PET Imaging for The Early Evaluation of Ocular Inflammation in Diabetic Rats by Using [ 18 F]-DPA-714

doi:10.21203/rs.3.rs-3119028/v1

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PET Imaging for The Early Evaluation of Ocular Inflammation in Diabetic Rats by Using [ 18 F]-DPA-714

https://doi.org/10.21203/rs.3.rs-3119028/v1

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Ocular complications of diabetes mellitus (DM) are the key leading cause of vision loss worldwide. Ocular inflammation is estimated to occur in the early stage of DM; however, there is no effective quantitative method for evaluating the inflammatory status in eye under diabetic status. The 18 kDa translocator protein (TSPO), an evolutionary conserved cholesterol binding protein localized in the outer mitochondrial membrane, is the biomarker of activated microglia/macrophages, while its role in ocular inflammation is rarely reported. Primary microglia/macrophages (PMs) extracted from the cornea, retina, choroid and sclera of neonatal rats were treated with or without high glucose (50 mM) and used as in vitro model, and SD rats with intraperitoneal administration of streptozotocin (STZ, 60 mg kg⁻¹ once) were used as in vivo model. Then, fluorine-18-DPA-714 ([¹⁸F]-DPA-714), as a specific TSPO tracer, was used for cell uptake and cell binding experiments as well as micro positron emission tomography (microPET) imaging. Increased cell uptake and high binding affinity of [¹⁸F]-DPA-714 were observed in primary PMs under hyperglycemia stress, which was consistent with morphological changes, cell activation and TSPO overexpression (p<0.05). Then, [¹⁸F]-DPA-714 uptake and biodistribution in eye of DM rats was found to be significantly increased at early stage (3 week and 6 week), which was consistent with microglia/macrophages activation and up-regulated TSPO in eye (p<0.05). [¹⁸F]-DPA-714 microPET imaging may be an important method for the early evaluation of ocular inflammation in DM.

Diabetes mellitus

Microglia/macrophage

Ocular Inflammation

TSPO

[18F]-DPA-714

Positron emission tomograph

Diabetes mellitus (DM) constitutes an unrelenting global epidemic affecting more than 422 million people worldwide, and its prevalence is expected to reach 592 million by 2035 (Saeedi et al. 2019). DM is a metabolic disorder of complex etiology. Characterized by chronic hyperglycemia, DM results in damage to various organs, particularly the eyes, kidneys, nerves, heart, and blood vessels (Liang et al. 2022). DM can cause ocular diseases (Skarbez et al. 2010), ultimately leading to diabetic keratopathy, diabetic retinopathy (DR), diabetic macular edema (DME), choroidal changes, cataract, and glaucoma (Hamadneh et al. 2020; Mesquida et al. 2019; Priyadarsini et al. 2020; Zhou et al. 2022). These complications can potentially induce severe vision loss or even blindness. among which, DR has been widely studied (Zhou and Lee. 2022). By contrast, other ocular diseases have been rarely reported. DM presents as a low-grade chronic inflammatory pathology. Inflammation plays a pivotal role in the development and progression of diabetic eye disease (DED), in conjunction with increases in proinflammatory mediators, apoptosis, and vascular permeability (Asiedu et al. 2022; Liu et al. 2023). Current research on the link between inflammation and DED has focused on DR and DME. Inflammation mediates deleterious effects on the neuronal and vascular components of the retina, which have been observed from the asymptomatic early phases to the sight-threatening advanced stages of the disease (Alves et al. 2018; Mesquida and Drawnel. 2019). The pathogenesis of inflammation in other anatomical parts of a diabetic eye remains unclear, and further research is urgently needed.

Macrophages are present at all parts of the eye including cornea, iris and ciliary body, retina (presents as microglia), choroid and sclera, and act as resident immune cells for host defense and homeostasis (Chinnery et al. 2017; Pavlou et al. 2018). Numerous studies have indicated that macrophage-mediated inflammation plays an important role before neuronal apoptosis and activation of other glial cells in the eye (Park et al. 2021). During the pathological process of DM, macrophages are the key cellular component upon the inflammatory response, having roles in phagocytosis, antigen presentation, as well as potentiation of inflammation (Ayala et al. 2019; Kinuthia et al. 2020; Narayan et al. 2017; Pavlou and Lindsay. 2018). These studies have rendered macrophages as the critical target for the therapeutic intervention of early stage of DM. However, the spatiotemporal changes in the inflammatory response of the macrophages of the anterior and posterior eye segments in the early stages of DM are rarely reported.

Positron emission tomography (PET) imaging has emerged as a helpful tool to non-invasively visualize and provide insights into individual and temporospatial biology and grade of inflammation in vivo in a variety of diseases especially in a preclinical setting (Iking et al. 2021). It has recently been used to evaluate intraocular and blood pharmacokinetics (Luaces-Rodriguez et al. 2020) and to detect patients with oculopathy, including primary and metastatic ophthalmic tumors (Jiang et al. 2018), orbital inflammatory diseases (Laban et al. 2019), and Graves’ orbitopathy (Weber et al. 2021). The translocator protein (18 kDa) (TSPO) is a transmembrane protein of the outer mitochondrial membrane, also referred to as peripheral benzodiazepine receptor (PBR), a key factor in controlling the transport of cholesterol necessary for mitochondrial cholesterol translocation, porphyrin transport and heme synthesis, cell proliferation, apoptosis, immunomodulation, stress adaptation, and inflammation (Corsi et al. 2022; Zhou et al. 2020). TSPO is highly expressed in inflammatory cells, such as macrophages, microglia, astrocytes, and peripheral immune cells (monocytes, lymphocytes) and then used as a key target in PET imaging (Barca et al. 2022; MacAskill et al. 2021).TSPO PET imaging provides an innovative and useful tool to evaluate the prevention and possible therapy of DR and diabetic neuropathy (Trotta et al. 2022; Zhou and Ou. 2020). However, studies regarding TSPO PET imaging for ocular inflammation evaluation in the early stage of DM are limited. [¹⁸F]-DPA-714 presents advantages over other TSPO radioligands with regard to the high binding affinity, high signal-to-noise ratio and low lipophilicity for autoradiography or in vivo neuroinflammatory PET scan purposes, which has an excellent predictive value and a good correlation with the functional outcome and/or progression of the disease (prognostic value) (Boutin et al. 2013; Doorduin et al. 2009). [¹⁸F]-DPA-714 PET has been used previously for TSPO PET imaging in DM rats; however, which only evaluates the inflammation in the late stage of DR (Wang et al. 2018; Zhou and Ou. 2020). Numerous studies have confirmed that the dynamics of [¹⁸F]-DPA-714 tracer correlate with the Iba-1⁺TSPO⁺ cell number, supporting its use for tracking macrophage-targeting immunomodulatory therapies in vivo (Barca and Kiliaan. 2022). Researches have shown that confirmed that PET imaging with [¹⁸F]-DPA-714 reflected the dynamic of macrophage infiltration during the inflammatory reaction(Luu and Kim. 2022; Wu et al. 2014). In addition, [¹⁸F]-DPA-714 binds to TSPO with high specificity and provides a sensitive tool for assessing inflammation(James et al. 2008; Lavisse et al. 2015; Wang et al. 2014). In this study, [¹⁸F]-DPA-714 microPET imaging seems most promising as a predictive biomarker for early spatiotemporal changes in inflammation in DED using in vitro and in vivo models.

Primary macrophages isolation and culture

Primary macrophages (PMs) were isolated from neonatal Sprague Dawley (SD) rats aged 1–3 d as previously reported (Harrison et al. 2010; Hu et al. 2021; Qin et al. 2012) . All experimental protocols were approved by the Laboratory Animal Management and Ethics Committee of Jiangsu Institute of Nuclear Medicine (approval number: JSINM-2021-118). The tissues of cornea, retina, choroid, and sclera were dissected from the eyes of the neonatal rat and minced with scissors and which then were fully mixed with trypsin digestion liquid. Then tissue fragments were passed through the cell strainer, and seeded in poly-lysine-coated flasks with the medium DMEM/F-12 containing 15% FBS (Gibico, Newyork, USA).), 1% penicillin/streptomycin, and 25 ng/mL macrophage colony-stimulating factor (M-CSF) (Beyotime, Nantong, China). After 1 week of culture, the cells were used for the further experiments.

MTT assay

PMs were divided into three groups: CT group (normal glucose), HG group (50 mmol/L glucose), and Mannitol group (50 mmol/L Mannitol). The MTT assay was used to determine cell viability. After treatment, MTT solution (0.5 mg/mL, 100 µL) was added to the cell culture and incubated for 3 h at 37 ℃. After removing the medium, DMSO (150 µL) was added to the cell culture for 10 min with gentle shaking. The absorbance was detected with a microstrip reader (BioRad Laboratories, CA, USA) at 490 nm wavelength.

Establishment of diabetic rats

The SD rats (male, aged 5–6 weeks, 250–300 g) used in this study were purchased from Changzhou Cavens Laboratory Animal Co., Ltd. (Changzhou, China) and housed in the Laboratory Animal Center of Jiangsu Institute of Nuclear Medicine under temperature-controlled (23 ± 2 ℃) and humidity-controlled (~50%) conditions. The rats were randomly assigned to the DM group (n = 10, 20 eyes) and control group (CT group, n = 10, 20 eyes), respectively. The modeling process was as follows: after 7 d of acclimatization, the rats in the DM group were intraperitoneally injected with streptozotocin (60 mg kg⁻¹ once, Sigma, Saint Louis, USA) dissolved in a citrate buffer (0.1 mol L⁻¹, pH 4.5) to induce hyperglycemia. The CT group received intraperitoneal injections of the same volume of citrate buffer (0.1 mol L⁻¹, pH 4.5) on the same day. Blood glucose was measured and a random result ≥ 16.7 mmol/L was confirmed as the success of the DM model.

Hematoxylin–eosin staining and TUNEL assay

Histology was assessed by HE staining and cell apoptosis was assessed by Terminal deoxynucleotidyl transferase-mediated nick end labeling (TUNEL) staining. First, formalin was removed from the eyeball tissue samples. And the tissue samples were cleared in xylene and embedded in paraffin blocks. For hematoxylin and eosin (HE) staining, paraffin sample sections (8 µm) were stained with hematoxylin (5 min), followed by eosin (2 min). All slides were mounted using neutral resin. For the TUNEL assay, sample sections were processed for TUNEL staining using a previously reported method (Loo. 2011). DAPI (0.5 µg/mL) was used to stain the nuclei. Ultimately, sections were observed with an Olympus fluorescent inverted microscope (Olympus, Tokyo, Japan).

Immunofluorescence

Ocular samples of the rats were taken out, frozen, and cut into 8 μm sections. PMs were identified by immunofluorescence staining of Iba-1, a marker of macrophage as the previous literatures (Giambrone et al. 2019; Kaczmarek-Hajek et al. 2018; Kambhampati et al. 2015). Cells or sections were fixed with 4% paraformaldehyde, permeabilized, and blocked in an immunofluorescence-blocking fluid (Beyotime, Nantong, China). Then, cells or sections were incubated with primary antibodies overnight at 4 ℃. The primary antibodies, including anti-Iba-1 (Abcam, Mass, United States; dilution 1:100) and TSPO (Abcam, United States; dilution 1 :100). Secondary antibodies, including the species-appropriate Cy3-conjugated donkey anti-rabbit IgG (Beyotime, Nantong, China; dilution 1:1000) and Alexa Fluor 488-conjugated donkey anti-goat conjugated IgG (Beyotime, Nantong, China; dilution 1:1000), were used for cells or sections for 1 h in the dark. DAPI (0.5 µg/mL) was used to stain the nuclei. Ultimately, cells or sections were observed with an Olympus fluorescent inverted microscope (Olympus, Tokyo, Japan).

Synthesis of [¹⁸F]-DPA-714

The radiolabeling precursor DPA-714 was provided by Jiangsu Institute of Nuclear Medicine. ¹⁸F was produced on the cyclotron (Sumitomo Heavy Industries, Tokyo, Japan) of Jiangsu Institute of Nuclear Medicine. The ¹⁸F produced by the cyclotron was captured on a QMA cartridge (Waters, Mass, United States). and eluted into a 5 mL reaction vessel. The mixed solution was dried 3 times under a nitrogen flow at 100 ℃. Subsequently, 0.5 mg of DPA-714 was dissolved in 0.5 mL of extra-dry acetonitrile, added to the completely dried ¹⁸F, and reacted at 105 ℃ for 10 min. The reacted solution was cooled to room temperature and injected into a semipreparative high performance liquid chromatography (HPLC) (Waters, Mass, United States). The mobile phase consisted of deionized water with 0.1% triethylamine (SCR, Chengdu, China) and acetonitrile ((SCR, Chengdu, China) with 0.1% triethylamine (50/50, v/v), and the flow rate was 5 mL/min. The collected [¹⁸F]-DPA-714 fraction was injected into the C18 cartridge (Waters, Mass, United States). and then eluted using 0.5 mL of ethanol (SCR, Chengdu, China) to obtain the final product. The entire synthesis process was completed within 1 h, and the radiochemical purity is ≥99%.

In vitro characterization

1. In vitro stability

[¹⁸F]-DPA-714 (radiochemical purity > 99%) was incubated in human plasma and PBS for 2 h at 37 ℃. After 0, 1, and 2 h, a sample was analyzed on the HPLC instrument (Waters, Mass, United States).

2. Cell uptake assay

Cell uptake was performed in accordance with the following protocol. On day 1, PMs were seeded into a 24-well plate (5×10⁴ cells/well) and then incubated overnight. On day 2, the medium was added with or without 50 mmol/L glucose. On day 3, the cells were rinsed with PBS, and the medium with [¹⁸F]-DPA-714 (2 μCi/well) was added to the wells. The incubation time was set at 5 time points (0, 30, 60, 120, and 150 min) in triplicate. At each given time point, the cells were washed 3 times with PBS and subsequently lysed with NaOH (1M NaOH). The cell lysates were collected in measurement tubes and counted by a γ-counter.

3. Cell binding assay

The binding affinities and specificities of [¹⁸F]-DPA-714 were determined, with DPA-714 as a competitive ligand. On day 1, PMs were seeded into a 24-well plate (5×10⁴ cells/well) and incubated overnight. On day 2, the medium was added with or without 50 mmol/L glucose. On day 3, DPA-714 (2*10^-9–2*10^-1mM) were administered into the wells in triplicate. The medium with [¹⁸F]-DPA-714 (2 μCi /well) was added into each well. After incubation for 2 h, the plates were washed with PBS and subsequently lysed with NaOH (1M NaOH). The cell lysates were collected and then counted by a γ-counter. The best-fit 50% inhibitory concentration (IC₅₀) was calculated by fitting the data via nonlinear regression.

In vivo characterization

1. [¹⁸F]- DPA-714 PET imaging

Small-animal PET was performed with a micro-PET scanner (Siemens Medical Solutions In, TN, USA). The animals were weighed before each imaging session to assess their health condition. During all experimental procedures, the rats were anesthetized with 1.5%–2% isoflurane in 100% O2. A radiotracer was prepared as previously described (James and Fulton. 2008), with >99% radiochemical purity. Under anesthesia, the rats were placed prone at the center of the field of view of the scanner and then injected with [¹⁸F]-DPA-714 via the lateral tail vein. Eye scanning was performed at 120 min after IV injection, and 10-min static PET images were acquired. The PET images were quantitatively analyzed using a previously reported method (Hu et al. 2020). Based on the anatomy of the CT, the region of interest (ROI) in the eyes was sketched to estimate the mean and maximum standardized uptake value (SUV) corrected for body weight in the eyes volume.

2. Ex vivo biodistribution

[¹⁸F]-DPA-714 (1.1-2.0 Mci/rat, 0.5 mL) was injected into the rats. The rats were anesthetized using ether and sacrificed by cervical vertebra dislocation 2 h after injection, and the biodistribution of [¹⁸F]-DPA-714 was determined. Tissue samples of the eye (cornea, lens + vitreous body, retina, choroid + sclera) were dissected and weighed, and the radioactivity was measured using a γ-counter.

Quantification of TUNEL (+), TSPO (+), and Iba-1(+) cell and Tissue thickness

The TUNEL (+), TSPO (+), and Iba-1(+) cell and tissue thickness of the cornea, retina, choroid, and sclera were quantified by ImageJ software. NIH ImageJ Software (National Institutes of Health, Bethesda, Maryland; http://imagej.nih.gov/ij/) was used to quantify the intensity and the length of each parameter. The mean of 3 samples was recorded as the representative value for each group.

Statistical Analysis

Statistical analysis was conducted using Graphpad Prism (8.0 version). All experiments were repeated for three independent replications. Data for multiple experiments are expressed as means ± SD. Statistical comparisons between two groups were conducted with the Student’s t-test and p < 0.05 was considered statistically significant.

Isolation, culture and identification of PMs

Primary macrophages (PMs) were isolated from neonatal Sprague Dawley (SD) rats aged 1–3 d as previously reported (Harrison and Ferreira. 2010; Hu and Zhao. 2021; Qin and Yeh. 2012). Figure 1A showed the critical steps during the extraction and purification of primary macrophages. After extraction of PMs from SD rats’ cornea, retina, choroid and sclera separately, after then 1 week of culture, the primary PMs model were employed. Then characters of PMs were identified by immunofluorescence using Iba-1. No available studies had been reported about the distinct morphology of the three PMs. The study first revealed the main difference between the PMs of cornea, retina, choroid and sclera (CHO&SCL) regarding to the branch length as well as the size of cell bodies. Three PMs exhibited ramified morphology in normal medium. The shortest branch was observed for corneal PMs, whereas the longest one was found for retinal PMs. The smallest was observed for retinal PMs, whereas the largest one was found for CHO&SCL PMs (Fig. 1).

PMs activation and up-regulated TSPO under hyperglycemia stress

The morphological changes as well as cell viability of PMs in CT, HG and mannitol groups were assessed. The results showed that the hypertonicity did not affect the viability of cells (Supplementary Fig. 1). For this reason, the mannitol group was not used in the further experiments. M0 and M2-polarized PMs exhibit morphological similarities, and M1-polarized macrophages generally show a flattened, rounded morphology (McWhorter et al. 2013). As shown in Fig. 2A, in the CT and mannitol groups, PMs displayed a ramified morphology with small cell bodies and elongated cell neurites, which are all characteristic features of M0 or M2 PMs. PMs in HG group had fewer and shorter branches but a larger cell body diameter, which are all characteristic features of M1 PMs. Our data indicated that high glucose could promote the transformation from M0/M2 to M1 macrophages in vitro (Fig. 2A) (Du et al. 2017; Okamura et al. 2021; Zhou et al. 2020). Immunofluorescence staining exhibited increased TSPO expression in PMs of HG group, indicating that TSPO may plays a critical role in macrophage proinflammatory processes under hyperglycemia stress (Fig. 2B).

Establishment of the diabetic rat model

STZ induced diabetic rats is a validated model of DED (Long et al. 2019; Shi et al. 2009). The initial stages of DED have occurred early retinal circulation disturbances and changes of ocular oxygen distribution, an decrease innervation of the cornea epithelium(Davidson et al. 2012), axoglial alterations of the distal optic nerve(Fernandez et al. 2012; Park and Lee. 2021) and an increase in microglia/macrophages immunoreactivity(Ibrahim et al. 2011; Park and Lee. 2021; Schallner et al. 2012), within 1–6 weeks after the onset of diabetes in rats. Based on the findings, this study chose the 3 and 6 week time points for investigating microglia/macrophages activation and proliferation during the early stage of experimental DED before the induction of chronic damage—to obtain insights into the contribution of inflammation to DED.

Diabetic rat model was established by intraperitoneal injection of STZ (60 mg kg^− 1 once). After diabetes onset, over a 3/6- week period, blood glucose levels were significantly up-regulated and body weight (BW) was significantly down-regulated in DM group compared to CT group (n = 5 for each group). The elevated blood glucose level and the decreased BW indicated the successful establishment of diabetic rat model (Fig. 3).

Presentation of ocular structure and injury in diabetic rats

Histological analysis indicated no apparent morphological and cell density changes, inflammatory cell infiltration and tissue structure changes in the eye (cornea, retina, choroid, and sclera) in the early stages of diabetes, that is, 3 or 6 weeks after induction of DM. TUNEL analysis demonstrated the occurrence of cell apoptosis at 3 Weeks after the diabetic status. The apoptosis of the cells was significantly increased at Week 6, relative to that in CT group. Changes of corneal cell in DM group mainly occurred in the epithelial layer at Week 3 and in the stromal layer at Week 6. Increased apoptosis was observed in the inner nuclear layer of the retina (Fig. 4).

Activated ocular macrophages and up-regulated TSPO in diabetic rats

The number of activated macrophages within the cornea, retina, choroid, and sclera for both groups at Weeks 3 and 6 were evaluated by using Iba-1 (a general marker of macrophages; green) and TSPO (a marker of inflammation; red) staining. Larger numbers of Iba-1⁺TSPO⁺cells were markedly detected in the cornea, retina, choroid, and sclera of rats in DM group, than in those of CT group. The number of macrophages in the DM rats tended to increase in the epithelium and the stromal layer of the cornea, choroid, and sclera after diabetes onset, with increasing number of cells over time. In the early stages of DM, no qualitative assessment of Iba-1⁺ cell morphology ramification was found in the eyes of DM and CT groups. In addition, Iba-1⁺TSPO⁺ cells exhibited high fluorescence intensity and density in the cornea, retina, and choroid of the DM rats at Weeks 6 and 3, relative to those in CT group. During the identification of activated macrophages, TSPO expression was significantly increased in the eyes of rats at Week 6 after diabetes onset. Subsequently, the colocalization of TSPO and Iba1 expression was examined: the overwhelming majority of TSPO-positive cells that were mainly Iba-1 positive. Colocalization immunofluorescence confirmed that TSPO expression was mostly exhibited in Iba-1⁺ cell bodies. Thus, we speculate that ocular macrophages are the main cell population expressing TSPO in the early stages of diabetes. Inflammation and macrophage activation present structural changes in blood vessels and tissues (Figs. 5).

Radioligand synthesis of [¹⁸F]-DPA-714

The stability of [¹⁸F]-DPA-714 in human plasma and PBS was analyzed by HPLC. After purification, the radiochemical purity was > 99% (Supplementary Fig. 2), and the stability was > 99% at Hours 1 and 2 (Fig. 6).

3.1. Cell uptake and cell binding assay and of [¹⁸F]-DPA-714 in PMs under hyperglycemia stress

The results of the in vitro cell uptake study showed that [¹⁸F]-DPA-714 was retained at favorable levels in PMs. [¹⁸F]-DPA-714 uptake in PMs increased over time. In addition, after incubation with high glucose for 24 h, the uptake values of cornea, retina, choroid, and sclera PMs were higher in HG group than those of CT group in the plateau phase (2 h). At Hour 2 during incubation, HG group exhibited significantly higher uptake values, compared with CT group (cornea: 5.95%/10⁴ and 3.67%/10⁴ cells, p < 0.0001; retina: 0.87%/10⁴ and 0.55%/10⁴ cells, p༜0.0001; choroid and sclera: 6.92%/10⁴ and 4.96%/10⁴ cells, p༜0.0001) (Fig. 7A). The TSPO binding affinity of [¹⁸F]-DPA-714 was evaluated using DPA-714 as a competitive ligand. The half maximal inhibitory concentration (IC₅₀) represents the TSPO binding affinity of [¹⁸F]-DPA-714 in substituting a high affinity molecule DPA-714 for binding to TSPO. Co-incubation with DPA-714 blocked the binding of [¹⁸F]-DPA-714 to cells in a dose-dependent manner. The IC₅₀ of [¹⁸F]-DPA-714 displacement with DPA-714 is shown in Fig. 7B. The TSPO binding affinity of [¹⁸F]-DPA-714 in descending order are as follows: HG > CT; cornea > choroid and sclera > retina PMs (Fig. 7B). The TSPO binding affinity assay of all groups displayed high binding affinity (estimated IC₅₀ < 1µM). The IC₅₀ of HG group is smaller than that of CT group, suggesting the TSPO binding affinity of PMs to DPA-714 is increased after exposed to high glucose for 24 hours.

[¹⁸F]-DPA-714 PET imaging and biodistribution in diabetic rats

The radiochemical purity of [¹⁸F]-DPA-714 in this experiment was > 99% (Supplementary Fig. 2). PET imaging studies using [¹⁸F]-DPA-714 of CT and DM groups were performed followed by animal euthanasia for biodistribution studies. In vivo PET imaging of [¹⁸F]-DPA-714 provided clear inflammation visualization, reflecting specificity and retention of [¹⁸F]-DPA-714 in eyes at 2 h post-injection (Fig. 8A). The representative PET images show that uptake values of radiotracer in PET images of the CT groups were higher than those in the DM groups (Fig. 8A). The whole eye was delineated as regions of Interest (ROIs). ROIs quantification quantified [¹⁸F]-DPA-714 uptake value of ROIs in PET images and the maximum uptake value (%ID/g-max) was used for calculation (Fig. 8B/D). As the results show, [¹⁸F]-DPA-714 uptake was significantly increased in the whole eye in both 3 and 6 weeks after the DM model was established. The %ID/g-max of ROIs in the CT and DM rats were 0.38 and 0.53 (means ± SD) at Week 3 and 0.25 and 0.48 (means ± SD) at Week 6. The student’s t-test indicated significantly increased tracer uptake in the DM group relative to the CT group at Weeks 3 and 6 (p < 0.0001) (Fig. 8B/D).

In order to validate the accuracy of the PET data and to provide a more comprehensive biodistribution profile of [¹⁸F]-DPA-714 in different anatomical structures of the eyeball, ex vivo biodistribution experiments were further carried out. Consistent with our results above, [¹⁸F]-DPA-714 efficiently accumulated in the whole eye. The maximal uptake values of [¹⁸F]-DPA-714 in the DM group were higher than those in the CT groups in the eye, cornea, lens + vitreous body, retina, choroid + sclera. [¹⁸F] The DPA-714 uptake values in the eye, cornea, lens + vitreous body of the rats in the DM group were significantly different from those in the control group at Weeks 3 and 6 (for the CT and DM rats at Week 3: 0.37%ID/g and 0.55%ID/g in the eye, p < 0.01; 0.14%ID/g and 0.21%ID/g in the cornea, p < 0.01, means ± SD; for the CT and DM rats at Week 6: 0.23%ID/g and 0.47%ID/g in the eye, p < 0.05; 0.08%ID/g and 0.21%ID/g in the cornea, p < 0.05; 0.003%ID/g and 0.007%ID/g in the lens + vitreous body, p < 0.05, means ± SD).) (Fig. 8C/E).

This study is the first to quantitatively evaluate temporal and spatial changes of ocular inflammation in the early stage of DM by using [¹⁸F]-DPA-714 microPET imaging. We revealed that TSPO in ocular macrophages served as a biomarker in early stage of DM and PET imaging of TSPO tracer [¹⁸F]-DPA-714 could evaluate different temporal and spatial changes of ocular inflammation. The results were confirmed by assessing the activation and TSPO expression of ocular primary macrophages in vitro and macrophages in ocular tissues in vivo.

Inflammation occurs in the whole process of DM, especially in DR (King. 2008), and the activation, polarization and function of macrophages/microglia is the most important events in this diseases (Kinuthia and Wolf. 2020). Recent advances displayed that hyperglycemia acts as signaling molecules (Wu et al. 2021) to induce the accumulation of macrophages and microglia with M1 phenotype(Dissanayake et al. 2021; Jia et al. 2015; Pavlou and Lindsay. 2018), which further aggravates DED (diabetic cataract, DR) (Cheng et al. 2019; Jain et al. 2002). In our study, primary macrophage was isolated from neonatal SD rat and used to establish an in vitro diabetic model. The current study found an increase in the number of macrophage/microglia under diabetic stress, not just in the retina, but elsewhere in the eye, including the cornea, sclera and choroid. In keeping with previous studies, we found that HG induced the differentiation of ocular primary macrophages or microglia from M0/M2 type to M1 type, which mainly rely on glycolysis and oxidative phosphorylation (Weinberg et al. 2016). TSPO has been widely used as an effective marker of inflammation (Carlezon et al. 2019; Endres et al. 2009; MacAskill and Stadulyte. 2021). Currently, epidemiological studies on the role of TSPO on DM has still not been explored much. TSPO represents a novel therapeutic target for depression in DM by Qiu et al (Qiu et al. 2016). TSPO/VDAC complex and NLRP3 inflammasomes may play an important role in the development and progression of DR by Guo et al (Guo et al. 2022). TSPO as a serum marker of microglial activation positively correlate with early retinal neurovascular alterations of DR patients by Maria Consiglia et al (Trotta et al. 2022). These findings led us to further investigate the role of the increased TSPO in early stage of DED. Our studies discovered and confirmed high TSPO expression in HG-induced PMs and 3 and 6 weeks of DM rats by immunofluorescence and [¹⁸F]-DPA-714 microPET imaging. Zhou et al found that the uptake of [¹⁸F]-DPA-714 was significantly reduced in the STZ-induced diabetic rats compared with that of healthy rats 12 weeks after modeling (Zhou and Ou. 2020). Given the massive activation of anterior and posterior ocular macrophages and high expression of TSPO in 3 and 6-weeks diabetic rats, we suggest that macrophage dysfunction and cell death may have occurred at 10 and 12 weeks of diabetes, resulting in decreased TSPO expression and decreased uptake of [¹⁸F]-DPA-714. In general, our study further complements basic experiments on changes in macrophage activation in vitro and identifies the underlying mechanism of TSPO in the early stage of DM.

Recent years non-invasive and simple methods were developed for macrophages imaging in living humans. Some studies have used OCT to visualize in vivo macrophage-like cells (MLCs), which is thought to be microglia or hyalocytes, located at the vitreoretinal interface. OCT found MLC showed increased density, nonuniform distribution and altered morphology in eyes of patients with proliferative diabetic retinopathy (PDR) (Corsi and Baglini. 2022; Hammer et al. 2020; Ong et al. 2021; Rajesh et al. 2022; Wang et al. 2023; Zhang et al. 2022). But these studies are limited in several aspect, such as restrict requirements of the imaging protocol and which can’t distinguish the origin and lineage cells (Corsi and Baglini. 2022; Hammer and Agrawal. 2020; Ong and Nesper. 2021; Rajesh and Droho. 2022; Wang and Sun. 2023; Zhang and Nesper. 2022). PET is a diagnostic imaging technique that enables targeting, visualization and quantification of biochemical processes at the cellular and sub-cellular level, via the three-dimensional reconstruction of the bio-distribution of several molecules radiolabeled with positron-emitting isotopes. The PET technique offers the possibility to retrieve anatomic, functional, and metabolic information in one examination(Boellaard et al. 2015). Developing PET imaging agents for ocular degeneration disease is a multidisciplinary research field. It has been developed for evaluate different molecular processes underlying the pathophysiology of retinal degeneration (Xie et al. 2012) and age-related macular degeneration (Luaces-Rodríguez et al. 2020; Wang and Fei. 2011). In ocular PET imaging studies using rabbits(Wang et al. 2002) and monkeys(Lacivita et al. 2010), anatomical regions such as the cornea, iris-ciliary body, retina, choroid and sclera can be visualized clearly. But imaging different ocular tissues of small animal models by using micro-PET imaging is not clear. Therefore, we delineate the whole eye as the ROIs and calculate the maximal uptake value of ROIs. To compensate for this, we conducted a [¹⁸F] DPA-714-biodistribution study and immunofluorescence staining of each site in rats’ eyes after the imaging study. N, N-Diethyl-2-(2-[4-(2-Fluoroethoxy)-Phenyl]-5,7-Dimethyl-Pyrazolo[1,5-a] Pyrimidin-3-yl)-Acetamide (DPA-714) is one of TSPO-specific pyrazolopyrimidine ligands and [¹⁸F] DPA-714 is a high-affinity radioligand specific for the TSPO. Its suitable in vivo properties and ability to bind to the TSPO with high specificity, along with its significant effect on steroid synthesis(James and Fulton. 2008), make this ligand an attractive tool for evaluating the TSPO in neuroinflammatory processes in many clinical trials (Arlicot et al. 2012; Hamelin et al. 2016). In our study, the increased binding of DPA-714 to TSPO in the whole eyes of DM rats, particularly the cornea, indicating substantial inflammatory cell infiltration was occurred in the anterior and posterior segments of the eye during the very early period of DED. These observations suggest that activation of ocular macrophages in the early period of DED could be a crucial therapeutic target for pharmacological intervention, and the [¹⁸F]-DPA-714 PET imaging allows quantitative evaluation of spatiotemporal changes in activation of ocular macrophages in the early phase after diabetes.

Our data show that [¹⁸F]-DPA-714 correlates well with changes in diabetic eye inflammation and provides useful information on macrophage-mediated pathological changes during disease progression and in response to therapy. The data may suggest that TSPO PET imaging has the potential for the accurate assessment of early-onset DED.

Considering these findings, we suggest that a crucial contribution of activated macrophages at different anatomical sites of the eye in DED. Moreover, the cellular origin of TSPO signal appears to represent the changes in macrophage function and [¹⁸F]-DPA-714 targeting TSPO exhibits potential as an imaging method for monitoring inflammatory microenvironment of diabetic eye. In addition, and the exact molecular function of TSPO in DED requires further research.

Diabetes mellitus

[¹⁸F]-DPA-714

Fluorine-18-DPA-714

PET

Positron emission tomography

TSPO

Translocator protein (18 kDa)

PMS

Primary macrophages

Diabetic retinopathy

DME

Diabetic macular edema

Sprague Dawley

M-CSF

Macrophage colony-stimulating factor

TUNEL

Terminal deoxynucleotidyl transferase-mediated nick end labeling

Hematoxylin and eosin

HPLC

High performance liquid chromatography

PBS

Phosphate-buffered saline

IC50

Best-fit 50% inhibitory concentration

EPI

Epithelium

STR

Stroma

INL

Inner plexiform layer

ONL

Outer plexiform layer

CHO

Choroid

SCL

Sclera

ROI

Region of Interest

DPA-714

N, N-Diethyl-2-(2-[4-(2-Fluoroethoxy)-Phenyl]-5,7-Dimethyl-Pyrazolo[1,5-a] Pyrimidin-3-yl)-Acetamide

Data availability statement

The datasets generated and analyzed during the current study are available from the corresponding author upon reasonable request.

Acknowledgments

The authors gratefully acknowledge the financial supports by Major Project of Wuxi Commission of Health (Z202009, Z202014, Z202110), Young and Middle-aged Top Medical and Health Talents Project of Wuxi Commission of Health (BJ2020031), Postdoctoral Science Foundation of Jiangsu Province (2021K196B)，Innovative application of medical and public health technology of Wuxi Bureau of Science and Technology (N20192020), Medical Research Project of Jiangsu Provincial Health Commission (ZD2021005), and Science and Technology Development Fund Foundation of Wuxi (Y20212050), Jiangsu Traditional Chinese Medicine Science and Technology Development Program project (MS2022145).

Author contributions

Conceptualization: W.J.Z. and K.W.; methodology: W.J.Z., K.W., and P.C.; formal analysis: P.C. and N.N.D.; investigation: P.C. and N.N.D.; resources: D.H.P., Y.P.X. and X.Z.; funding acquisition: W.J.Z. and K.W.; data curation: S.Y.L. and Z.Q.C.; writing–original draft preparation: P.C. and N.N.D.; writing–review and editing: W.J.Z. and K.W.; visualization: X.L.C. and Y.D.L.; project administration: W.J.Z. and K.W.; All authors have read and agreed to the published version of the manuscript.

Ethics statement

All animal experiments were approved by the Ethics Committee of the Jiangsu Institute of Nuclear Medicine for the studies involving animals (approval number: JSINM-2021-118).

Conflict of interest

The authors have stated explicitly that there are no conflicts of interest in connection with this article.

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No competing interests reported.

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PET Imaging for The Early Evaluation of Ocular Inflammation in Diabetic Rats by Using [ 18 F]-DPA-714

Status:

Version 1

Abstract

Figures

Introduction

Materials and methods

Results

Isolation, culture and identification of PMs

PMs activation and up-regulated TSPO under hyperglycemia stress

Establishment of the diabetic rat model

Presentation of ocular structure and injury in diabetic rats

Activated ocular macrophages and up-regulated TSPO in diabetic rats

Radioligand synthesis of [¹⁸F]-DPA-714

3.1. Cell uptake and cell binding assay and of [¹⁸F]-DPA-714 in PMs under hyperglycemia stress

[¹⁸F]-DPA-714 PET imaging and biodistribution in diabetic rats

Discussion

Abbreviations

Declarations

References

Additional Declarations

Status:

Version 1

PET Imaging for The Early Evaluation of Ocular Inflammation in Diabetic Rats by Using [ 18 F]-DPA-714

Status:

Version 1

Abstract

Figures

Introduction

Materials and methods

Results

Isolation, culture and identification of PMs

PMs activation and up-regulated TSPO under hyperglycemia stress

Establishment of the diabetic rat model

Presentation of ocular structure and injury in diabetic rats

Activated ocular macrophages and up-regulated TSPO in diabetic rats

Radioligand synthesis of [18F]-DPA-714

3.1. Cell uptake and cell binding assay and of [18F]-DPA-714 in PMs under hyperglycemia stress

[18F]-DPA-714 PET imaging and biodistribution in diabetic rats

Discussion

Abbreviations

Declarations

References

Additional Declarations

Status:

Version 1

Radioligand synthesis of [¹⁸F]-DPA-714

3.1. Cell uptake and cell binding assay and of [¹⁸F]-DPA-714 in PMs under hyperglycemia stress

[¹⁸F]-DPA-714 PET imaging and biodistribution in diabetic rats