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Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results and Discussions
  6. Conclusions
  7. Acknowledgement
  8. References

Recently, we have proposed that the fluorescence spectra of sheep retina can be well correlated with the presence or absence of scrapie. Scrapie is the most widespread TSE (transmissible spongiform encephalopathy) affecting sheep and goats worldwide. Mice eyes have been previously reported as a model system to study age-related accumulation of lipofuscin, which has been investigated by monitoring the increasing fluorescence with age covering its entire life span. The current work aims at developing mice retina as a convenient model system to diagnose scrapie and other fatal TSE diseases in animals such as sheep and cows. The objective of the research reported here was to determine whether the spectral features are conserved between two different species namely mice and sheep, and whether an appropriate small animal model system could be identified for diagnosis of scrapie based on the fluorescence intensity in retina. The results were consistent with the previous reports on fluorescence studies of healthy and scrapie-infected retina of sheep. The fluorescence from the retinas of scrapie-infected sheep was significantly more intense and showed more heterogeneity than that from the retinas of uninfected mice. Although the structural characteristics of fluorescence spectra of scrapie-infected sheep and mice eyes are slightly different, more importantly, murine retinas reflect the enhancement of fluorescence intensity upon infecting the mice with scrapie, which is consistent with the observations in sheep eyes.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results and Discussions
  6. Conclusions
  7. Acknowledgement
  8. References

Fluorescence spectroscopy has been commonly used as a tool in a variety of biological applications due to its high sensitivity [1-5]. It is used to detect fecal contamination in meat by exploiting the fluorescence from chlorophyll metabolites in the fecal matter that can be present on meat during the slaughter [6]. A similar approach can be used to detect central nervous system (CNS) tissues in meat that can be contaminated during slaughter. The CNS tissues are reported to be highly fluorescent compared with non-CNS tissues, and thus can be detected even when present in small quantities in meat [7]. The increased fluorescence of CNS tissues is most likely due to the enrichment of lipofuscin, which is a highly fluorescent, heterogeneous, high-molecular-weight material that has been shown to accumulate in high concentrations in neural tissues [8-12].

Recently, we have proposed that the fluorescence spectra of sheep retina can be well correlated with the presence or absence of scrapie [13]. Scrapie is the most widespread TSE affecting sheep and goats worldwide. It is characterized by a gradual onset; advanced cases show typical disease characteristics including lethargy, compulsive itching, balance and ambulatory abnormalities, convulsions and eventual death. The disease has been observed for centuries. At present, it is incurable and the most common form of control is quarantine, euthanasia and proper disposal of the carcass. Scrapie is not considered infectious to humans. The TSE of most concern for the food supply is bovine spongiform encephalopathy (BSE), a fatal neurodegenerative transmissible disease in cattle that is thought to be associated with variant Creutzfeldt–Jakob disease (vCJD) in humans [14]. The oral route of infection is considered the most probable path for transmission of BSE to humans [15, 16]. To reduce the risk of human exposure, specified risk material (SRM, e.g. brain and spinal cord) from cattle is removed during slaughter and processing.

Recently, Smith et al. have shown that the function and morphology of retinas are altered in TSE-infected cattle and sheep infected with scrapie [17, 18]. Hortells et al. concluded that PrPSc which is an abnormal form of the prion protein, in the retina was highly correlated with the progression of scrapie during preclinical and clinical stages [19]. Rubenstein et al. used ultraviolet fluorescent spectroscopy to detect different forms of PrPSc in the eye [20]. Fundus autofluorescence has been investigated for possible diagnostic use in a large number of retinal diseases [21, 22] including the “conformational diseases” caused by accumulation of proteinaceous aggregates [23]. In addition, autofluorescence has been used to map lipofuscin distribution in the retina of humans [21]. In this study, we have not determined the specific structures that are responsible for the fluorescence but others have suggested that lipofuscin and other macromolecular aggregates are responsible [19, 23].

Mice eyes have been previously reported [24, 25] as a model system to study age-related accumulation of lipofuscin, which has been investigated by monitoring the increasing fluorescence with age covering its entire life span. The current work aims at developing mice retina as a convenient model system to diagnose scrapie and other fatal TSE diseases in animals such as sheep and cows. The objective of the research reported here was to determine whether the spectral features are conserved between two different species namely mice and sheep, and whether an appropriate small animal model system could be identified for diagnosis of scrapie based on the fluorescence intensity in retina.

Materials and Methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results and Discussions
  6. Conclusions
  7. Acknowledgement
  8. References

Collection of murine eye samples

The animal work was approved by the National Animal Disease Center animal care and use committee and the mice were housed and fed according to Federal Animal Welfare Act Guidelines. Litters of mice were raised from birth and weaned at 3 weeks of age. Mice were housed in cages with littermates of the same sex with no more than six female or four male per cage. All mice were fed a standard ration of mouse chow (Harlan Teklad, Madison, WI). Artificial fluorescent lights were cycled on and off every 12 h during the entire course of the rearing period. The animals were reared behind double door entryways and never exposed to sunlight. Mice were euthanized by exposure to 100% carbon dioxide (CO2) until cessation of breathing and for a minimum of 15 additional minutes until necropsy. Mice were not decapitated so as to avoid intracranial pressure which could cause hemorrhages in the eyes which might interfere with the subsequent analysis.

Collection and dissection of murine retinas

Healthy (n = 25) C57BL/DK mice and scrapie-infected mice (n = 74) were evaluated in these studies. Mice were inoculated with well-characterized Rocky Mountain Laboratory (RML) scrapie, which is a mouse-adapted scrapie strain. Fluorescence experiments were done with only those mice that showed positive clinical signs (neurological signs such as poor coordination, inability to walk, ataxia or an inability to stand if placed on their side or back etc.). All of the scrapie-affected mice with typical scrapie clinical signs were also scrapie positive by Western Blot (WB). The eyes from the mice were removed at necropsy. Both eyeballs were collected, stored in an Eppendorf tube and placed on ice until dissection of the retina. The retina from each eyeball was dissected using a dissecting stereo microscope (Stereo Star Zoom; Reichert Scientific Instruments, NY) for magnification. Lens and vitreous body were removed after circular cutting in the ora serrata. The retina was removed from the eye cup and placed on a microscope slide (25 mm × 75 mm × 1 mm), which was prepared with a drop of distilled water. Each sample was covered with a cover slip for additional enlargement of the surface area of the retina. The retina was oriented as a thin layer on the microscope slide enabling the entire area of the sample to be exposed to the excitation light. To avoid dehydration of the samples, they were stored in a moist chamber and placed at ≤ +7°C until fluorescence spectra were collected within the next 12 h.

Steady-state measurements

Steady-state fluorescence spectra were obtained on a SPEX Fluoromax-2 (ISA Jobin-Yvon/SPEX, Edison, NJ) with a 5 nm band pass unless otherwise specified and corrected for lamp spectral intensity and detector response. Fluorescence spectra were collected in the front-faced orientation. For the experiments reported in this study, the samples were excited at λex = 350, 410, 470 and 520 nm with an interference filter on the excitation side, and emission was collected using appropriate cutoff filters before the detector to eliminate scattered light. All the spectra were recorded after identical number of postinfection days.

Hyperspectral fluorescence imaging microscopy

The hyperspectral images were captured on a system located in the Roy J. Carver Laboratory for Ultrahigh Resolution Biological Microscopy, Iowa State University. This system was based on a NIKON ECLIPSE TE 2000-E microscope. The illumination source was an X-Cite 120 PC from EXFO. Samples were excited at 470 nm using an interference filter, and fluorescence was collected at ≥500 nm with a long-pass filter and a Nikon Plan Fluor 10X/0.30 PH1-DL objective. The collected fluorescence was dispersed using a Spectral-DV spectrometer from Optical Insights and captured by a Photometrics Cascade 512 B CCD camera (Roper Scientific). The software used for image capture was Mélange V3.7.

Results and Discussions

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results and Discussions
  6. Conclusions
  7. Acknowledgement
  8. References

Steady-state fluorescence spectra were collected by illuminating the entire retina of scrapie-infected mice, exciting at 350, 410, 470 and 505 nm, and were compared with those from healthy mice, which served as a control. The fluorescence intensity of the retina samples excited at 410 and 470 nm was 2–3 orders of magnitude higher than those excited at other wavelengths, thus experiments focused upon 410- and 470-nm excitation. The representative fluorescence spectra from several age-matched mice retina exciting at 410 and 470 nm are shown in Figs. 1 and 2 respectively. The gender of the mice is not considered for comparison because the fluorescence from retina is invariant with sex, but it increases with age [24]. Thus, for accurate comparison all the selected mice samples are of the same age. The statistical average of integrated intensities (area under the fluorescence curves) of the retinas from the scrapie-infected mice was five times higher than the uninfected control mice retinas. This observation is consistent irrespective of the excitation wavelengths (410 or 470 nm). The difference in the spectral intensity varied from one sample to the other, which may be due to the heterogeneous distribution of the accumulated lipofuscin pigment in the scrapie-infected mice eyes.

image

Figure 1. Fluorescence intensity of retina from 12 representative age-matched (~178 days) RML (Rocky Mountain Laboratory) scrapie strain infected and uninfected C57BL/DK mice. All the spectra were recorded after identical number of postinfection days (146 days). The samples were excited using a 410-nm interference filter, and the fluorescence was collected using a 425-nm long-pass filter. The retina was placed on a microscope slide, and the fluorescence was collected in a front-faced geometry. The comparative plot clearly indicates higher fluorescence from the retinas obtained from scrapie-infected mice, compared with the healthy ones. All the 12 spectra on both panels are not distinctly visible because of the overlap of intensity among them.

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image

Figure 2. Fluorescence intensity of retina from 10 representative age-matched (~178 days) RML (Rocky Mountain Laboratory) scrapie strain infected and uninfected C57BL/DK mice. All the spectra were recorded after identical number of postinfection days (146 days). The samples were excited using a 470-nm interference filter, and the fluorescence was monitored using a 505-nm long-pass filter. The retina was placed on a microscope slide, and the fluorescence was collected in a front-faced geometry. The comparative plot clearly indicates higher fluorescence from the retinas obtained from scrapie-infected mice, compared with the healthy ones. All the 10 spectra are not distinctly visible because of the overlap of intensity among them.

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The heterogeneity of the accumulated pigment in the retina was also tested by monitoring the excitation wavelength dependence of the fluorescence of retina of the scrapie-infected mice. The retina samples were excited using four different wavelengths (350, 410, 470 and 505 nm), and the fluorescence peak maxima was found to shift with increase in the excitation wavelengths. This observation has been shown in Fig. 3. This observation is consistent with previous reports. Delori et al. observed a redshift of emission spectra (both in the ex vivo and in vivo measurements) of human fundus autofluorescence using excitation wavelengths ranging from 430 to 550 nm [26]. Sparrow and coworkers conducted a detailed study of human fundus autofluorescence and attributed the inherent fluorescence to a mixture of bisretinoid fluorophores [27].

image

Figure 3. Excitation wavelength dependence study of retina from scrapie-infected mice. Representative normalized fluorescence spectra of a single retina collected by exciting at four different wavelengths (350, 410, 470 and 520 nm). All the spectra were collected using appropriate long-pass emission filters (λem > 370, 425, 505 and 550 nm). Distinct spectral shift upon different excitation wavelengths indicates the heterogeneity of the fluorescent pigment (most likely lipofuscin) accumulated in the eye.

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Besides steady-state fluorescence studies of murine retina, where the entire retina is illuminated, hyperspectral imaging microscopy was used to study localized fluorescence from the retinas of RML scrapie-infected mice by comparing the images with those from healthy (control) mice. Four adjacent areas of the retinas were scanned (labeled as zone 11, 12, 21, 22) to cover the entire sample placed on the glass slide. Representative images of retinas from two age-matched healthy and two scrapie-infected age-matched mice are shown in Fig. 4. The images of retina from scrapie-infected mice showed significantly higher intensity than those from healthy mice. Also, images from the retinas of scrapie-infected mice appeared substantially heterogeneous, with localized highly fluorescent (shown by arrows) regions. The fluorescence spectra obtained from these images are shown in Fig. 5. Fluorescence intensity from different regions of the retina of scrapie-infected mice was also significantly different, as opposed to those in healthy retina, where the fluorescence intensity was weak irrespective of the focal spots. The fluorescence of the retina of scrapie-infected mice was believed to be primarily due to the accumulation of lipofuscin, which itself is a heterogeneous pigment.

image

Figure 4. Hyperspectral fluorescence images of the retina of mouse. Representative images from four adjacent regions (labeled as 11, 12, 21 and 22) of retinas from two different scrapie-infected (upper panel) and uninfected control (lower panel) age-matched (~5.5 months) mice. The samples were excited at 470 nm using an interference filter, and fluorescence was collected at wavelengths greater than 500 nm using a long-pass filter. The images shown are 800 μm by 800 μm. All images were captured under identical conditions. High intensity is represented by red; low intensity by black on a color gradient. The localized highly intense fluorescence and aggregation of fluorescent material in the images of scrapie-infected mice are shown by white arrows.

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image

Figure 5. Fluorescence spectra obtained from the different hyperspectral imaging regions (labeled as zone 11, 12, 21, 22) of retina from (a) uninfected (control) and (b) RML scrapie- infected mice under identical conditions as mentioned in Fig. 4. Numbers (1 and 2) represent representative samples from two different mice. Fluorescence from the retinas of scrapie-infected mice is 3–4 times higher compared with the uninfected control mice. In contrast, the retinas from control uninfected mice had nearly identical fluorescence intensities in all the scanned regions. This demonstrated the heterogeneity of the accumulated pigment in the scrapie-infected samples.

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Conclusions

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results and Discussions
  6. Conclusions
  7. Acknowledgement
  8. References

Our results are consistent with the previous reports on fluorescence studies of healthy and scrapie-infected retina of sheep [13]. The retinal fluorescence from the scrapie-infected mice showed substantially higher fluorescence and more heterogeneity in the distribution of lipofuscin, compared with retinas of uninfected mice. Although the structural characteristics of fluorescence spectra of scrapie-infected sheep and mice eyes are slightly different, more importantly, murine retinas reflect the enhancement of fluorescence intensity upon infecting the mice with scrapie, which is consistent with the observations in sheep eyes. Thus, our goal of finding a convenient model system to study scrapie in animals and its facile detection exploiting the fluorescence of accumulated lipofuscin due to scrapie was achieved using mice as a model animal. Mice are more easily available and more conducive to experimental work than larger animals, such as sheep and cows.

Acknowledgement

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results and Discussions
  6. Conclusions
  7. Acknowledgement
  8. References

We thank Matt Inbody, Bob Morgan, and Deb Lebo for their assistance in providing the animal tissue samples.

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results and Discussions
  6. Conclusions
  7. Acknowledgement
  8. References
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