Combination of pathological and spectroscopic characterization to promote diagnosis of retinal pigment epithelium-Bruch’s membrane complex in a diabetic rat model

: Diabetic retinopathy (DR) is a common condition of diabetes, and approaches to detecting early DR using the unique characteristics of the retinal pigment epithelium-Bruch’s membrane complex (RBC) have increasingly attracted attention. A diabetic model was established in Sprague-Dawley rats via streptozocin (STZ) injection for 1 (DM1) and 6 months (DM6), confirmed by weekly blood glucose measurement. Serum and retinal tissue-based advanced glycation endproducts (AGE) levels significantly elevated in diabetic rats, and RBC was evaluated by transmission electron microscopy and Raman spectroscopy. The results showed that whole Raman spectra and all marked band intensities could respectively achieve almost equal and accurate discrimination of all animal groups, along with the determination of important molecules from the band data. Further quantitative analyses indicated series of metabolic disturbance due to hyperglycemia were involved while the body self-regulation mechanism still played a role with different effects during the disease progression. Given this, Raman can reliably distinguish the early characterization of DR in addition to providing intrinsic key molecules that is sensitive to identify the early


Introduction
Diabetic retinopathy (DR) is a common complication in patients with diabetes mellitus (DM) and a leading cause of blindness worldwide. The prevalence of DM has been increasing rapidly over years. According to a report of the World Health Organization [1], there were approximately 422 million adults living with DM in 2016 globally. With the growing number of DM patients, the number of people with DR was estimated to reach 191 million by 2030 [2]. It is widely believed that elevated blood glucose level induces injury in retinal vessel network, resulting failure of retinal blood supply, and eventually it progresses to retinal neovascularization. Previous studies have been shown accumulation of advanced glycation end products (AGE) due to oxidative stress and hyperglycemia is associated with blood vessel injuries in DR patients [3]. AGE accumulation, which is generated through nonenzymatic glycation of plasma proteins, lipids and nucleic acids, has been suggested to be involved in inflammatory responses and epithelium apoptosis in DM patients [4,5]. In animals studies, increased AGE level was found in diabetic models, and it also found reduced AGE levels in diabetic animals with successful blood glucose control [6][7][8]. Given this, AGE level may be a useful marker to indicate the development of DM and DR. In the eye, retinal pigment epithelium-Bruch's membrane complex (RBC) has been considered the major site impaired by chronic high blood glucose induced oxidative stress [9,10]. In addition, AGE accumulation has been proved to be appeared in both Bruch's membrane and retinal pigment epithelium (RPE) [11]. Using transmission electron microscopy (TEM), previous studies show DR is associated with the breakdown of blood-retinal barrier and retinal endothelial cells, and more recent findings suggest the tight junction of RPE is essential for basal transcellular transport and more importantly maintaining the barrier function [12]. Laboratory in vivo studies have pointed out that RPE injuries, either dysfunction or morphological changes, may be an early sign of the development of DR [13,14].
At present there is no cure for DM, and therapeutic approach for DR is also limited. For this reason early management is crucial to prevent the progression of DM complications [15,16]. However, the detection of both DM and DR is also challenging. The fact is that up to 45% of DM patients remain undiagnosed, and the number for DR patients was 25% in 2016 [17][18][19]. Therefore, there is need to seek new approaches for DR detection, especially early detection. Diabetic retinopathy is commonly diagnosed using fluorescein angiography and ocular coherence tomography in the clinic. Although fluorescein angiography provides sensitive detection of retinal vessel blockage and leakage, it requires fluorescent reagent injection. Ocular coherence tomography is a non-invasive approach while it often fails to pick up early changes [20,21]. Vibrational spectroscopy, such as Raman spectroscopy (RS), has exhibited substantial promise due to its exquisite molecular specificity and minimal interference of water as potential approach of non-invasive diagnosis with capability of early detection. The RS exploits the inelastic scattering of photons to probe the structure and dynamics of molecules through their vibrational transitions. The shift in the initial and final vibrational states of the molecule is manifested in the form of characteristic and fingerprint patterns [22]. Studies have shown RS can distinguish between normal and pathological tissues [23][24][25]. Notably, with minimum sample preparation, this technique has received considerable attention in the medical community for monitoring pathophysiological processes. The current study was aimed to evaluate if RS could probe early alterations in RBC of DM rats and explored the underlying mechanisms.

Animals
In total 30 male Sprague-Dawley rats at an age of 6 weeks were purchased from Slaccas Animal Company (Shanghai, China). Animals were handled in accordance with the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research, and animal ethics were approved by the Animal Care and Use Committee of Fujian Medical University and conformed to the guidelines for ophthalmic and vision research. These rats were housed in a pathogen-free environment with an alternating 12:12 h of light/dark cycle at an ambient temperature of 25°C. After 1 week of acclimatization, rats were randomized to either the age-matched normal control group (NC, n=10) or the DM groups (1-month, DM1 and 6-month, DM6, n=10/group). Rats in the NC group were injected intraperitoneally with 0.1 mmol/L citric acid buffer (pH=4.2∼4.5) and fed with routine diet. DM model was induced by injection of STZ as described before [26]. In brief, rats in the DM group were injected intraperitoneally with 1% streptozocin (150 mg/kg in citric acid buffer, STZ, Sigma, USA) for 3 days. After STZ injection, the rats were fed with high-glucose water and diet. Fasting blood glucose (FBG, after 12 h) were measured 12-hour post-injection and then weekly till the endpoint in all groups. FBG was measured via the tail venous blood using a portable glucose monitor (Sinocare, Changsha, China). Animals with FBG values of ≥16.7 mmol/L were considered successful DM establishment [27]. Rats from each group were euthanized at one month (DM1, n=10) and six months (DM6, n=10) after DM model was established, and their eyes were subsequently enucleated for the following assessments, with 20 eyeballs in each group.

Measurement of AGE levels by an enzyme-linked immunosorbent assay
Both serous and retinal AGE levels were measured. About 5 mL of cardiac blood was collected from the heart directly, and then it was centrifuged at a speed of 3000 r/min for 10 min at 4°C to achieve supernatants for serous AGE measurement. To quantify retinal AGE, the retina (10 eyeballs were used for each group) was isolated and homogenized with a homogenizer. Thereafter, the intermediate product was centrifuged at a speed of 10,000 r/min for 15 min at 4°C. Finally, the supernatants were collected for retinal AGE assessment. An ELISA kit (Shanghai Enzyme-linked Biotechnology Co., Ltd., Shanghai, China) was utilized to quantify serous and retinal AGE levels.

Transmission electron microscopy
Eyes (4 eyeballs were used for each group) were fixed in 2.5% glutaraldehyde and 2% formaldehyde in 0.1 M of cacodylate buffer (pH=7.4) at 4°C overnight. Next, samples were post-fixed in 2% osmium tetroxide, dehydrated in a graded ethanol series, and embedded in epoxy resin for sectioning. Sections were stained with a saturated aqueous uranyl acetate solution and Sato lead stain (Structure Probe, Inc., West Chester, PA), and images were acquired via Philips EM208S (Philips, Eindhoven, The Netherlands) microscope. To minimize error and bias, TEM images were taken at the central and examined by three independent pathologists who were blinded to the treatment.

Raman spectra acquisition
The retinal tissues from the remaining 6 eyeballs for each group were separated and cut into four pieces. The RPE layer was exposed using an ophthalmic scissors, and then the RBC spread on a rectangle-aluminum plate for Raman spectroscopy. To prepare the sample for Raman spectra acquisition, a fine ophthalmic scissors was utilized to separate the retina from eyecup, and then the RBC was carefully exposed with RPE attached. Raman spectra were acquired using a Renishaw InVia micro-Raman system with a 50× objective (numerical aperture of 0.75). Laser beam from a 785 nm multimode high-power diode light source (about 20 mW) was used for signal excitation. Raman spectra were taken in exposure time of 10 seconds, and two accumulations were used at a spectral resolution of 1 cm −1 over the spectral window from 400 to 1800cm −1 . For each piece of sample, two spectra were acquired satisfactorily and the duration for each measurement was less than 2 minutes (including scan time of grating). To note, several spectra were excluded due to poor signal-to-noise ratio. Finally, a total number of 30 spectra were acquired from each group (NC, DM1, and DM6).

Data preprocessing and pattern recognition
To remove the prominent autofluorescence from the raw Raman spectra, background subtraction was carried out by using an automated algorithm for autofluorescence removal by a fifth-order polynomial function [28]. All procedures of spectral data preprocessing were performed consistently, and each spectra was normalized according to the area under the curve. We implemented algorithms of principal components analysis (PCA) and decision trees (DT) built in the SPSS software package (version 19.0, IBM, Armonk, USA) for statistical analysis. Detailed protocols have been described in our previous work [29]. Notably, both whole Raman spectra and all marked band data sets were used in PCA and DT analyses. The band data-based tree illustrates the band 'importance' during the cross-validation procedure. Such importance means a contribution of each band to the establishment of the model as a variable. During cross-validation, the tree quantified the band importance in correct classification. It is important to discover those bands with key roles in classification. Finally, posterior probability distributions from the DT model, calculated using the classification and regression trees (CRT) algorithm, were used to estimate receiver operating characteristic (ROC) curves. The data were presented as mean ± standard deviation. Student's t-test was used and P<0.05 was considered statistically significant.

Hyperglycemia associated anatomical changes with DM progression
In treated groups, opacification of the lens was found to be progressively developed. In the DM1 group, the size of RPE cells of DM group remained similar to those in the NC group. However the axial length of RPE cells significantly decreased in the DM6 group, and the intercellular transport structures, such as the basal membrane infolding labyrinth and the apical fringe of microvilli, were significantly reduced. Besides, basal membrane infoldings in DM1 rats lost regular architecture, and some mitochondrial structures were destroyed, resulting in an appearance of vacuoles. The elastic lamina of Bruch's membrane (BM) in DM1 could not be clearly identified due to abundant dense deposits in the RPE and disturbed structure of BM. Inflammatory cells could also be seen in the blood vessels of choriocapillaris. Furthermore, we noted that the RPE in DM6 rats became atrophic attributed to loss of regular cell shape. To the same degree, the photoreceptor outer segments were found to be altered with sectorial loss of regular disc arrangements, while more heterogeneous thickening and shapes of BM in DM6 rats were found with discontinuities in contrast to the continuous elastic lamina of the BM.

Precise characterization of RBC by RS
Raman spectra were able to be repeatedly measured the region of RBC as showing in Fig. 2(g). To be more specific, the laser beam was focused on the surface of RBC during each Raman spectra acquirement (Fig. 2(h)). The full range of Raman spectra responses (400∼1800cm −1 ) was expressed as group mean (solid trace) and standard deviation (gray area) in Fig. 3. It is clear that spectra of all groups show similar profiles (peaks and troughs at certain spectra). The difference (black traces in Fig. 3) of Raman spectra responses between the NC group and treated groups (DM1 and DM6), and the variation was relatively small in both groups. This is important as it guaranteed our spectra band assignment analysis and following post-hoc analysis was not biased by altered tissue composition due to STZ injection. Several characteristic Raman bands were identified (Table 1), in consistence with previous publications [30][31][32][33]. Most signals were assigned to proteins, nucleic acids, collagen or lipids. Signals located at 941 and 1587 cm −1 were assigned to heme and melanin, respectively. According to the difference spectra, changes of intensities in some bands may be related to the disease progression of DM. Six bands belonging To identify the characteristic differences for the Raman spectra of RBC between groups, we then performed PCA to explore any intimation and reliable markers to discriminate the histological type and DM status. Two types of data source were fed into the software for factor analysis, including whole Raman data ( Fig. 4(a)∼(d)) and all marked band-intensity data (Fig. 4(e)∼(h)). In the PCA process, given the first two principal components (PCs) contain primary effects and explain the most variance, while the variance explained by PCs>2 gradually decline, we used scores and projections of the first two PCs to represent the most amount of original spectral information. Notably, for both data sources, each group exhibited clear division. These results confirmed that there were significant differences between the all groups. Meanwhile, with the additional combination of disease progression, the ability of time-dependent effects represented some relationship with pathological findings that is described above. Furthermore, we implement DT model based on CRT algorithm that is a commonly employed statistical classifier using the concept of information entropy. The detailed methods have been described in our previous publications [34,35]. Briefly, we applied method called 'Gini index' to build the DT model, and a 20-fold cross-validation procedure was used to evaluate the predictive error of the model. The input data were the same as PCA, and the whole Raman data and all marked band-intensity data of RBC samples were used to construct DT models by CRT algorithm. The classification results indicated that only few samples were misclassified, and the obtained diagnostic ability derived from ROC analyses were put together in Fig. 5 for comparison. Interestingly, the outcomes of these models were not affected by the input data sources and the areas under the curves were almost equal, suggesting that models derived from whole spectra and marked band intensities have acquired similar performance.  Figure 6 shows the top ten bands with the most importance in each model ranked from the post-hoc analysis. The model with the data from all three groups indicated the most importance of bands for result prediction (Fig. 6(a)). Notably, our post-hoc analysis results returned six bands that shared importance in both CRT models (NC vs. DM1, 1449, 1247, 1269, 1587, 1064, and 538 cm −1 ; NC vs. DM2, 538, 715, 1380, 1449, 1401, and 1269 cm −1 ), as shown in Fig. 6(b)∼(c). When we overviewed the importance analysis of all CRT models, all these three models, it can be seen band positions at 538, 1449, and 1269 cm −1 were highlighted by all three models. Thus, Fig. 6. Importance of independent Raman bands in the CRT models. Variable importance used were initiated from (a) all three groups, (b) the NC and the DM1 groups, (c) the NC and the DM6 groups, (d) the DM1 and the DM6 groups, respectively. The importance of each band means a contribution to the establishment of the model as a variable.
it is possible to differentiate the early and late stage RBC alterations during the development and progression of DM.

Increased RS ratio intensities correlate to DM progression
Based on previous RS studies that applied to several biological samples [36,37], peak-intensityratio measurements returns a simple and effective approach for RS analysis, and thus it was employed in this study. After CRT screening, the most 'important' bands were classified into 4 categories based on their assigned biological identities, namely heme/collagen, nucleic acid, lipids, and amino acids. The correlation between bands within the same categories was further compared via the combination of band-intensity ratios. The progressively up-regulation of ratio intensities in both NC and DM groups were shown in Table 2. There were 3 and 6 ratios were detected significant different to the NC group in the DM1 group and in the DM6 group (all P<0.05), respectively. In the DM1 group, ratio intensity of 1449/715 referring to lipids was significantly higher than that in the NC group (DM1 vs. NC, P<0.05), and similar trend was noticed at the ratio intensities of 1208/1005 (DM1 vs. NC, P<0.05) that indicates amino acids. Although ratio intensity of 1380/715 referring to lipids also showed significant difference between DM1 and NC groups (P<0.05), the mean value in DM1 group was only slightly higher than that in the NC group, probably due to intra-class differentiation at these two bands in DM1 or NC group. In comparison, 6-month of STZ injection induced elevation of ratio intensities in 4 tissue components shown in Table 2 (all P<0.05). Moreover, an increasing trend of ratio intensities (1401/1247, 1380/715, 1449/715, and 1247/715) with DM progression (DM1 vs. DM6) was found (all P<0.05), especially those indicating lipids (1380/715 and 1449/715). This data suggests a positive correlation between certain ratio intensities and DM progression. The ratio values were presented as mean ± SE, and P<0.05 was considered with statistical significance versus: a NC group b DM1 group.

Discussion
Diabetic retinopathy is common in DM patients due to chronic hyperglycemia. The STZ Rat model has been widely used for diabetic studies given it is capability to mimic high blood glucose induced impairment as seen in humans [38,39]. Our data is in line with other studies that are with sustained hyperglycemia, accumulated AGE in both serum and retina, and progress optic conditions. However, the underlying mechanisms for early Raman characterization of diabetic complications are yet fully understood. High blood glucose has broken the glucose metabolism balance in the body, resulting in an increase of AGE accumulation [40], as well as a fluctuation of heme to that of melanin. It has been shown the correlation between the AGE accumulation in serum and retina tissue and DM progression. If without intervention, sustained AGE accumulation will exacerbate the injury and thus result in further pathological structural abnormalities of RBC [41]. Because of the anatomic contacting of RPE and BM, AGE deposition on the BM may be associated to RPE dysfunction and subsequent atrophy and photoreceptor degeneration [42]. It is believed that series of metabolic disturbances are involved in this process, including amino acid metabolisms, lipid metabolisms, glucose metabolism, and protein biosynthesis [43]. However, it is hard to make a clear division from the number or type of molecules with characteristic changes, if just according to the results of semi-quantitative analysis of the selected categories of key molecules. Therefore, the balance of carbohydrate-related metabolisms in the body is happening, but due to certain self-regulating functions or mechanisms in the early stage of diabetes, it seems that some of the inter-molecular relative contents and the duration of the disease have formed a special relationship, which together constitute the early onset characteristics of the DM that we propose. Nevertheless, we still feel that the work of this study is not sufficient to determine the details of the specific molecular content alteration, as well as the internal structural modifications corresponding thereto. The results of this preliminary study confirm the possible early pathophysiological changes of DM, and the more detailed mechanism of the action will still need to be addressed in the future work. By using RS, we found the ratio intensities of key molecules from four categories including heme/collagen, nucleic acid, lipids, and amino acids in the RBC tissues were obviously elevated in the DM groups. Specifically, the ratios intensities from lipids signals of both 1380 cm −1 (symmetric CH 3 bending) and 1449 cm −1 (C-H vibration) to that of 715 cm −1 (C-N in membrane phospholipid head) increased significantly from 1 month to 6 months in STZ rats, which implies the relative content of different structures in lipids may be impaired. Such changes may be explained by our morphological examination given the basal membrane infolding labyrinth and the apical fringe of microvilli were significantly reduced. Other studies also show that lipid metabolism is first regulated in the early stage of DM [44], and the RBC is defeated by enhanced lipid metabolism along with DM progression [41]. The content of phenylalanine increased relative to its symmetric ring breathing configuration and continued to 6 months, indicating some altered configuration in phenylalanine while the meaning of its indication is still unclear. It was assumed that because the 1208 cm −1 band came from phenylalanine and tyrosine, and tyrosine metabolism is regulated by phenylalanine metabolism. Those altered configuration may also be due to the disorder of phenylalanine metabolism, which may lead to abnormal tyrosine metabolism. In general, the metabolic behavior of amino acids has occurred from 1 month to 6 months after DM, suggesting some alterations due to early DR may be involved, and the degree of abnormality was further exacerbated at 6 months.
Special attention is worthwhile to be paid to spectral signals related to heme/collagen, i.e., 941 cm −1 (C-C bonds) and 1401 cm −1 (bending modes of methyl groups) to that of 1247 cm −1 (amide III, CH 2 wagging and C-N stretch). Our data showed the increase of those two ratio intensities (941/1247 and 1401/1247) only appeared in the DM6 group but other two groups, which indicated a potential biomarker that for diabetic changes in a late stage. To detect he changes of collagen mainly occurred in late stage diabetes (DM6) may be important, as the major component in matrices and tissues, collagen represents a key target of this spontaneous reaction which leads to the modification of collagen spatial structure and by this way to tissue damages [45]. Such alternation of collagen arrangement caused by the change of molecular ultrastructure can be seen by morphological examination, which was reflected in our result of electron microscopy examination. Another intensity ratio was identical in the DM6 group, pyrimidine to purine (1247/715) that showed significant difference comparing to either the NC group or DM1 group. However it is still unclear whether purine metabolism is more active than pyrimidine, and further studies are required.
Great efforts have been made to develop tailored optics-based biosensors, applying to the tissue interface [46,47]. For example, Barman and colleagues demonstrated the pilot application of a drop coating deposition RS for label-free and selective detection of glycated hemoglobin and albumin, without addition of exogenous dyes or reagents [48,49]. The glycation of hemoglobin or albumin-induced subtle changes were acquired from significant Raman spectral features with help of conventional multivariate techniques. Emerging report also demonstrated quantitative, continuous, and rapid glucose sensing at physiologically observed levels by tracking the shifts in the Raman-emission peaks of specific molecule such as the bonding of glucose molecules [50]. Therefore, the RS has been proven the capability to directly highlight the effect of diabetic alterations, and advances in noninvasive prognostic monitoring have also been put forth and tested with varying degrees of success. We showed the feasibility to detect the tissue molecular changes in DM rat RBC, and more importantly to differentiate such changes in early and late DM. However, besides quantify the changes in RBC, further studies to examine the change in retinal blood vessels and capillaries using RS will be necessary. It is widely believed that the DR is associated with retinal-blood barrier breakdown and expression of vascular endothelial growth factor due to capillary injury [51]. Evidence showing changes of lipids, protein and collagen in retinal vascular network has been documented [52,53], thus it leaves us the gap to further investigate the structural and molecular changes in retinal capillaries during the development of DR. The significance of this study is that it sheds a light on seeking novel non-invasive approaches to detect DR in the early stage. Although this study did not directly assess the diabetic changes in the eye by using RS, our ex vivo study may point out future direction of detecting specific RS band ratio intensities, and our optimized analysis protocol may build a fundamental stone for in vivo studies or clinical application. Based on this, the next movement to establish a non-invasive assessment in vivo is possible. We acknowledge this study has limitations. More detailed mechanisms of those response and correlation need to be investigated, and the potential of the proposed characteristic bands cross different categories also need to be further concerned. In addition, we used albino Sprague-Dawley rats which are adequate for ex vivo assessment in this study, however rats with pigmented retina such as Long-Evans rats may be used for in vivo examination.

Conclusions
In summary, this study used RS to identify the indicators reflecting the early characterization of the RBC in a STZ induced diabetic rat model. The results demonstrated characteristic alterations of RBC attributed to DM. The AGE levels of serum and retina tissue were increased with STZ injection, along with appearance of pathological and morphological onsets in RBC. We propose that Raman spectroscopy can reliably distinguish different phases of DM. The important band extracted from the whole Raman data sets can be determined in combination with CRT algorithm, and it highlights potential biomarkers indicating the intrinsic key molecules that cause disease progress. The early changes of four key molecules, including heme/collagen, nucleic acid, lipids, and amino acids were quantitatively evaluated. Our findings lay the basis and experimental reference for both understanding the molecular mechanisms underlying early DR and exploiting noninvasive biomarkers for the detection and treatment.