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Lycium barbarum glycopeptide promotes neuroprotection in ET-1 mediated retinal ganglion cell degeneration

Journal of Translational Medicine volume 22, Article number: 727 (2024) Cite this article

Abstract

Background

Vascular dysregulation is one of the major risk factors of glaucoma, and endothelin-1 (ET-1) may have a role in the pathogenesis of vascular-related glaucoma. Fruit extract from Lycium Barbarum (LB) exhibits anti-ageing and multitarget mechanisms in protecting retinal ganglion cells (RGC) in various animal models. To investigate the therapeutic efficacy of LB glycoproteins (LbGP) in ET-1 induced RGC degeneration, LbGP was applied under pre- and posttreatment conditions to an ET-1 mouse model. Retina structural and functional outcomes were characterised using clinical-based techniques.

Methods

Adult C57BL/6 mice were randomly allocated into four experimental groups, namely vehicle control (n = 9), LbGP-Pretreatment (n = 8), LbGP-Posttreatment (day 1) (n = 8) and LbGP-Posttreatment (day 5) (n = 7). Oral administration of LbGP 1 mg/Kg or PBS for vehicle control was given once daily. Pre- and posttreatment (day 1 or 5) were commenced at 1 week before and 1 or 5 days after intravitreal injections, respectively, and were continued until postinjection day 28. Effects of treatment on retinal structure and functions were evaluated using optical coherence tomography (OCT), doppler OCT and electroretinogram measurements at baseline, post-injection days 10 and 28. RGC survival was evaluated by using RBPMS immunostaining on retinal wholemounts.

Results

ET-1 injection in vehicle control induced transient reductions in arterial flow and retinal functions, leading to significant RNFL thinning and RGC loss at day 28. Although ET-1 induced a transient loss in blood flow or retinal functions in all LbGP groups, LbGP treatments facilitated better restoration of retinal flow and retinal functions as compared with the vehicle control. Also, all three LbGP treatment groups (i.e. pre- and posttreatments from days 1 or 5) significantly preserved thRNFL thickness and RGC densities. No significant difference in protective effects was observed among the three LbGP treatment groups.

Conclusion

LbGP demonstrated neuroprotective effects in a mouse model of ET-1 induced RGC degeneration, with treatment applied either as a pretreatment, immediate or delayed posttreatment. LbGP treatment promoted a better restoration of retinal blood flow, and protected the RNFL, RGC density and retinal functions. This study showed the translational potential of LB as complementary treatment for glaucoma management.

Background

Glaucoma is one of the world’s leading causes of irreversible blindness [1], characterised by retinal ganglion cell (RGC) death [2]. Damage to RGCs can result from many factors, including raised intraocular pressure (IOP) levels [3,4,5,6,7], vascular insufficiency [8,9,10,11], neurotoxicity [12,13,14], neurotrophic factor deprivation [15,16,17], inflammation [18,19,20] and dysregulation of pro-apoptotic, pro-survival or neuroprotective signalling pathways [21,22,23]. Despite its complex aetiology, glaucoma is mainly managed by lowering IOP [3,4,5,6,7]. This may explain why a subset of the treated population continues to lose useful vision even though target IOP levels are attained [24]. To address this issue, a decades-long, ongoing expedition is underway to identify therapies that target one or more IOP-independent mechanisms [25,26,27]. Whilst some of these approaches have shown preclinical success, none are yet available for clinical use [28,29,30].

Vascular dysregulation is one of the major risk factors for glaucoma [11, 31, 32]. The vasoconstrictor peptide endolthelin-1 (ET-1) and its G-protein coupled receptors, namely endothelin receptor A (ET-A) and endothelin receptor B (ET-B), are present abundantly in ocular structures. They play important roles in regulating IOP [33, 34], ocular blood flow [35,36,37] and neuronal functions [38]. Dysregulation of ET-1 and its receptors has been associated with clinical glaucoma [37, 39,40,41,42]. Elevated ET-1 levels in plasma [37, 41, 43, 44], aqueous humour [36, 45, 46] or following coldinduced vasospasm [47, 48] have been reported in patients with normal tension or open angle glaucoma. Experimental studies also showed that retinas exposed to high levels of ET-1 resulted in a transient reduction in blood flow and significant RGC loss [49,50,51]. Recently, our team reported that intravitreal ET-1 injection induced a transient reduction in ocular blood flow, leading to RNFL thinning, RGC and retina functional loss in a dose-dependent manner [52]. As the induced effects on the retina overlap with the clinical presentation of normal tension glaucoma, it opens up the possibility of using the ET-1 induced RGC neurodegeneration model as a platform to test the neuroprotective effects of therapeutic agents against vascular dysregulation. Some studies applied vascular-targeting therapies such as ET receptor antagonist [53,54,55], vasodilators [56, 57] and drugs that inhibit vascular oxidative stress and inflammation [58,59,60] to ET-1 and ocular hypertension model, and have shown RGC neuroprotection.

Lycium Barbarum (LB) is a traditional Chinese medicinal herb, largely consumed to improve physical well-being, promote mental health, increase longevity, and treat ailments [61,62,63,64]. Polysaccharides extracted from the fruits of LB exhibits neuroprotective properties in a range of experimental neurodegeneration models [65,66,67,68,69,70,71]. LB pretreatment, which always commences one week before the onset of injury, was able to protect RGCs in various models of injuries, including rodent models of experimental glaucoma [72,73,74,75,76], acute ocular hypertension [77,78,79,80], optic nerve transection [81,82,83,84] and ischemic reperfusion injuries [85]. While recent studies demonstrated the posttreatment neuroprotective effects of LB in IOP-dependent models [76, 79], with treatments initiated after the onset of injury, its protective effects in non-IOP-related glaucoma models remain largely unknown. Given the therapeutic effects of LB in improving the tissue perfusion [86, 87], regulating ET levels and its receptors [75], combating reperfusion-induced oxidative stress [78, 85] and inflammations [77, 85, 88], and protecting RGCs in IOP-dependent models [89], it holds great promise in protecting RGCs against ET-1 induced neurodegeneration.

In this study, we investigated the therapeutic efficacy of LB in the non-IOP-related, ET-1 mouse model of RGC degeneration. Purified glycoproteins extracted from LB glycoproteins (LbGP) was applied to treat ET-1 mice in both pre-and posttreatment manners. As ET-1 induced RGC degeneration is a slow, progressive process, wherein clinically significant RNFL loss occurs at day 28 [52], it offers a wide timeframe for the investigation of the therapeutic efficacy of LbGP and how LbGP, administered at different time points over the course of neurodegeneration, impacts the treatment outcome. Treatment effects on different retinal layers and functions were examined using clinically relevant, in vivo retinal structure-function tools. RGC survival was evaluated using a histological approach.

Methods

Animals

Adult C57BL/6 mice aged 8–10 weeks were purchased from the centralized animal facility services of The Hong Kong Polytechnic University and housed in a centralized animal facility at a room temperature of 20ºC under a 12-hour light/dark cycle. Animals received food (PicoLab diet 20 (5053); PMI Nutrition International, Richmond, IN, USA) and water ad libitum. All experimental procedures and care involving animals were approved by the Animal Ethics Sub-committee of The Hong Kong Polytechnic University and conformed with the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research.

Experimental design

All animals underwent baseline examinations of retinal structure, blood flow and function using spectral domain optical coherence tomography (SD-OCT), doppler OCT and electroretinogram. 40 animals were then randomly allocated into 4 treatment (Tx) groups, namely vehicle control, LbGP-PreTx, LbGP-PostTx (day 1) and LbGP-PostTx (day 5). At day 0, animals received intravitreal injections of ET-1 (1 nmol dosage) in one eye and PBS in the other eye to serve as the fellow sham control. The animals were fed daily once either with PBS for vehicle control or 1 mg/kg of LbGP mixed in PBS for LbGP treatment groups, via oral gavage. The treatment was commenced on day − 7 (i.e. 7 days prior to the injections) for the LbGP-PreTx group, day 1 for the vehicle control and LbGP-PostTx (day 1) groups, and day 5 for the LbGP-PostTx (day 5) group. The treatments were continued up to the end of the experiment at day 28. All animals underwent follow up examinations of ERG, OCT and doppler OCT at post-injection days 10 and 28. At the end of the experiment, animals were sacrificed by exposure to carbon dioxide gas until complete cessation of breathing was observed for a minimum of 2 minutes. Visual inspection for the absence of movement and respiration, and confirmation of a lack of heartbeat were performed. The eyes were then collected for retinal whole mounts for further quantifications of RGC survival. Carcasses were disposed of in a hygienic and ethical manner, according to the regulations of the University’s Centralised Animal Facilities. Briefly, carcasses were placed in designated clinical waste bags and stored in the freezer, which were later disposed of by the university’s animal facility service using an incinerator.

Intravitreal injection of ET-1

For intravitreal injection, mice were anaesthetized with an intraperitoneal injection of 100 mg/kg of ketamine (Alfasan International B.V., Woerden, Holland) and 20 mg/kg of xylazine (Alfasan International B.V.). The eyes were then dilated using Mydrin-P eye drops (Santen Pharmaceutical Co, Ltd, Osaka, Japan). A drop of short-term anaesthetic for the ocular surface (Provain-POS 0.5%w/v eye drops; URSAPHARM, Saarbrucken, Germany) was also applied. The cornea was kept hydrated using a lubricating gel (Lacryvisc gel, Alcon, Rueil-Malmaison, France). After making an incision at the supero-temporal quadrant of the conjunctiva, another small incision just behind the limbus was made into the vitreous using a 30-gauge needle. A clean cotton tip was gently pressed against the incision site for 10 s. Intravitreal injection was then performed using a 5 µL Hamilton syringe (Hamilton Company, 7633–01) with a blunt, 33-gauge needle. The syringe was introduced at a 45-degree angle. 2 µL of PBS (for the sham group) or ET-1 (Sigma, E7764) in PBS, at a concentration of 500 µM/µL (1.0 nmol), was slowly injected into the vitreous over the course of 2 min. After completing the injection, the needle was held in place for 30 more seconds before being slowly removed. Gentamycin antibiotic eye drops (Gibco, Thermo-Fisher Scientific) were applied after the procedure. Mice with moderate back flow following injection (n = 1), lens injury (n = 3), vitreous haemorrhage (n = 1) and lost during follow up (n = 2) were excluded from the study. Mice included for final analysis were 8 in vehicle control, 9 each in LbGP-PreTx, LbGP-PostTx (day 1) and 7 in LbGP-PostTx (day 5).

Electroretinogram

A full-field Ganzfeld (Q450; RETI Animal, Roland Consult, Brandenburg an der Havel, Germany) with light-emitting diode light source was used to measure the positive scotopic threshold response (pSTR), scotopic b- and a-wave from ganglion, bipolar and photoreceptor cells respectively. The recording protocol was described in detail elsewhere [79]. Under dim red light, overnight dark-adapted animals were anesthetized using a ketamine-xylazine mixture. Pupils were dilated and the cornea was kept hydrated through application of lubricating gel. Two needle electrodes were inserted into the base of the tail and the scalp to serve as the ground and the reference. A pair of gold ring electrodes were placed on the corneal surface to serve as the active electrodes. Impedance was below 10 KΩ for all recordings. All procedures were undertaken under dim red light. The animal platform was connected to a hot water bath of 37oC. pSTR responses were first measured at very low light densities that ranged from − 4.8 to -4.2 log U with an interstimulus interval (ISI) of 2 s, and 30 responses were averaged per step. This was followed by measuring scotopic response using a bright light of 1.3 log U. The amplitudes and implicit times of pSTR (-4.35 log U), b- and a-wave responses were extracted for further analysis.

Optical coherence tomography

All animals underwent SD-OCT imaging immediately after ERG and were therefore under the same anaesthetic and mydriatic effects. A 0.8 mm diameter annular B-scan consisting of 1000 A-scans, with a 5-frame averaging, was performed. Manual segmentation of the retinal layers was carried out later using the FIJI software (https://imagej.net/software/fiji/). The peripapillary retinal thicknesses of retinal nerve fibre layer (RNFL), inner retinal layer (IRL; including the inner plexiform and the inner nuclear layers), and outer retinal layer (ORLT; including the outer plexiform layer and the RPE) were quantified as described previously [52].

Following OCT scan, doppler OCT using annular B-scan of 0.5 mm diameter (1000 A scans, Envisu R2210, Bioptigen, Morrisville, NC, USA) was performed. The scan allowed the direct visualization of retinal blood flow, wherein the arterial and venular circulation were represented in red or blue pixels respectively. The colour threshold algorithm available in FIJI software (https://imagej.net/software/fiji/) was then used to quantify the number of red and blue pixels in the RNFL, serving as surrogate measurements of the retinal arterial (red pixels) and venous (blue pixels) calibres and their flows as described previously [52]. For final analysis, the measurement of arterial or venular pixels numbers from ET-1 eyes were normalised to their fellow sham eyes (ET-1 eye/ fellow sham), which were then compared among treatment groups.

Retinal whole mount

Animals were sacrificed by CO2 asphyxiation. Eyes were immediately collected and fixed in 4% PFA at 4ºC for 20 min. Retinal whole mounts were prepared under a dissection microscope and incubated in blocking buffer (10% normal goat serum (NGS), 1% bovine serum albumin (BSA), 0.05% sodium azide, 0.5% Triton X-100 in PBS) for 1 h at room temperature on a shaker. The samples were then incubated in primary antibodies against RBPMS (rabbit, 1:500, 1830, PhosphoSolutions) for 5 days at 4ºC, followed by PBS washes and further incubation in secondary antibody (goat anti-rabbit, Alexa Flour 488, 1:1000, ab150077, abcam) at 4ºC overnight. All antibodies were diluted in a buffer solution containing 3% NGS, 1% BSA, 0.05% sodium azide, 0.5% Triton X-100 in PBS. After washing the retinas in PBS, samples were mounted onto microscopic slides for imaging using a confocal laser scanning microscope (LSM800, Carl Zeiss). Three regions of 250 μm x 250 μm from the central, middle and peripheral retina were randomly selected from each quadrant. These regions were taken from 1/4, 2/4 and 3/4 the distance from the optic nerve head to peripheral edge respectively. Automated RGC counting was then performed using SimpleRGC, a FIJI plugin [90]. The central, middle and peripheral RGC counts were calculated by averaging the corresponding values from all quadrants.

Analysis

Data collection and analysis were performed in a blinded manner. Data were expressed as mean ± standard deviation (SD) and all analyses were performed using SPSS 26.0 (IBM Corp., Armonk, NY, USA). Relative changes in OCT, ERG and RGC data in ET-1 injected eyes were defined as the percentage change from the fellow sham eye. Then, mixed model ANOVA with Bonferroni post hoc correction was used to compare the difference within (time main effect) and among (Tx main effect) and the four treatment groups over time. Also, two-way repeated measure (RM) ANOVA was used to compare the treatment outcomes between ET-1 and fellow sham eyes over time for each experimental group. For RGC density analysis, one-way ANOVA with Bonferroni post-hoc test was used to find the difference among the treatment groups.

Results

LbGP treatment facilitated earlier restoration of retinal blood flow in ET-1 injected eyes

Following ET-1 injection, all retinas showed an arterial constriction. This observation was irrespective of treatment conditions (vehicle or pretreatment with LbGP, see Additional File.1 showing arterial constriction). For ET-1 injected eyes, the normalized arterial blood flow showed both significant temporal and treatment differences (time main effect: P = 0.001; Tx main effect: P = 0.04; interaction effect: P = 0.92). As for the normalized venous blood flow, a significant temporal difference was detected (time main effect: P = 0.02). Figure 1 shows the representative doppler B-scans of each experimental group imaged at baseline, and post-injection days 10 and 28. The arterial (Fig. 2a) and venous (Fig. 2b) blood flows of ET-1 and fellow sham eyes as well as the corresponding normalized blood flow (Fig. 2c, d) are presented in Fig. 2.

Fig. 1
figure 1

ET-1 induced a transient constriction in retinal vessels. a-e Representative images of annular doppler B-scans from one mouse per group, showing temporal changes in vessel calibre / blood flow (red and blue pixels represent arterial and venous flow respectively), captured at baseline, post-injection days 10 and 28 for (a) PBS injected sham eye, (b-e) ET-1 injected eyes treated with (b) vehicle control, (c) LbGP-PreTx, (d) LbGP-PostTx from day 1, and (e) LbGP-PostTx from day 5 groups

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Fig. 2
figure 2

LbGP treatment facilitated better restoration in arterial flow following ET-1 injection. a-b Mean (a) arterial, and (b) venous flows measured at baseline, post-injection days 10 and 28 from ET-1 injected eyes and their fellow sham eyes are presented for groups treated with vehicle control (n=8), LbGP-PreTx (n=9), LbGP-PostTx from day 1 (n=9) and LbGP-PostTx from day 5 (n=7). All data are presented as the mean ± SD and were analysed using RM ANOVA followed by Bonferroni post-hoc test. Each circle in the bar chart represents an individual data point. * p<0.05 when compared with baseline, † p<0.05 when compared with day 10, ǂ p<0.05 when compared with fellow sham. c-d Percentage changes from baseline are presented for (c) arterial, and (d) venous blood flows following ET-1 injection and were compared among groups treated with vehicle control (n=8), LbGP-PreTx (n=9), LbGP-PostTx from day 1 (n=9) and LbGP-PostTx from day 5 (n=7). All data are presented as the mean ± SD and were analysed using mixed model ANOVA followed by Bonferroni post-hoc test. * p<0.05 when compared with baseline, † p<0.05 when compared with day 10, ǂ p<0.05 when compared with LbGP-PreTx

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ET-1 injection reduced the arterial calibre, per se the arterial flow, in all treatment groups until day 10 (Fig. 2d), and the reduction was statistically significant in the vehicle control group (P = 0.04 as compared with baseline). At day 28, the recovery in arterial calibre/flow of all LbGP treatments was better than the vehicle control. As for the venous calibre/ flow, a reduction was detected in the LbGP-PreTx (P = 0.01 as compared with baseline) and LbGP-PostTx day 5 (P = 0.15 as compared with baseline) groups at day 10, which was maintained at similar levels at day 28. On the other hand, the LbGP-PostTx day 5 showed an increase in venous calibre/flow at day 28 (P = 0.01 as compared with day 10) (Fig. 2d).

LbGP treatment retained the thickness of RNFL in ET-1 injected eyes

The retinal layer thicknesses of ET-1 injected eyes receiving vehicle, LbGP pre- and posttreatments (day 1 or 5) were compared. For RNFL thickness (RNFLT), a significant difference between the treatment groups was detected over time (interaction effect: P = 0.03). The other retinal layer thicknesses, including IRLT (time main effect: P = 0.001; Tx main effect: P = 0.08; interaction effect: P = 0.36), ORLT (time main effect: P = 0.001; Tx main effect: P = 0.52; interaction effect: P = 0.53) and TRT (time main effect: P = 0.001; Tx main effect: P = 0.25; interaction effect: P = 0.49), all showed a significant temporal difference. Figure 3 presents the representative OCT B-scan images of each treatment group at baseline and post-injection days 10 and 28. The retinal layer thicknesses of the four treatment groups are presented in Fig. 4. Temporal changes in the retinal layer thicknesses compared among treatment groups are shown in Fig. 5.

Fig. 3
figure 3

LbGP treatment protected the retina from ET-1 induced RNFL thinning. a-e Representative images of SD-OCT from one mouse per group, showing cross sections of retinal layers captured at baseline, postinjection days 10 and 28 for (a) PBS injected sham eye, (b-e) ET-1 injected eyes treated with (b) vehicle control, (c) LbGP-PreTx, (d) LbGP-PostTx from day 1, and (e) LbGP-PostTx from day 5

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Fig. 4
figure 4

LbGP treatment preserved the RNFL thickness following ET-1 injection. a-d Mean peripapillary retinal thicknesses of (a) RNFL, (b) IRL, (c) ORLT, and (d) TRT quantified at baseline, postinjection days 10 and 28 from ET-1 injected eyes and their fellow sham eyes are presented for groups treated with vehicle control (n=8), LbGP-PreTx (n=9), LbGP-PostTx from day 1 (n=9) and LbGP-PostTx from day 5 (n=7). All data are presented as the mean ± SD and were analysed using RM ANOVA followed by Bonferroni post-hoc test. Each circle in the bar chart represents an individual data point. * p<0.05 when compared with baseline, † p<0.05 when compared with day 10, ǂ p<0.05 when compared with fellow sham. RNFL, retinal nerve fibre layer; IRL, inner retinal layer; ORL, outer retinal layer and TRT, total retinal thickness

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Fig. 5
figure 5

LbGP treatment prevented RNFL loss following ET-1 injection. a-d Percentage losses from baseline for the retinal layer thickness of (a) RNFL, (b) IRL, (c) ORLT, and (d) TRT following ET-1 injection. They were compared among groups treated with vehicle control (n=8), LbGP-PreTx (n=9), LbGP-PostTx from day 1 (n=9) and LbGP-PostTx from day 5 (n=7). All data are presented as the mean ± SD and were analysed using mixed model ANOVA followed by Bonferroni post-hoc test. * p<0.05 when compared with baseline, † p<0.05 when compared with day 10, ǂ p<0.05 when compared with LbGP-PreTx. RNFL, retinal nerve fibre layer; IRL, inner retinal layer; ORL, outer retinal layer and TRT, total retinal thickness

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For RNFLT, ET-1 injected eyes of the vehicle control group showed a gradual thinning over the course of the study, which reached statistical significance at day 28 (-17.5 ± 15.6% from baseline, P = 0.01 as compared with baseline and day 10) (Fig. 5a). Pretreatment with LbGP prevented this loss, and resulted in a significantly higher RNFLT than the vehicle control at day 28 (-0.6 ± 5.2% from baseline, P = 0.03), despite an insignificant transient reduction at day 10 (-4.6 ± 8.4% from baseline). Posttreatment with LbGP starting from day 1 or 5 also protected the RNFL, despite a mild, non-significant thinning detected at day 10 (-5.9 ± 9.6% and − 8.8 ± 8.1% from their respective baselines respectively) and day 28 (-6.6 ± 6.8% and − 7.5 ± 6.4% from their respective baselines respectively) (Fig. 5a). However, when compared with the contralateral sham eyes, the RNFLT of ET-1 injected eyes was significantly lower at days 10 (P = 0.05) and 28 (P = 0.04) in LbGP-PostTx day 5 group (Fig. 4a).

As for IRLT, the ET-1 injected and vehicle treated eyes showed a significant thinning at day 10 (-8.4 ± 4.4% from baseline, P = 0.003 as compared with baseline), which was mildly improved at day 28 (-5.1 ± 4.3% from baseline, P = 0.03 as compared with baseline and day 10) (Fig. 5b). Whereas in LbGP treatment groups, the reduction was delayed. A significant thinning was detected at day 28 in the LbGP-PreTx (-5.2 ± 5.6% from baseline, P = 0.02) and LbGP-PostTx day 1 (-5.3 ± 3.1% from baseline, P = 0.02) groups but not in the LbGP-PostTx day 5 group (-3.0 ± 3.3% from baseline, P = 0.42).

For ORLT and TRT, the vehicle treated ET-1 injected eyes developed a significant thinning in both thicknesses (Fig. 5c, d) (ORL: -9.3 ± 5.5% from baseline, P = 0.001; TRT: -8.5 ± 4.0% from baseline, P = 0.001). LbGP treatment did not prevent this loss in both LbGP-PreTx (ORL: -8.3 ± 7.3% from baseline, P = 0.003; TRT: -6.4 ± 5.7% from baseline, P = 0.002) and LbGP-PostTx day 1 (ORL: -9.6 ± 8.1%, P = 0.001; TRT: -7.7 ± 5.6%, P = 0.001) at day 10. Although a mild recovery was detected at day 28, they were still significantly thinner than their baselines. As for the LbGP-PostTx day 5 group, a non-significant thinning in both thicknesses was observed at day 10 (ORL: -4.9 ± 3.7% from baseline; TRT: -4.9 ± 2.4% from baseline) which later showed a recovery at day 28 that was comparable to the baseline (ORL: -3.2 ± 4.4% from baseline; TRT: -3.1 ± 3.5% from baseline).

LbGP treatment assisted in the better recovery of retinal functions in ET-1 injected eyes

Significant temporal differences in pSTR (time main effect: P = 0.001; Tx main effect: P = 0.52; interaction effect: P = 0.88), scotopic b-wave (time main effect: P = 0.002; Tx main effect: P = 0.51; interaction effect: P = 0.56) and scotopic a-wave (time main effect: P = 0.001; Tx main effect: P = 0.86; interaction effect: P = 0.57) responses were detected in ET-1 eyes. Figure 6 presents the traces of averaged pSTR (Fig. 6a, b) and scotopic (Fig. 6c, d) ERG responses of all treatment groups. ERG responses of ET-1 eyes (Fig. 6a, c) and fellow sham eyes (Fig. 6b, d) were recorded at baseline, post-injection days 10 and 28. The magnitude of ERG responses (Fig. 7a, b, c) and their corresponding changes from respective baselines (Fig. 7d, e, f) are presented in Fig. 7.

Fig. 6
figure 6

ET-1 induced a transient decrease in retinal functions. a-d Averaged ERG traces of (a, b) pSTR and (c, d) scotopic responses from ET-1 injected eyes (a, c) and their fellow sham eyes (b, d) treated with vehicle control (n=8), LbGP-PreTx (n=9), LbGP-PostTx from day 1 (n=9) and LbGP-PostTx from day 5 (n=7) were presented for baseline and post-injection days 10 and 28

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Fig. 7
figure 7

LbGP treatment assisted in better recovery of retinal functions. a-c Mean amplitudes of (a) pSTR, (b) scotopic b- and (c) a-waves responses measured at baseline, postinjection days 10 and 28 from ET-1 injected eyes and their fellow sham eyes from the vehicle control (n=8), LbGP-PreTx (n=9), LbGP-PostTx from day 1 (n=9) and LbGP-PostTx from day 5 (n=7) groups. All data are presented as the mean ± SD and were analysed using RM ANOVA followed by Bonferroni post-hoc test. Each circle in the bar chart represents an individual data point. * p<0.05 when compared with baseline, † p<0.05 when compared with day 10, ǂ p<0.05 when compared with fellow sham. d-e Percentage losses from baseline for (d) pSTR, (e) scotopic b- and (f) a-waves responses following ET-1 injection. They were compared among groups treated with vehicle control (n=8), LbGP-PreTx (n=9), LbGP-PostTx from day 1 (n=9) and LbGP-PostTx from day 5 (n=7). All data are presented as the mean ± SD and were analysed using mixed model ANOVA followed by Bonferroni post-hoc test. * p<0.05 when compared with baseline, † p<0.05 when compared with day 10, ǂ p<0.05 when compared with LbGP-PreTx

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Regardless of treatment conditions, all ET-1 injected eyes developed a significant reduction in pSTR magnitude at day 10 (vehicle control: -49.8 ± 12.2% from baseline, LbGP-PreTx: -45.6 ± 14.7% from baseline, LbGP-PostTx day 1: -33.8 ± 33.9% from baseline, LbGP-PostTx day 5: -36.8 ± 12.1% from baseline, P = 0.01 for all treatment groups as compared with their baselines), followed by different degrees of recovery at day 28. (Fig. 7d). Both LbGP-PreTx and LbGP-PostTx day 1 groups (LbGP-PreTx: -3.2 ± 23.8% from baseline, LbGP-PostTx day 1: -0.7 ± 16.2% from baseline, P = 0.001 for both treatment groups as compared with day 10) showed an almost full recovery at day 28 as compared with LbGP-PostTx day 5 group (-15.8 ± 10.8% from baseline) and vehicle control group (-10.9 ± 26.4% from baseline).

For scotopic b-wave (Fig. 7e), the vehicle treated ET-1 injected eyes showed a significant reduction in magnitude at day 10 (-44.9 ± 22.7% from baseline, P = 0.01 as compared with baseline), followed by a partial restoration at day 28 (-22.2 ± 35.5% from baseline). As for the LbGP treatment groups, a milder reduction was observed at day 10 (LbGP-PreTx: -22.9 ± 17.3% from baseline, LbGP-PostTx day 1: -7.8 ± 42.7% from baseline, and LbGP-PostTx day 5: -26.0 ± 20.2% from baseline), followed by a varying degree of recovery at day 28. While the LbGP-PreTx and LbGP-PostTx day 1 groups recovered to baseline levels (LbGP-PreTx: 0.6 ± 36.4% from baseline, LbGP-PostTx day 1: -3.6 ± 24.4% from baseline), the LbGP-PostTx day 5 group exhibited only a marginal recovery (-18.8 ± 14.6% from baseline) at day 28.

Similar to scotopic b-wave, ET-1 injection resulted in a reduction in a-wave responses of all treatment groups at day 10 (vehicle control: -43.4 ± 16.4% from baseline, LbGP-PreTx: -28.5 ± 16.2% from baseline, LbGP-PostTx day 1: -20.4 ± 26.7% from baseline, and LbGP-PostTx day 5: -27.6 ± 16.7% from baseline), with that in the vehicle control (P = 0.001) and the LbGP-PreTx groups (P = 0.02) reaching statistical significance (Fig. 7f). At day 28, a recovery was observed in both the LbGP-PreTx (1.2 ± 30.6% from baseline, P = 0.03) and the LbGP-PostTx day 1 groups (-8.8 ± 18.0% from baseline, P = 0.08). However, only partial recovery was detected in both the LbGP-PostTx day 5 group (-20.9 ± 11.0% from baseline) and the vehicle control group (-20.1 ± 35.8% from baseline).

No significant difference was detected in the implicit times of pSTR and scotopic a-wave. On the other hand, a significant temporal difference in the b-wave implicit time was observed (mixed model ANOVA: time: P = 0.04; between Tx groups: P = 0.54; interaction effect: P = 0.47). The LbGP-PreTx (13.1 ± 12.4% from baseline, P = 0.03) and LbGP-PostTx day 1 (11.1 ± 22.2% from baseline, P = 0.07) groups showed a mild delay in response at day 28 when compared with the baselines (see the implicit times for all treatment groups provided in Additional File.2).

LbGP treatment preserved RGC survival in ET-1 injected eyes

Significant differences in RGC survival at the central (P = 0.01) and peripheral (P = 0.02) retinal regions were detected among treatment groups. Representative immunohistochemical RGC staining at the central, middle and peripheral retinal regions are presented in Fig. 8 (a-c). The number of RGCs were quantified (see the raw data of RGC densities provided in Additional File.3) and the percentage change from the fellow sham eyes are presented in Fig. 8 (d-f).

Fig. 8
figure 8

LbGP treatment improved RGC survival following ET-1 injection. a-c Representative images of retinal flat mounts with RBPMS immunostaining from one mouse per group are presented from (a) centre, (b) mid-peripheral and peripheral retinal locations for (a) PBS injected sham eye, (b-e) ET-1 injected eyes treated with (b) vehicle control, (c) LbGP-PreTx, (d) LbGP-PostTx from day 1, and (e) LbGP-PostTx from day 5 groups. d-e Percentage losses in RGC densities following ET-1 injection for (a) centre, (b) mid-peripheral and peripheral retinal locations. They were compared among groups treated with vehicle control (n=6), LbGP-PreTx (n=6), LbGP-PostTx from day 1 (n=6) and LbGP-PostTx from day 5 (n=6). All data are presented as the mean ± SD and were analysed using one-way ANOVA followed by Bonferroni post-hoc test. * p<0.05. Each circle in the bar chart represents an individual data point

Full size image

While a significant RGC loss was detected in the vehicle control, all LbGP treatments were able to improve RGC survival in ET-1 injected eyes. Compared with the vehicle control, LbGP-PreTx resulted in better RGC retention at the central (vehicle: -10.3 ± 6.3%, LbGP-PreTx: 0.9 ± 5.9%, P = 0.01) and peripheral retina (vehicle: -10.4 ± 8.0%, LbGP-PreTx: 1.4 ± 6.0%, P = 0.03). LbGP-PostTx day 5 also showed a better retention at the central retina when compared with the vehicle control (LbGP-PostTx day 5: -0.4 ± 4.8%, P = 0.02) (Fig. 8d).

Discussion

Dysregulation of ET-1 and its receptors has been suggested to play a role in the pathophysiology of glaucoma [91,92,93]. LbGP, which is a further purified glycoprotein from LBP extract comprising of arabinose, galactose and glucose with 30% protein [94, 95], has been shown to ameliorate neurodegeneration [96,97,98], modulate neuroinflammatory response [97, 99], and increase longevity and general well-being [98], is an attractive candidate to complement the current glaucoma treatment. This study investigated the pre- and posttreatment effects of LbGP on a non-IOP-related, ET-1 mouse model using clinical-based techniques. We demonstrated that orally administered LbGP treatment, either as preventive (i.e., pretreatment) or therapeutic (i.e., posttreatment) intervention, offered protection against ET-1 induced retinal neurodegeneration using a mouse model. This study is the first to compare the prolonged structure-function outcomes of both pre- and posttreatment with LbGP in protecting RGC fibres (RNFLT), its soma (RGC density) and retinal cell functions in an IOP-independent model of RGC neurodegeneration.

Previous preclinical studies often investigated the therapeutic efficacy of neuroprotective drugs by administering treatments before or at the time of model induction (i.e., the onset of injury). Although they were able to offer insights into the potential efficacy of different candidates, the findings may not be directly translatable to bedside as treatments in human patients often start after confirming the disease status and severity. In view of this, this study investigated the efficacy of LbGP against ET-1 induced RGC degeneration using both pre- and posttreatment approaches. ET-1 induced RGC degeneration is a slow, progressive model wherein clinically significant RNFL loss occurs at day 28 [52], and this allowed an adequate time window to investigate how the neuroprotective therapies administered at various time points during the course of degenerative process affect the treatment outcomes. For assessing posttreatment intervention, LbGP was administered on post ET-1 injection days 1 or 5. These time points were selected to represent early and late phases of neurodegeneration, based on the study of Marola et al. [51]. For pretreatment, animals were treated with LbGP from 7 days prior to 1 nmol ET-1 injection.

Intravitreal ET-1 injection in mice was shown to induce vasoconstriction immediately after the administration, resulting in a compromised retinal flow that took almost 28 days to restore to full reperfusion [52]. The doppler OCT data in the present study demonstrated that LbGP treatment did not prevent the immediate vasoconstriction effects of ET-1, but facilitated the earlier restoration of retinal blood flow (in terms of increase in the vessel calibre), and promoted RGC density retention. Similar to vasoconstrictive effects, ET-1 induced a transient decline in retinal cell responses (pSTR, b- and a-waves) up to day 10, which could not be prevented by both pre- and posttreatment with LbGP. This is expected as LbGP interventions did not seem to affect the immediate, ET-1 induced vasoconstriction. However, all retinal responses recovered subsequently at day 28 with the restoration of blood flow. Interestingly, functional recovery was also observed in the vehicle control, which did not correspond with the structural loss in RNFL/RGC. We hypothesised that the improvement in electrophysiological measurement of retinal functions is associated with the enrichment in retinal metabolism, resulting from the restoration of blood flow. Another possible explanation is that the decline in RGC density was below the detection limit of flash ERG. As such, alternative functional assessment approaches, such as pattern ERG, can be applied in future studies to improve tests sensitivity and therein, the structure-function relationship [100, 101]. Nevertheless, both LbGP pretreatment and early posttreatment from day 1 achieved better retinal function recovery than delayed LbGP treatment from day 5 or the vehicle treatment.

To enhance the translatability of our findings, longitudinal OCT evaluation of RNFL thickness, a standard clinical approach used to monitor glaucoma progression and treatment effects, is applied in the current study. Changes in the thicknesses of other retinal layers and RGC cell counts by immunohistochemical staining were also performed for investigating the protective effects of LbGP. The corroborating OCT and RGC findings in the present study suggested that pretreatment with LbGP was able to avert the ET-1 induced RNFL loss and retained the RGC density. Similarly, posttreatments from days 1 or 5 also alleviated both RNFL thinning and RGC loss. Although both posttreatments ended up with a mild RNFL loss (-6.6 ± 7%; -7.5 ± 7% from their respective baselines), their protective effects were comparable with the pretreatment group (-0.6 ± 5% from baseline) and were superior to the vehicle control group (-17.5 ± 16% from baseline). The findings that LbGP pretreatment or immediate posttreatment were more efficacious was consistent with previous reports [76, 79]. Moreover, the present study showed that delayed posttreatment from day 5 was able to retain RNFL thickness and RGC density comparable to pretreatment or immediate posttreatment conditions, which was consistent with a previous study on chronic hypertension model [76].

With regard to inner and outer retinal layers, both LbGP pre- or posttreatment were unable to restore the mild losses in IRL and ORL thicknesses induced by ET-1. As the IRL is a highly metabolic layer that is very sensitive to hypoxia, we speculated that the hypoxic damages resulted from the initial transient vasoconstriction were drastic for the currently tested treatment approaches to work. As for the outer retinal layers, which relied mainly on the choroidal supply and were therefore more resistant to hypoxia, another mechanism may be involved. It is possible that the ET-1 receptors present in choroidal vessel [102, 103] were affected by the injected ET-1, and the transient vasoconstriction resulted in a disruption in choroidal supply to outer retina which led to the degenerative changes observed. In short, our data suggested that LbGP exerted its neuroprotective effects against transient, ischemic changes in the retina mainly through preserving RNFL and RGCs, but not the IRL and ORL.

Previous studies showed that ET-1 induced RGC death is mainly triggered by vascular ET-A mediated vasoconstriction through JNK-JUN signalling pathway [50, 51, 104]. Therefore, modulating ET-1 levels or its receptors in the retina can potentially help to restore vascular function and regulate certain signalling pathways involved in RGC death. An early recovery in vessel calibre measurement together with the protective effect on RGC axons (RNFL) and soma (RGC density) in the treated groups indicated that LbGP facilitated restoration of retinal arterial flow and counteracted distinct cellular signalling pathways involved in RGC degeneration. However, the work has not assessed the expressions of ET receptors or evaluated cellular signalling pathways in the treated groups. Evidence from earlier studies suggested that LB has ability to modulate the expressions of ET-1 levels and its receptors differently in retinal vasculature and neurons [75], preserve blood-retinal-barrier, vascular density and decrease glial activation [77, 85, 88]; combat reperfusion induced oxidative stress by enhanced Nrf2/HO-1 oxidative pathway [78], decrease the expression of pJNK2/3 and phosphorated c-jun [81], thereby offering RGC protection. Further studies are warranted to directly investigate how the LB treatment influenced the ET-1 induced changes in blood vessels and its receptors levels, together with the neuroprotective cellular mechanisms in ET-1 treated retina.

Conclusions

The present work demonstrated that the use of LbGP either as preventive (pretreatment) or therapeutic (posttreatment) interventions facilitated early restoration in retinal blood flow, protected the RGCs fibres (RNFL), its soma (RGC density) and retinal functions in ET-1 mouse model. Effective protection was observed even with delayed treatment, with therapeutic efficacy comparable with pretreatment. As LB was able to exert RGC neuroprotection in both IOP-dependent and IOP-independent models under both pre-and posttreatment conditions, it has great translational potential as an adjuvant therapy for glaucoma management.

Data availability

All data generated or analysed during this study are included in this published article (including additional files).

Abbreviations

LB:

Lycium Barbarum

LbGP:

Lycium Barbarum Glycoprotein

IOP:

Intraocular pressure

RGC:

Retinal ganglion cell

ET-1:

Endothelin-1

ET-A:

Endothelin receptor A

ET-B:

Endothelin receptor B

Tx:

Treatment

SD-OCT:

Spectral domain optical coherence tomography

RNFL:

Retinal nerve fibre layer

IRL:

Inner retinal layer

ORL:

Outer retinal layer, ERG: electroretinogram

pSTR:

Positive scotopic threshold response

PBS:

Phosphate-buffered saline

SD:

Standard deviation

RM-ANOVA:

Repeated measures ANOVA

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Acknowledgements

The authors also thank the University Research Facilities in Behavioural and Systems Neuroscience (UBSN), The Hong Kong Polytechnic University, for technical and facility support.

Funding

Funding supported by the General Research Fund from the Research Grants Council (PolyU 15100222), PolyU Research Grants (UALH), the funding of Research Centre for SHARP Vision (RCSV) (1-BBC1), and the InnoHK initiative by the Hong Kong Special Administrative Region Government.

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Authors and Affiliations

  1. Laboratory of Experimental Optometry (Neuroscience), School of Optometry, The Hong Kong Polytechnic University, Hong Kong, China

    Yamunadevi Lakshmanan, Francisca Siu Yin Wong & Henry Ho-Lung Chan

  2. Centre for Eye and Vision Research (CEVR), 17W Hong Kong Science Park, Hong Kong, China

    Yamunadevi Lakshmanan, Francisca Siu Yin Wong & Henry Ho-Lung Chan

  3. Guangdong-Hongkong-Macau (GHM) Institute of CNS Regeneration, Jinan University, Guangzhou, China

    Kwok-Fai So

  4. Department of Ophthalmology, LKS Faculty of Medicine, The University of Hong Kong, Hong Kong, China

    Kwok-Fai So

  5. Research Centre for SHARP Vision (RCSV), The Hong Kong Polytechnic University, Hong Kong, China

    Henry Ho-Lung Chan

  6. Research Centre for Chinese Medicine Innovation (RCMI), The Hong Kong Polytechnic University, Hong Kong, China

    Henry Ho-Lung Chan

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  1. Yamunadevi Lakshmanan
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  2. Francisca Siu Yin Wong
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Contributions

YL – Conception of work, performed the study, data collection, analysis, interpretation, manuscript preparation. FW – Conception of work, performed the study, data collection, analysis, interpretation, critical revision of manuscript. HC - Conception of work, interpretation, substantively revised the manuscript. KFS – provided LbGP, substantively revised the manuscript. All authors read and approved the final manuscript.

Corresponding author

Correspondence to Henry Ho-Lung Chan.

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All experimental procedures and care involving animals were approved by the Animal Ethics Sub-committee of The Hong Kong Polytechnic University prior to commencement of experiments (16–17/32-SO-OTHERS).

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Additional file 1:

Intravitreal ET-1 injection induced retinal arterial constriction. Representative fundus photographs from one mouse eye, captured before (baseline) and after the intravitreal injection of PBS or ET-1. The retinal blood vessels appeared normal in the PBS injected sham group (black arrows), and 1 nmol ET-1 injection induced narrowing of retinal arteries (white arrows) in both vehicle control and LbGP-PreTx groups

Additional file 2: 

a-c Mean implicit times of (a) pSTR, (b) scotopic b- and (c) a-waves responses measured at baseline (BL), postinjection days 10 and 28 from ET-1 injected eyes and its fellow sham eyes are presented for groups treated with vehicle control (n=8), LbGP-PreTx (n=9), LbGP-PostTx from day 1 (n=9) and LbGP-PostTx from day 5 (n=7). All data are presented as the mean ± SD and were analysed using RM ANOVA followed by Bonferroni post-hoc test. Each circle in the bar chart represents an individual data point. * p<0.05 when compared with BL, † p<0.05 when compared with day 10, ǂ p<0.05 when compared with fellow sham

Additional file 3:

a-c Mean RGC densities from (a) centre, (b) mid-peripheral and peripheral retinal locations from ET-1 injected eyes (colored bars) and their fellow sham eyes (open bars) for groups treated with vehicle control (n=6), LbGP-PreTx (n=6), LbGP-PostTx from day 1 (n=6) and LbGP-PostTx from day 5 (n=6). All data are presented as the mean ± SD and were analysed using RM ANOVA followed by Bonferroni post-hoc test. Each circle in the bar chart represents an individual data point. * p<0.05 when compared with fellow sham, BL, † p<0.05 when compared with the fellow sham of vehicle control

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Lakshmanan, Y., Wong, F.S.Y., So, KF. et al. Lycium barbarum glycopeptide promotes neuroprotection in ET-1 mediated retinal ganglion cell degeneration. J Transl Med 22, 727 (2024). https://doi.org/10.1186/s12967-024-05526-8

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Journal of Translational Medicine

ISSN: 1479-5876

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