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. 2023 May 24;243(4):697–705. doi: 10.1111/joa.13889

Hyper‐reflective dots in optical coherence tomography imaging and inflammation markers in diabetic retinopathy

Mohd N Mat Nor 1,2, Cindy X Guo 1, Colin R Green 3, David Squirrell 4, Monica L Acosta 1,5,✉
PMCID: PMC10485584  PMID: 37222261

Abstract

The aim of this study is to correlate small dot hyper‐reflective foci (HRF) observed in spectral domain optical coherence tomography (SD‐OCT) scans of an animal model of hyperglycaemia with focal electroretinography (fERG) response and immunolabelling of retinal markers. The eyes of an animal model of hyperglycaemia showing signs of diabetic retinopathy (DR) were imaged using SD‐OCT. Areas showing dot HRF were further evaluated using fERG. Retinal areas enclosing the HRF were dissected and serially sectioned, stained and labelled for glial fibrillary acidic protein (GFAP) and a microglial marker (Iba‐1). Small dot HRF were frequently seen in OCT scans in all retinal quadrants in the inner nuclear layer or outer nuclear layer in the DR rat model. Retinal function in the HRF and adjacent areas was reduced compared with normal control rats. Microglial activation was detected by Iba‐1 labelling and retinal stress identified by GFAP expression in Müller cells observed in discrete areas around small dot HRF. Small dot HRF seen in OCT images of the retina are associated with a local microglial response. This study provides the first evidence of dot HRF correlating with microglial activation, which may allow clinicians to better evaluate the microglia‐mediated inflammatory component of progressive diseases showing HRF.

Keywords: HRF, hyper‐reflective spot, microaneurysm, microglia, retina

Small dot hyper‐reflective foci (HRF) observed in optical coherence tomography images of the retina in a diabetic rat model. These dot HRF correlated with microglial activation and reduced local retinal function.

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1. INTRODUCTION

In both animal and human optical coherence tomography (OCT) studies three principal types of hyper‐reflective lesions are observed: blood vessels, microaneurysms and small dot hyper‐reflective foci (HRF; Fragiotta et al., 2021). Blood vessel lesions are recognised as large (over 200 μm) and are associated with shadowing of the underlying tissues. Microaneurysms are typically located near the capillary plexus (inner retina), frequently adjacent to areas of capillary nonperfusion (Salmon, 2020) and plasma leak. However, small dot HRF are frequently observed in retinal OCT scans obtained from patients with a diverse range of conditions including age‐related macular degeneration (AMD), hypertension and diabetic retinopathy (DR; Ahn et al., 2014; Pengo et al., 2022; Vujosevic et al., 2017, 2023). The histopathological description of small dot HRF has been carried out in AMD donors and models where they are observed close to the retinal pigmented epithelium (Schuman et al., 2009). We have also observed these HRF in rodent models of retinal diseases, including rodent models of DR located in inner retina, where we investigated their histopathological properties (Mat Nor et al., 2020).

Although large population‐based studies have demonstrated that factors such as poor glycaemic control, duration of diabetes and increased blood pressure, influence the rate of progression of DR (Hove et al., 2006), it is well recognised that other factors must also influence progression, as some patients with good metabolic control may worsen rapidly and develop severe retinopathy. And yet others, with poor systemic metabolic control, exhibit little progression (Lobo et al., 2004). Increasingly, it is recognised that inflammation may have a central role in the development and subsequent progression of DR (Forrester et al., 2020) and that differences in how the inflammatory process is initiated and expressed may be responsible for the different rates of disease progression that are observed.

Small dot HRF have been described in all stages of retinal disease (Coscas et al., 2013; Vujosevic et al., 2017). In human retina OCT scans, these were proposed to be markers of activated microglial cells. Here we provide evidence of the co‐relation between microglia and single dot HRF, reinforced by previous studies in other models (Miller et al., 2019). Clinical reports have proposed that these small dot HRF represent aggregated microglial cells, and small HRF are used in many clinical studies as an in vivo biomarker of local inflammation (Pilotto et al., 2020, 2022; Turgut & Yildirim, 2015; Vujosevic et al., 2013, 2017, 2023).

In our laboratory, we discovered an animal model of DR that spontaneously arose in a colony of albino Sprague–Dawley rats. These animals exhibited hyperglycaemia, as well as progressive degeneration of the retina, which was characterised by the presence of microaneurysms correlated with the expression of inflammatory markers, and small dot HRF in OCT scans. The characterisation of the animal model and the description of the microaneurysms was conducted and documented in a study by Mat Nor et al. (2020).

In this study, we report the characteristics of the dot HRF, which were solitary, less than 30 μm in diameter, located mostly in the inner nuclear layer (INL) but also in the outer nuclear layer (ONL) and around enlarged retinal blood vessels in inner retina. These small dot HRF in the OCT images of the retina did not correspond to any visible lesion in the corresponding fundus image. Clinical assessment using fundus imaging and OCT of these animals has allowed us to detect pathological signs in the eye in correlation with the histological findings (Mat Nor et al., 2020). Hence, we have developed and applied an ophthalmic‐histopathology evaluation of these small dot HRF. We assessed the clinical features of the retina using fundus imaging and OCT. The procedure was followed by a functional assessment of the retina using focal electroretinography (fERG) and histological evaluation and immunolabelling of activated inflammatory cells and retinal stress. Retinal stress is usually detected in total retina or a sector of the retina upregulated in response to stress due to injury or disease (Carpi‐Santos et al., 2022). The in vivo generated optical image has a resolution of 2 microns, which when correlated with the histological analysis allows accurate assessment of retinal lesions. The aim of this histopathological study was to describe the correlation of the small dot HRF observed in OCT with immunohistopathology and functional findings in a unique animal model.

2. MATERIALS AND METHODS

2.1. Animal model of DR

All experimental procedures were approved by the University of Auckland Animal Ethics Committee (001462) and comply with the Association for Research in Vision and Ophthalmology (ARVO) statement for the use of animals in eye research. Clinical signs of DR were found in a spontaneous hyperglycaemic strain of Sprague–Dawley (S‐D) rats, housed in the Vernon Jansen Animal Research Unit, Faculty of Medical and Health Sciences at the University of Auckland. Ten 6‐ to 8‐week‐old female and male rats were used in this study. Rats were deeply anaesthetised via intraperitoneal injection using a combination of Ketamine (75 mg/kg, Parnell Technologies) and Domitor (0.5 mg/kg, Pfizer).

2.2. Focal electroretinography

fERG is a diagnostic test used to measure the electrical activity of the retina in response to light stimulation of small retinal areas. The fERG test requires an overnight period of dark adaptation of the rats before them being anaesthetised. On anaesthetised rats, a gold ring electrode attached to the micron IV objective lens is placed in contact with the cornea to detect the electrical signals generated by the retina in response to flashes of light. A dim red light generated by a light‐emitting diode (λ max = 650 nm) was used during manipulations. Body temperature was kept at approximately 37°C to avoid temperature‐driven fERG amplitude fluctuation. Under red illumination, the objective lens of the micron IV attachment allows visualisation of the retinal and guides the area used to record the fERG. A reference electrode was placed under the skin, to eliminate background noise interference as described by Guo et al. (2016). The cornea was kept hydrated with 1% carboxymethylcellulose sodium (Celluvisc, Allergan) throughout the fERG recording. This fERG technique was used to evaluate the function of specific retinal regions with and without HRF. A visual stimulus is presented to a specific region of the retina using the micron IV (Phoenix Research) light‐emitting diode (LED) light source. The fERG recordings were in response to white‐light flashes from a LED source (5‐ms duration) and were recorded using the LabScribe ERG 3 software (Phoenix Research Laboratories). The light intensity used to elicit the fERG response ranged from −0.40 to 3.20 log candela seconds per square metre (log cd s/m2), with 20 sweeps and multiple individual responses from each sweep were averaged to obtain an improved signal‐to‐noise ratio. One eye (left or right) per animal was used in the study. The fERG waveform was recorded over discrete areas of 0.75‐mm diameter and analysed using the micron IV in‐built programme to isolate the a‐wave and b‐wave components. Briefly, the a‐wave reflects the rod photoreceptor response in the outer retina, whilst postphotoreceptoral response in the inner retina is reflected by the b‐wave. Six HRF areas in four hyperglycaemic animals were used to record fERG data, six areas adjacent to the HRF and data compared with six areas in control normal rats.

2.3. Ophthalmic examination

Hyperglycaemic rats and control S‐D rats were anaesthetised for ophthalmic examination. A Phoenix Micron IV imaging system with a fundus camera and SD‐OCT was used. The imaging procedures were executed immediately after fERG recordings under anaesthesia were taken and pupil dilation. A retinal camera (Micron IV; Phoenix Research Laboratories) was used to capture photos of the fundus of anaesthetised rats as previously described (Guo et al., 2016). Rats were placed on a 37°C heating pad to maintain body temperature. The eyes were dilated with 1.0% tropicamide (Bausch and Lomb New Zealand Ltd.). Dilated eyes were lubricated with Poly Gel (containing 3 mg/g Carbomer; Alcon Laboratories, Pty Ltd.). The eye was centred facing the camera lens and the posterior pole was visualised by making contact between the fundus lens and the gel. Subsequently, the software StreamPix 6 (Phoenix Research Laboratories) was utilised to acquire the fundus retinal photographs for each eye once the camera had been centred on the optic nerve head.

Immediately after fundus photography, the OCT imaging technique was used. The SD‐OCT (Micron IV; Phoenix Research Laboratories) was employed. The retina was visualised by contacting the OCT lens to the gel. StreamPix 6 software, version 7.2.4.2 (Phoenix Research Laboratories) was used in image acquisition. The SD‐OCT ultra‐broadband (160 nm) light source was used to obtain 1024 pixels per A‐scan and 10 frames per horizontal B‐scan with 2‐μm axial resolution. The retina was serially scanned for the presence of classic signs of nonproliferative DR including hard exudates, microaneurysms and dot/blot haemorrhages in the diabetic rat retina, according to guidelines for monitoring human diabetic changes in the eye (Ministry of Health, 2016).

2.4. Immunohistochemistry

At the conclusion of the last SD‐OCT, rats were deeply anaesthetised via intraperitoneal injection of Ketamine and Domitor. Eyes were enucleated, processed and sectioned as previously described (Mat Nor et al., 2020). Briefly, superior/inferior, nasal/temporal coordinates of the retina were maintained during the dissection and the cornea and lens were removed and the eyecup was fixed in 4% paraformaldehyde for 30 min followed by a wash in 0.1 M phosphate‐buffered saline (PBS), pH 7.4. The eyecups were immersed in sequential 10% and 20% sucrose solutions every 30 min and lastly in 30% sucrose at 4°C overnight. The location of dot HRF in the dissected retina was determined through a combination of methods. First, the location of the HRF was identified in a SD‐OCT scan. Then, the location was estimated by mapping the SD‐OCT coordinates onto the fundus image. Finally, the location was extrapolated to the dissected eyecup to confirm its position in the tissue. To further evaluate the HRF, 1 mm of the retinal area enclosing the HRF was dissected, embedded in optimum cutting temperature compound (SakuraFinetek), serially sectioned at 12 μm thickness in the vertical plane to the retina using a Leica CM3050 S cryostat (Leica). Sections of the retina were collected using Superfrost Plus Slides (Labserv), and the slides were stored at −20°C for subsequent immunohistochemistry using a microglial marker (mouse anti‐ionised calcium‐binding adapter molecule‐1, iba‐1; 1:250, Cat Ab5076, Abcam) and cell stress (mouse anti‐glial fibrillary acidic protein, GFAP; 1:1000, Cat C9205, Sigma‐Aldrich) markers and processed using standard immunolabelling techniques (Mat Nor et al., 2020). Briefly, the immunohistochemical labelling was conducted by using the indirect immunofluorescence technique. Tissue sections were blocked with a solution containing 6% normal goat serum, 1% bovine serum albumin (BSA) and 0.5% triton X‐100 in 0.1 M PBS for 1 h at room temperature. The primary antibodies were prepared and diluted in 3% normal goat serum, 1% BSA and 0.5% triton X‐100. The primary antibodies were applied overnight at room temperature in a volume sufficient to cover the tissue. Slides were then washed in 0.1 M PBS four times for 5 min each. The secondary antibody, goat anti‐rabbit and goat anti‐mouse conjugated with Alexa TM 488 or Alexa TM 594 was diluted 1:500 in the primary antibody buffer and applied to the sections for 3 h at room temperature. After the incubation period, the sections were washed four times for 15 min each in 0.1 M PBS in order to remove unbound antibodies. In the last 15‐min wash, sections were incubated with 4′,6‐diamidino‐2‐phenylindone (Sigma‐Aldrich) prepared as 1 μg/mL in 0.1 M PBS in order to stain the cell nuclei. Slides were mounted in a non‐fluorescence‐fading medium (Citifluor Ltd) and coverslips were sealed with nail polish to avoid leakage and evaporation of the mounting medium. For imaging, a confocal laser scanning microscope, Olympus FluoView FV1000 (Olympus Corporation) with excitation 473 and/or 559 nm wavelength was used. A series of four to eight optical slices at 1 μm intervals were collected through each specimen and image analysis was performed on average intensity projection images using plain ImageJ software (National Institutes of Health; Schneider et al., 2012).

2.5. Data analysis

The number of Iba‐1‐labelled cells corresponding to microglia in normal S‐D retinas and hyperglycaemic retinas was counted per unit area in at least six retinas per group. Morphological assessment confirmed activation status as reported in human DR stages (Zeng et al., 2008). The reactivity of GFAP was measured as percentage reactivity per unit area in normal retina and areas associated with HRF. The immunohistochemical data were statistically analysed using one‐way repeated‐measures analysis of variance (ANOVA) and a Bonferroni post hoc test with an alpha value of 0.05. ERG data in the hyperglycaemic model compared with control normal rats were analysed using a two‐way ANOVA followed by a Bonferroni post‐test to compare the effects of stimulus intensity. Graphing and statistical analyses were performed using GraphPad Prism 9 (GraphPad Software). All data are presented as the mean ± the standard error of the mean.

3. RESULTS

HRF were observed in the OCT images of the retina of animals with hyperglycaemia as early as 4 weeks of age (Mat Nor et al., 2020). Although in humans most blood vessel problems are seen in a fundus image, this is not the case for the albino rat model. One important feature of albino rats is that the lack of pigmentation in the eye can mask or conceal blood leakage making it more difficult to detect vascular damage with fundus examination alone (Figure 1a,b). However, OCT clearly shows these areas of vascular damage (Figure 1c,d) as round well‐demarcated isolated intraretinal dot HRF. We did not observe the simultaneous occurrence of HRF in both the INL and ONL of a single animal among the hyperglycaemic rats studied. In some animals, HRF accompanied lesions of 100–200 μm in the outer plexiform layer‐INL intersection. These big HRF cast a shadow in the OCT scan suggesting these are enlarged blood vessel and were surrounded by small HRF (arrows in Figure 1d). Small isolated dot HRF were also observed in the ONL. We then isolated those areas with HRF by consecutive and serial sectioning and immunolabelling.

FIGURE 1.

FIGURE 1

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Hyperglycaemic rat model showing dot hyper‐reflective foci in OCT scans. (a, b) Fundus images do not reveal obvious vascular changes in the albino rat. The white circles in (a, b) indicate the location of the HRF observed in the OCT images. (c) OCT image corresponding to the green line in (a) shows a hyper‐reflective dot in the outer retina (red arrow). See inset (d). OCT image corresponding to the green line in (b) shows a large blood vessel (white arrow); note the shadowing and elevation of the surface of the retina over it and small hyper‐reflective dots above it (red arrows). Scale bar 100 μm. GCL, ganglion cell layer; INL, inner nuclear layer; ONL, outer nuclear layer; RPE, retinal pigmented epithelium.

Both control S‐D rats and hyperglycaemic rats were screened for markers of inflammation using immunohistochemistry techniques. In Figure 2a,b, we show the fundus image and corresponding OCT of a big HRF surrounded by small dot HRF. The big HRF was histologically identified as an enlarged blood vessel in a DAPI‐stained section (Figure 2c), but there were no obvious anatomical distinctions for the small HRF. These enlarged blood vessels are reported to trigger an inflammatory response elicited by serum exuding from leaky vessels (Bolz et al., 2009). Therefore, retinal stress and inflammatory response were analysed. GFAP labelling was used to detect gliosis or Müller cell hyper‐reactivity in this area. GFAP is a protein that is commonly found in astrocytes and at very low levels in Müller cells. However, Müller cells express large quantities of GFAP in response to various insults such as retinal injury or ocular inflammation, with GFAP expression becoming visible in an immunohistochemical reaction. The expression of GFAP in Müller cells is considered a marker of retinal injury. GFAP upregulation in Müller cells was detected as thin, elongated filamentous structures that run down the length of the Müller cell processes perpendicular to and extending beyond the normal astrocyte reactivity seen in the nerve fibre layer. There is also a thickening of the GFAP label pattern in the nerve fibre layer, indicative of new reactivity in Müller cell end feet (Figure 2d). We observed an increase in GFAP reactivity from the nerve fibre layer up to the INL in hyperglycaemic rats (Figure 2d). Quantification of the GFAP labelling in HRF areas and non‐HRF normal areas is shown in Figure 1f, indicating that GFAP hyper‐reactivity is a localised event related to HRF. In addition, we labelled sections for microglia using Iba‐1 and found that around the HRF areas, microglia in the inner retinal layers were characterised by enlarged soma and numerous elongated branches, in the inner retina (Figure 2e). Morphological alterations in response to injury or inflammation are highly stereotyped, and in this model, the initial response of microglia to hyperglycaemia appears to be rapid process extension and reorientation within the IPL. In HRF areas, microglial cells had an increased soma size and the number of process branches per microglia was higher. In comparison, normal S‐D rats immunolabelled as control tissue in previous publications never showed this level of Iba‐1 labelling and activation in the retina or choroid (Mat Nor et al., 2020). The Iba‐1 labelling also detected activated microglia in discrete areas in the choroid; however, we do not know whether these are related to the HRF in inner retina. We then conducted further histological examination of animals with single dot HRF in the ONL (Figure 3). The OCT image showed that in comparison with normal S‐D rats (Figure 3a), the hyperglycaemic rat retina showed several dot HRF (two HRF seen in Figure 3b). Microglia had a normal resting morphology in the control retina (Figure 3a′). Immunolabelling using Iba‐1 on serial sections corresponding to the isolated HRF area, showed an increase in the number of activated microglia with branching morphology in the inner retina (Figure 3b′). A moderate increase in microglial cells labelled by the Iba‐1 marker was mostly seen in the inner retinal layers (Figure 3c). We determined that these were activated microglia as they appeared hypertrophic and showed numerous nodular processes and labelling intensity of the microglia were markedly increased. To determine whether the presence of dot HRF causes changes to retinal activity, we conducted fERGs (Figure 4). For this, dot HRF were first identified in hyperglycaemic rats using OCT (Figure 4a), the area was further located in the fundus image, and the function of the retina was assessed using fERG. Recordings were collected from light‐stimulated HRF areas (Figure 4c) and from areas within 1 OD—distance from the HRF (Figure 4b). The fERG waveform was also collected from normal retinas. The ERG response over HRF areas showed a reduction in both a‐wave (Figure 4d) and b‐wave (Figure 4e) deflection of the ERG when compared with normal control (p < 0.05). Adjacent areas to the dot HRF also displayed attenuated b‐wave of the ERG compared with control animals (Figure 4e).

FIGURE 2.

FIGURE 2

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Big and small hyper‐reflective foci observed in the OCT images. (a) Fundus imaging does not reveal obvious changes. (b) OCT image corresponding to the green line in (a) shows a 100 μm thick lesion in inner retina. (c) Section of the HRF area in B and staining with DAPI defines an enlarged capillary lumen area (asterisk) distorting the INL. (d) Immunohistochemical labelling with anti‐glial fibrillary acidic protein (GFAP) shows stress areas (white encircled area) around the vascular lesion. (e) Shows increased labelling of inflammatory cells (Iba‐1, white encircled areas) around the location of the HRF lesion and in the RPE‐choriocapillaris interface. The asterisks in (c)–(e) show the histological appearance of a large HRF in (b), corresponding to an enlarged blood vessel. (f) Quantification of GFAP labelling. Scale bar 100 μm.

FIGURE 3.

FIGURE 3

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Histological examination of the small dot HRF in the hyperglycaemic rat model. (a) Retinal area without HRF does not show microglial activation in the corresponding retinal section (a′). (b) OCT image showing two dot HRF in outer retina (arrows). (b′) Retinal section of the area of HRF in (b) shows an increased number of branched microglia (Iba‐1) in the inner retina. (c) Quantification of Iba‐1 labelling. Scale bar 30 μm. INL, inner nuclear layer; IPL, inner plexiform layer; ONL, outer nuclear layer; RPE, retinal pigmented epithelium.

FIGURE 4.

FIGURE 4

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Retinal function on the dot HRF areas in the hyperglycaemic rat model. (a) OCT scan showing several dot HRF in the ONL and INL (red arrows), two of them visible surrounding a blood vessel in the GCL. (b) fERG results (blue line, average value) of areas adjacent to the HRF. Black double arrow indicates differences in a‐wave and in the b‐wave values compared with normal retina. (c) fERG results on HRF areas (blue line is average result) showing reduced a‐wave and b‐wave (indicated by double arrow) compared with normal retina. (d) a‐wave of the ERG is unaffected in low light levels compared with normal retinal areas but is significantly reduced in dot HRF areas when high light stimulus is used. (e) b‐wave of the ERG is significantly reduced in both HRF and HRF‐adjacent retinal areas compared with normal areas. The asterisk (*) denotes that a post hoc analysis revealed significant differences in fERG function between normal area and the HRF area, with a p value <0.05. GCL, ganglion cell layer; INL, inner nuclear layer; ONL, outer nuclear layer.

4. DISCUSSION

In humans, it has been proposed that HRF are linked to early signs of diabetic eye disease, present even before clinical retinopathy is detectable (Turgut & Yildirim, 2015; Vujosevic et al., 2013). Hyper‐reflective dots in an OCT scan have also been associated with exudates in macular oedema, AMD and hypertension (Coscas et al., 2013; Lammer et al., 2014). Using serial sectioning and an immunohistological approach we have been able to correlate HRF in OCT scans of the retina with microglial activation, evidence for the first time that dot HRF are indicative of an underlying inflammatory problem, where single small dot HRF are proposed to correspond to those HRF lesions seen in humans (Turgut & Yildirim, 2015; Vujosevic et al., 2013). Moreover, the literature suggests that dot HRF may be key clinical indicators for a range of conditions (Brand, 2012). Their number has been used as indicators of the effectiveness of treatment before and after anti–vascular endothelial growth factor injection into the eyes of patients with macular oedema (Li et al., 2021).

Small hyper‐reflective lesions have rarely been described in animal models. In DR models, the large HRF lesions have been variably identified as microaneurysms, microvascular lesions and hard exudates (Jiang et al., 2015; Lai & Lo, 2013; Robinson et al., 2012). In the hyperglycaemic model presented here, it has been possible for the first time to evaluate the histopathological significance of the smaller dot HRF. We have demonstrated that these small HRF can be identified by size and retinal location. The solitary, small dot (<30‐μm diameter) HRF are mainly located in the outer retina and inner retina. They do not correspond to any specific lesion on fundus images but are indicative of a focal inflammatory response. OCT and immunohistochemistry demonstrated that small dot HRF observed adjacent to large blood vessels may be associated with extravasated lipoproteins and proteins and indicate a localised inflammatory reaction (Bolz et al., 2009). In a previous publication (Mat Nor et al., 2020) we showed that some of the HRF were leaky microaneurysms located close to the retinal capillary plexus and that these elicited retinal stress.

Although there are several hypotheses for the development of HRF, we have provided support for the theory that could be lipid exudates/deposits due to the breakdown of the blood–retinal barrier (Vinores et al., 1999), eliciting microglial activity. Bolz et al. (2009) indicated that this is lipoprotein extravasation that could act as precursors of hard exudates when in the outer retina. Zeng et al. (2008) described in the human exudative retinopathy that microglia clustered around microaneurysms and intraretinal haemorrhages, and infiltrated OPL and ONL, whilst in diabetic macular oedema, proliferating microglia extended throughout the retina and sub‐retinal space.

Whether HRF are due to the breakdown of the blood–retinal barrier or due to the accumulation of microglia, there is evidence to suggest that they are markers of early inflammatory states in the retina and may be correlated with the development of HRF. In a study of patients with macular oedema related to DR or retinal vein occlusion, the levels of inflammatory factors (IL‐8) and vascular cell adhesion molecule‐1 in the aqueous humour were analysed and the numbers of HRF shown by SD‐OCT. There was a marked correlation between the presence of HRF and the levels of inflammatory factors (Li et al., 2021). Studies have also shown that inflammatory markers such as cytokines and chemokines are elevated in the retina of individuals with HRF in the brain. These markers may contribute to the development of HRF through their effects on the small blood vessels in the brain. One proposed mechanism for the link between retinal inflammation and HRF involves damage to the blood–retinal barrier, which can allow inflammatory cells and molecules to enter the bloodstream and travel to the brain. Inflammation in the retina may be related to the development of other conditions that are associated with HRF, such as hypertension and diabetes. Conversely, inflammation in the brain may well lead to elevated inflammatory markers in the retina and lead to the onset of retinal HRF and retinal disease.

Not all small HRF in the inner retina may be associated with blood vessels (when in the middle of the ONL or INL), but we have shown that they nevertheless corresponded with an inflammatory response and were associated with reduced focal retinal function. Reduced inner retinal function and photoreceptor function were both detected over the HRF areas. This was done using fERG, a technique that when showing abnormality has been proposed to predict the location of future microaneurysms according to McAnany et al. (2022).

5. CONCLUSION

We have provided evidence that a single small dot HRF corresponds to an area with clusters of activated microglia. This has implications for the monitoring of treatment effectiveness in individual patients or monitoring novel treatment efficacy. More importantly, HRF may be a useful clinical biomarker for inflammatory reactions in patients at an early stage of disease development, prior to more severe clinical signs becoming apparent (De Benedetto et al., 2015; Niu et al., 2017; Vujosevic et al., 2017).

AUTHOR CONTRIBUTIONS

Monica L. Acosta, David Squirrell and Colin R. Green contributed to concept/design; Mohd N. Mat Nor and Cindy X. Guo involved in the acquisition of the data; all authors contributed to data analysis/interpretation; Mohd N. Mat Nor drafted the manuscript; Monica L. Acosta, David Squirrell and Colin R. Green applied the critical revision of the manuscript and approval of the article.

CONFLICT OF INTEREST STATEMENT

The author reports no conflicts of interest in this work.

ACKNOWLEDGMENTS

We thank the New Zealand Optometric Vision Research Foundation (NZOVRF) for the funds received for this project. Open access publishing facilitated by The University of Auckland, as part of the Wiley ‐ The University of Auckland agreement via the Council of Australian University Librarians.

Mat Nor, M.N. , Guo, C.X. , Green, C.R. , Squirrell, D. & Acosta, M.L. (2023) Hyper‐reflective dots in optical coherence tomography imaging and inflammation markers in diabetic retinopathy. Journal of Anatomy, 243, 697–705. Available from: 10.1111/joa.13889

DATA AVAILABILITY STATEMENT

The data that support the findings of this study are openly available within the manuscript and further details can be requested from the corresponding author.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Data Availability Statement

The data that support the findings of this study are openly available within the manuscript and further details can be requested from the corresponding author.

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