that take place between the lens and the aqueous and vitreous humor and the aggregation of crystallins proteins in the fibers of the lens nucleus. The lens epithelium is fundamental in the opaque process since it contains a region with a high metabolic level, which is responsible for the vital transport of different electrolytes, amino acids and metabolic product. The reactions pump introduces potassium into the lens as sodium is expelled. The transparency of the lens is partly due to the correct functioning of this pump, and when it stops with the death of the subject potassium levels fall in the lens, favouring the entry of chlorine, accompanied by water and calcium. Together with the cell necrosis that occurs after death, in which lysosomal enzymes play a part, this leads to an increase in opacity, i.e. loss of transparency [21,22]. The in vivo process of opacification, known as cataracts, is produced by different mechanisms to those which act postmortem. The oxidising agents alter the protein structure and affect the mechanisms of aggregation, cross-linking, peptide cleavage and the introduction of active carbons in the protein chains [7,23–25]. Besides these processes, crystallins appear in the fibers a protein fraction of high molecular weight [26]. Protein–protein disulphides are responsible for the increase in protein of high molecular weight, found in only in cataractous lenses [27]. It has been hypothesized that lens proteins aggregate to large particles that scatter light, which causes lens opacity [28]. Autopsies performed within 10–30 h of death can confidently use this method for the accurate estimation of postmortem a time. However, with shorter and longer times after death, the accuracy may diminish. Corneal opacity can also occur due to the transpiration of fluid and the degeneration of endothelial cells, and may therefore be accelerated in high temperatures [29]. Upon death or removal of an eye the cornea absorbs the aqueous humour, thickens, and becomes hazy. A hazy cornea presents edema, paleness, obvious thickening and tiny frills on the surface, and becomes concave due to dehydration and consequent shrinkage; finally, its endodermis adheres to the crystalline lens. 4.2. Decrease in absorbance with PMI Absorbance was seen to decrease with the length of the PMI, reaching a minimum at 96 h. This decrease was due to the loss of orientation of the fibers in the lens [7]. The damage suffered by
In the anatomopathological study the PMI-dependent destructuralization was also clear in the epithelium central zone or nucleus and the capsule. At 24 and 48 h after death, the lens structure was conserved as if it had been enucleated immediately after death just as it was after enucleation, preserving all its cell layers and fibers intact. However, after 48 h the anterior capsule was looser than the posterior since the latter was more hydrated through contact with the vitreous humour and so was less affected by postmortem phenomena. The integrity of the crystallin proteins of the fibers depends on vital metabolic processes that involve their aggregation, oxidation, proteolysis, deamination, glycosylation and transpeptidation [32,33]. The cessation of metabolic exchange processes increases the quantity of free glucose within the lens, provoking excessive glycosylation of the crystallin proteins. Consequently the lens attracts water, changing the permeability of the sodium and so altering the distribution of ions [34]. This is accompanied by an increase in the concentration of sodium and a loss of potassium and amino acids. Moreover, the high sorbitol concentration attracts water, provoking the breakage, liquefaction and separation of the fibers, and asymmetry in the lens sutures, creating vacuoles in the cortex and affecting the lens structure. As a result of the loss of the intracytoplasmic content during the cell degeneration process, a dense material, known as multilamellar bodies, appears, producing changes in the density of the cytoplasmic membranes and separating the fibers [35,36]. Something similar occurs with cornea. The change in corneal opacity is believed to be secondary to the change in hydration, while the increased water content of the stroma is the main cause of corneal swelling. Other causes of loss of structure include architectural destruction of the collagen fiber network, functional alteration of corneal endothelia, dysregulation of proteoglycan hydration and the ion concentration of the corneal stroma. The part of the cornea covered by the eyelid is more likely to become hazy sooner. When the ambient temperature is relatively high, opacity tends to occur early [31]. One limitation of the study is that it only looks at the lens of presumably healthy eyes. It has been seen that temperature has a significant influence on protein degradation and the survival of endothelial cells [37,38], and so the ambient temperature should be taken into account. Light per se does not affect lens transparency, unlike the cornea in which changes in opacity are believed to be secondary to the change in hydration, while the increased water content of the stroma is the main cause of corneal swelling [31], both of which are closely related with the eye being open [29,39]. Lens and cornea opacity are two independent processes. Age is another factor to consider, since cataracts tend to be formed with age, while it is generally accepted that corneal opacity increases with age due to a decrease in the number of corneal endothelial cells, which results in an increase in the thickness of the cornea owing to inflow of aqueous humor to the cornea [29]. Other influential factors include the individual variation and presence of cataracts, in which case measurements of transparency cannot be used, and only sphericity can. Lastly, it should be mentioned that the procedures described can be applied in human autopsies so that the eyeball does not have to be removed, making ophthalmological endoscopy a valuable tool in forensic autopsy [40]. Another technique, postmortem
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monocular indirect ophthalmoscopy, consists of a light source attached to a headband along with a hand-held lens. This permits a wide view of the fundus after death. To facilitate skill acquisition in monocular indirect ophthalmoscopy, a simple and inexpensive teaching model can be constructed from hinged, cylindrical plastic containers [41]. When the Miyake-Apple posterior video/photographic technique is used (the most widely used technique), the eyes are bisected through the equator and glued to a glass slide. Posterior images can be obtained using an extra microscope connected to a camera recorder below the eye. Such procedures are usually carried out with an open sky preparation. This technique is the easiest and provides a wide spectrum of information [42–44].
[14]
[15]
[16]
[17]
[18]
[19]
5. Conclusion The results obtained in this study suggest that measurements of lens sphericity and absorbance and a histological analysis of the lens may be regarded as complementary tools that will provide useful information for estimating the PMI between 24 and 96 h. Further studies in human will provide more information on the proposed method. It is possible to apply radiocarbon dating of the human eye lens crystallines to reveal proteins [15] by means of a histopathological study of postmortem eyes. Lastly, complementary immunohistochemistry can be used for skill acquisition in monocular indirect ophthalmoscopy to detect lens protein and structural changes.
[20] [21]
[22] [23] [24]
[25]
[26] [27]
Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.legalmed.2015. 09.002.
[28]
[29]
[30]
References [1] H.V. Chandrakanth, T. Kanchan, B.M. Balaraj, H.S. Virupaksha, T.N. Chandrashekar, Postmortem vitreous chemistry – an evaluation of sodium, potassium and chloride levels in estimation of time since death (during the first 36 h after death), Int. J. Leg. Med 20 (2013) 211–216. [2] G.L. Lü, F.X. Jiang, X.S. Xu, Y.J. Jiang, Z.G. Li, X. Wang, H. Shi, L.C. Yu, C.C. Xu, Estimation of postmortem interval by detecting thickness of cornea using ultrasonic method, Fa Yi Xue Za Zhi 28 (2012) 89–91. [3] R.T. Matias, X.L. Rae, L. Ebiham, The localization of transport properties in the frog lens, Biophys. J. 48 (1985) 423–434. [4] Z. Mihailovic, T. Atanasijevic, V. Popovic, M.B. Milosevic, Could lactates in vitreous humour be used to estimate the time since death?, Med Sci. Law 51 (2011) 156–160. [5] S. Bassnett, Y. Shi, G.F. Vrensen, Biological glass: structural determinants of eye lens transparency, Philos. Trans. R Soc. Lond. B Biol. Sci. 366 (2011) 1250–1264. [6] W.K. Subczynski, M. Raguz, J. Widomska, L. Mainali, A. Konovalov, Functions of cholesterol and the cholesterol bilayer domain specific to the fiber-cell plasma membrane of the eye lens, J. Membr. Biol. 245 (2012) 51–68. [7] B.H. Toyama, M.W. Hetzer, Protein homeostasis: live long, won’t prosper, Nat. Rev. Mol. Cell Biol. 14 (2013) 55–61. [8] A.R. Clark, N.H. Lubsen, C. Slingsby, SHSP in the eye lens: crystallin mutations, cataract and proteostasis, Int. J. Biochem. Cell B 44 (2012) 1687–1697. [9] A. Gwon, The rabbit in cataract/IOL surgery, Anim. Model Eye Res. (2008) 184– 204. [10] S. Al-khudari, S.T. Donohue, W.M. Al-Ghoul, K.J. Al-Ghoul, Age-related compaction of lens fibers affects the structure and optical properties of rabbit lenses, BMC Ophthalmol. 7 (2007) 19. [11] J.R. Kuszak, J.G. Sivak, K.L. Herbert, S. Scheib, W. Garner, G. Graff, The relationship between rabbit lens optical quality and sutural anatomy after vitrectomy, Exp. Eye Res. 71 (2000) 267–281. [12] N. Braun, M. Zacharias, J. Peschek, A. Kastenmüller, J. Zou, M. Hanzlik, S. Weinkauf, Multiple molecular architectures of the eye lens chaperone aBcrystallin elucidated by a triple hybrid approach, Proc. Natl. Acad. Sci. 108 (2011) 20491–20496. [13] A. Laganowsky, J.L. Benesch, M. Landau, L. Ding, M.R. Sawaya, D. Cascio, D. Eisenberg, Crystal structures of truncated alphaA and alphaB crystallins reveal
[31]
[32] [33] [34]
[35] [36]
[37]
[38]
[39] [40] [41] [42] [43]
[44]
structural mechanisms of polydispersity important for eye lens function, Protein Sci. 19 (2010) 1031–1043. X. Liu, M. Zhang, Y. Liu, P. Challa, P. Gonzalez, Y. Liu, Proteomic analysis of regenerated rabbit lenses reveal crystallin expression characteristic of adult rabbits, Mol. Vis. 14 (2008) 2404. J.R. Kuszak, C.A. Enneser, J. Umlas, The ultrastructure of fiber cells in primate lenses: a model for studying membrane senescence, J. Mol. Struct. Res. 100 (1988) 60–66. J.R. Kuszak, H.G. Brown, Embryology and Anatomy of the Lens. Embryology and Anatomy of the Lens. Principles and Practice of Ophthalmology: Basic Sciences, Philadelphia Press, 1994. N. Lynnerup, H. Kjeldsen, S. Heegaard, C. Jacobsen, J. Heinemeier, Radiocarbon dating of the human eye lens crystallines reveal proteins without carbon turnover throughout life, PLoS One 3 (2008) e1529. V. Stemberga, A. Petaros, D. Kovacevic, M. Coklo, N. Simicevic, A. Bosnar, The assessment of lens opacity postmortem and its implication in forensics, J. Forensic Leg. Med. 2013 (20) (2013) 1142–1144. J. DiMattio, In vivo entry of glucose analogs into lens and cornea of the rat, Invest. Ophthalmol. Vis. Sci. 25 (1984) 160–165. L. Harris, J.B. Gehrsitz, Sodium pump in human lens, Am. J. Ophthalmol. 36 (1953) 39–44. S. Bassnett, S. Stewart, O. Duncan, Efflux of chloride from rat lens: influence of membrane potential and intracellular acidification, Exp. Physiol. 73 (1988) 941–949. O. Duncan, The site of the ion restricting membranes in the toad lens, Exp. Eye Res. 8 (1969) 406. O.A. Candia, P.J. Bentley, C.D. Milis, Asymmetrical distribution of the potential difference in the toad lens, Nature 227 (1970) 852. V.A. Lucas, J.R. Zigler, Identification of the monkey lens glucose transporter by photoaffinity labeling with cytochalasin, Invest. Ophthalmol. Vis. Sci. 29 (1988) 630. C.A. Paterson, N.A. Delamerre, L. Mawhorter, Na, K-ATPase in stimulated eye bank and cryoextracted rabbit lenses, and human eye bank lenses and cataracts, Invest. Ophthalmol. Vis. Sci. 24 (1983) 1534–1538. J.J. Harding, Disulphide cross-linked protein of high molecular weight in human cataractous lens, Exp. Eye Res. 17 (1973) 377–383. A. Spector, Debdutta Roy, Disulfide-linked high molecular weight protein associated with human cataract, Proc. Natl. Acad. Sci. 75 (1978) 3244–3248. R.E. Perry, M.S. Swamy, E.C. Abraham, Progressive changes in lens crystallin glycation and high-molecular-weight aggregate formation leading to cataract development in streptozotocin-diabetic rats, Exp. Eye Res. 44 (1987) 269–282. W. Kawashima, K. Hatake, R. Kudo, M. Nakanishi, S. Tamaki, S. Kasuda, A. Ishitani, Estimating the time after death on the basis of corneal opacity, J. Forensic Res. 6 (2015) 1. F. Malacaze, P. Chollet, E. Cavrois, Role of interleukin 6 in the inflammatory response after cataract surgery: an experimental and clinical study, Arch. Ophthalmol. 109 (1991) 1681–1683. L. Zhou, Y. Liu, L. Li, L. Zhuo, M. Liang, F. Yang, S. Zhu, Image analysis on corneal opacity: a novel method to estimate postmortem interval in rabbits, J. Huazhong Univ. Sci. Technol. [Med. Sci.] 30 (2010) 235–239. G.P. Ceschi, L.G. Artraria, Clear lens extraction for correction of high grade myopia, Klin. Monbl. Augenheilkd. 212 (1998) 280–282. J.L. Rae, J.R. Kuszak, The electrical coupling of epithelium and fibers in the frog lens, Exp. Eye Res. 36 (1983) 317–326. L.W. Andrew, G.J. Jin, Prospective evaluation of early visual and refractive effects with small clear corneal incision for cataract surgery, J. Cataract Refract. Surg. 22 (1996) 1456–1460. N. Brown, Slit-image photography, Trans. Ophthalmol. Soc. UK 89 (1969) 397– 408. C.M. Cunanan, P.M. Tarbux, P.M. Knight, Surface properties of the intraocular lens material and their influence on in vitro cell adhesion, J. Cataract Refract. Surg. 17 (1991) 767–773. Y.O. Poloz, D.H. O’Day, Determining time of death: temperature-dependent postmortem changes in calcineurin A, MARCKS, CaMKII, and protein phosphatase 2A in mouse, Int. J. Leg. Med. 123 (2009) 305–314. C.M. Xu, H.S. Zhang, S.F. Qian, The influence of temperature on the feature parameter of the endothelial cell living rate from corpse cornea, Chin. J. Forensic Med. 14 (1999) 33–34. C. Moreschi, U. Da Broi, P. Lanzetta, Medico-legal implications of traumatic cataract, J. Forensic Leg. Med. 20 (2013) 69–73. R. Amberg, S. Pollak, Postmortem endoscopy of the ocular fundus. A valuable tool in forensic postmortem practice, Forensic Sci. Int. 124 (2001) 157e62. P.E. Lantz, A simple model for teaching postmortem monocular indirect ophthalmoscopy, J. Forensic Sci. 54 (2009) 676e7. B.L. Davis, C.D. Nilson, N. Mamalis, Revised Miyake-Apple technique for postmortem eye preparation, J. Cataract Refract. Surg. 30 (2004) 546–549. D.J. Apple, E.S. Lim, R.C. Morgan, J.C. Tsai, T.D. Gwin, S.J. Brown, A.N. Carlson, Preparation and study of human eyes obtained postmortem with the Miyake posterior photographic technique, Ophthalmology 97 (1990) 810–816. K. Miyake, C. Miyake, Intraoperative posterior chamber lens haptic fixation in the human cadaver eye, Ophthalmic Surg. 16 (1985) 230–236.