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. 2025 Jan 3;39(1):132–145. doi: 10.21873/invivo.13811

Therapeutic Potential of Dimethyl Sulfoxide via Subconjunctival Injection in a Diabetic Retinopathy Rat Model

JIYI HWANG 1, JI SEUNG JUNG 1, DONGHEE KIM 1, MYEONGJEE KWON 1, JUNGYEON YONG 1, HAERIN YOON 1, KYUNG-MEE PARK 1
PMCID: PMC11705140  PMID: 39740902

Abstract

Background/Aim

Diabetic retinopathy (DR), a complication of diabetes, causes damage to retinal blood vessels and can lead to vision impairment. Persistent high blood glucose levels contribute to this damage, and despite ongoing research, effective treatment options for DR remain limited. Dimethyl sulfoxide (DMSO) has shown anti-inflammatory and antioxidant properties in both in vivo and in vitro studies; however, its potential as an anti-inflammatory agent in the context of DR has not yet been explored. This study aimed to assess the effects of subconjunctival injection of DMSO on the progression of DR.

Materials and Methods

DR was induced in rats using intraperitoneal injections of streptozotocin (55 mg/kg), confirmed by measuring blood glucose levels and electroretinography (ERG). The rats were divided into five groups: a normal control group (CON), a DR control group receiving PBS injections (DMSO 0), and three DR groups receiving different concentrations of DMSO (98%, 50%, and 10%). Retinal function was evaluated using ERG at weeks 10 and 14, and histological analysis at week 16.

Results

The DMSO 50 group had significantly higher B-wave amplitude in ERG compared to the DMSO 0 group (p<0.05). Flicker response amplitudes were also significantly greater in the DMSO 50 and DMSO 10 groups compared to DMSO 0 (p<0.05). Histological examination revealed thinner retinal layers in the DMSO 0 group compared to the CON group, while the DMSO-treated groups showed improved retinal thickness.

Conclusion

Subconjunctival injection of 50% DMSO appears to improve retinal function in a rat model of DR.

Keywords: Diabetic retinopathy, dimethyl sulfoxide, subconjunctival injection, anti-inflammatory

Diabetic retinopathy (DR) is a significant complication of diabetes mellitus (DM) and remains a leading cause of visual impairment among the working-age population worldwide (1,2). An analysis of review paper revealed that approximately 93 million individuals suffer from DR, with 17 million experiencing proliferative DR, 21 million affected by diabetic macular edema, and 28 million facing vision-threatening DR globally (3). Recent years have seen notable changes in the epidemiological characteristics of DR. A comprehensive analysis of 59 population-based studies found that 22.27% of individuals with diabetes worldwide have DR, and among these, 6.17% suffer from vision threatening DR. The prevalence of DR varies by region, with approximately 23.71% in Europe, 33.2% in the United States, 17.94% in Asia, and 17.6% in India (4-6).

In diabetes, retinopathy is the most common microvascular complication and is classified into two categories. The early stage of retinal involvement is known as non-proliferative diabetic retinopathy (NPDR), characterized clinically by microaneurysms, retinal hemorrhages, intraretinal microvascular abnormalities, and venous caliber changes. In NPDR, progressive retinal vascular perfusion impairment due to loss of vascular integrity leads to microvascular occlusion or degeneration. This results in ischemia, which progresses to proliferative diabetic retinopathy (PDR), characterized by hypoxia-induced expression of proangiogenic growth factors and subsequent preretinal neovascularization (7).

Treatments for mitigating DR include stringent management of blood glucose (BG) and blood pressure, laser photocoagulation, anti-vascular endothelial growth factor (VEGF) injections, such as ranibizumab, aflibercept, and bevacizumab, and steroid injections (8-10). Vitrectomy is also performed in cases of PDR when vitreoretinal traction and other complications arise. Additionally, new treatment modalities, such as stem cell therapy and exosome therapy, are being developed (11). The current treatment trends include the mechanisms previously described, but they also face several limitations and challenges. Moreover, treatment involves intraocular injections, which can lead to potential complications such as bleeding at the injection site and damage to the retina (12). In the case of stem cell therapy, tissue rejection and low survival rates are concerns (13). Furthermore, treatments that use steroids may exacerbate systemic conditions, such as diabetes and reduction of serum cortisol levels (14). Therefore, it is essential to investigate anti-inflammatory drugs that are administered safely with fewer systemic effects.

Dimethyl sulfoxide (DMSO) is an amphipathic substance known for its remarkable ability to dissolve both organic and inorganic compounds. This versatility makes DMSO a widely used solvent for various chemicals. Additionally, DMSO can penetrate the phospholipid bilayer of cell membranes, forming “water pores” that facilitate the transport of substances into the cell (15,16).

Therapeutically, DMSO exhibits diverse properties, including anti-inflammatory and analgesic effects. In the United States, it is used as a local treatment for conditions, such as osteoarthritis and interstitial cystitis, often in combination with analgesics. DMSO is an FDA-approved substance appreciated for its affordability and non-toxicity, serving both as a therapeutic agent and a solvent for medications. Building on the clinical application of DMSO in various conditions, it is important to note the therapeutic effects of DMSO which have been documented in various studies. DMSO modulates critical biological pathways by inhibiting NF-kB and MAPK signaling, which are integral in controlling the production of signaling molecules. Additionally, DMSO has been shown to reduce the levels of specific cytokines like TNF-α and IFN-γ, further contributing to its therapeutic properties. These mechanisms suggest potential benefits of DMSO in managing various medical conditions (17,18).

DMSO has been used as a therapeutic agent for ocular diseases such as superficial keratitis in dogs (19). DMSO has been applied to humans as an ophthalmic treatment to examine the progression of retinitis pigmentosa and macular degeneration (20-22). This study aimed to investigate the retinal functional improvements following the first application of DMSO via subconjunctival injection in a type 1 DR disease rat model. Our goal was to objectively describe these effects through clinical retinal function assessments using ERG and histological analysis of retinal changes associated with DR.

Materials and Methods

Animal housing condition. All procedures complied with the Institutional Animal Care and Use Committee of Chungbuk National University (CBNUA-2032-22-01). Thirty-five 7-week-old male Sprague-Dawley rats were obtained from Nara Biotech (Pyeongtaek, Republic of Korea). The rats weighed 200-250 g at study initiation. They were housed in a conventional environment with a standard 12-hour light/12-hour dark cycle, and room temperature was set to range from 20 to 26˚C, relative humidity of 30% to 70%. The rats had free access to normal pellet chow (Experimental Rat & Mouse Diet, Purina, St. Louis, MO, USA) and water.

DR animal model generation and experimental grouping. Diabetes (BG level >250 mg/dl) was induced with intraperitoneal injection of streptozotocin (55 mg/kg) (STZ, Sigma-Aldrich, St. Louis, MO, USA) in sodium citrate buffer (0.1 M, pH 4.5) (Sigma-Aldrich) while the normal control group was injected with the same volume of sodium citrate buffer at week 2 (23-27). The body weight (BW) and the BG measurements were conducted after a 6-hour fasting period. The initial data was measured just before the diabetes induction by a commercial BG meter (FORA G11, Fora Care, Moorpark, CA, USA). The final measurements were taken at week 16.

Following an acclimatization period, diabetes was induced in the rats at week 2. At 10 weeks of age, an ERG assessment showed no differences between the groups. The rats were then randomly divided into five groups as follows: Group 1, the normal control group (CON; nine rats, eighteen eyes); Group 2, the DR control group with PBS injection (DMSO 0; eight rats, sixteen eyes); Group 3, 98% DMSO injection group (DMSO 98; six rats, twelve eyes); Group 4, 50% DMSO injection group (DMSO 50; six rats, twelve eyes); and Group 5, 10% DMSO injection group (DMSO 10; six rats, twelve eyes) (Figure 1).

Figure 1.

Figure 1

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Scheme of the experiment.

Subconjunctival DMSO injection. The 10 μl of DMSO (Daejung, Siheung, Republic of Korea) was injected subconjunctivally using a 31G insulin syringe (Ultra-Fine II short needle, BD Biosciences, Franklin Lakes, NJ, USA) under anesthesia with isoflurane (Terrell, Primal Critical Care, Bethlehem, PA, USA). The DMSO was diluted in phosphate buffer saline (PBS) and then subjected to microfiltration to prepare injections. The subconjunctival injection was repeated every week with a total of two times at week 12 and 13 (28,29). The DMSO injected groups received 10 μl of different concentration of DMSO via subconjunctival injection, whereas the CON and DMSO 0 groups received the same volume of PBS. Before subconjunctival injection, eyes were disinfected with povidone-iodine 0.5% and topical anesthesia was applied with 0.5% proparacaine (Alcaine®, Alcon, Geneva, Switzerland).

Electroretinography (ERG). To confirm DR, the ERG measurements were conducted in the entire study group at week 10 prior to subconjunctival injection and to allow assessment of therapeutic effect of sub-conjunctival DMSO injection, ERG was performed again at week 14 (24,28,30). The ERG was performed using the RETevet™ (LKC, Gaithersburg, MD, USA) device after dark adaptation in all groups and all procedures were performed under dim red light and anesthesia with isoflurane. To achieve uniform stimulation across the retina, complete mydriasis was induced with 0.01% tropicamide and phenylephrine (Mydrin-P, Santen, Osaka, Japan). A contact electrode designed for rats was employed to make direct contact with the cornea, while a reference electrode was placed on the forehead and a ground electrode was inserted into the tail. Flash ERG was presented at a frequency of 2 Hz at 8.0 cd·s/m2 with a background at 30 cd·s/m2, and flicker ERG was presented at a frequency of 11 Hz at 8.0 cd·s/m2 with a background at 30 cd·s/m2 (31,32).

Slit lamp bio-microscopy examination. The slit lamp bio-microscopy (MW50D, Grand Seiko Co. Ltd., Hiroshima, Japan) was used to examine the anterior segment including inflammation at the injection site, cataract formation and uveitis. Due to the induction of diabetes, the severity of cataracts was advanced, making fundus imaging challenging. The progression of the diabetic cataract was assessed, and the cataract grade was classified by one to four stages according to the previous report (24,33).

Histological examination. After euthanasia, the eyes were harvested from rats. The excised eyes were fixed in the Biofix HD (BioGnost Ltd., Zagreb, Croatia) and then tissue processing and paraffin embedding were performed. After being sectioned to 5 μm, all tissues were analyzed histologically following Hematoxylin and Eosin (H&E) stain and DAB (3, 3’-diaminobenzidine) immuno-histochemistry (IHC). The thickness of the retinal tissue was measured at a magnification ×200. The thickness of the ganglion cell layer (GCL), nerve fiber layer (NFL), inner nuclear layer (INL), outer nuclear layer (ONL), and photoreceptor layer (PRL) were compared among groups to evaluate the degree of degeneration of the retinal structure.

To determine the anti-inflammatory effect of 50% DMSO based on the expression of glial fibrillary acidic protein (GFAP) in retinal slices, the retinal tissues were rehydrated and then incubated with rabbit polyclonal anti-GFAP primary antibodies (1:1,000, ab7260, Abcam, Cambridge, UK) for 1 h and 30 min at 26˚C. After rinsing with distilled water and blocking, the retinal slices were incubated with the appropriate secondary antibodies (ab62464, Abcam) and counterstained with hematoxylin. The processed samples were photographed using a microscope (KCS3-63S, Optinity, Korea Lab Tech, Seongnam, Republic of Korea).

Evaluation of systemic effects of subconjunctival injections. To evaluate the systemic effects of subconjunctival injection in three rats, a comprehensive blood chemistry analysis was conducted using the Vetscan V2 (Abaxis, Union City, CA, USA). Blood samples were collected from the tail vein according to the experimental schedule before the injections. The rats were then administered a 98% DMSO subconjunctival injection once a week. After a total of two injections, blood samples were collected one week later for comparison. The normal blood test values of rats before and after injection were compared by referring to the literature (34,35).

Statistical analysis. GraphPad Prism 10 software (GraphPad Software, Boston, MA, USA) was used for data analysis. Results are presented as mean±standard deviation. Statistical significance between groups was determined using an ordinary one-way analysis of variance (ANOVA) and the Wilcoxon signed-rank test. p-Values lower than 0.05 were considered statistically significant.

Results

Changes of BW and BG. At week 16, BW and BG data were compared across all groups. The average BW for the CON, DMSO 0, DMSO 98, DMSO 50, and DMSO 10 groups were 589.5±34.4 g, 234.2±33.5 g, 238.7±54.5 g, 240±33.4 g, and 229.3±26.3 g, respectively (Figure 2A). Likewise, the average BG levels for the CON, DMSO 0, DMSO 98, DMSO 50, and DMSO 10 groups were 98.8±10.9 mg/dl, 540.7±123 mg/dl, 468.7±59 mg/dl, 557±82 mg/dl, and 537.4±67.9 mg/dl, respectively (Figure 2B). Significant differences were observed in the average BW and BG levels between the CON group and the other groups. While the CON group exhibited an increase in average BW over the preceding two months, no such increase was noted in the DMSO groups. Notably, there were no statistically significant differences in BW and BG between the DMSO groups, indicating that subconjunctival injections did not elicit systemic treatment effects on diabetes.

Figure 2.

Figure 2

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Changes in body weight (BW) and blood glucose (BG) over time. (A) A graph for BW changes over time. (B) A graph for BG changes over time, subconjunctival administration was conducted at 11 and 12 weeks. Significant differences in BW were observed between the CON group and the diabetic retinopathy (DR) experimental groups at weeks 2, 10, and 16. For BG, significant differences were noted between the CON group and the DR experimental groups at weeks 10 and 16. There were no significant differences in BW and BG between the DMSO 0 and other DMSO groups at weeks 10 and 16. The CON group exhibited significantly higher BW and lower BG at all time points. *p<0.05, CON, n=9 rats; DMSO 0, n=8 rats; DMSO 98, n=6 rats; DMSO 50, n=6 rats; DMSO 10, n=6 rats. CON: Normal control group; DMSO 0: DR group with PBS injection; DMSO 98: DR group with subconjunctival 98% DMSO injection; DMSO 50: DR group with subconjunctival 50% DMSO injection; DMSO 10: DR group with subconjunctival 10% DMSO injection.

Evaluation of retinal function with ERG. The ERG was used to confirm the DR model before administering DMSO, and the measurement at 10 weeks assessed retinal dysfunction differences between the CON group and DR groups. Starting from the 10-week results, significant differences were noted in the implicit time and amplitude of the flash b-wave and flicker.

The implicit time of the flash b-wave was delayed in the DR group, showing a significant difference compared to the CON group (Figure 3A). The average flash b-wave amplitude for the CON and the DR groups were 143.28±50.07 μV and 76.26±34.1 μV, respectively (Figure 3B). However, no significant difference was observed between the CON and DR groups in the implicit time of the flicker wave (Figure 3C). The average flicker amplitude for the CON and the DR groups were 109.87±32.9 μV and 67.38±26.03 μV, respectively (Figure 3D). This measurement at 10 weeks indicates that the DR disease model was successfully established.

Figure 3.

Figure 3

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Comparison of electroretinogram results between normal control group (CON) and diabetic retinopathy (DR) groups at week 10. (A) Results of flash b wave implicit time, (B) flash b wave amplitude, (C) flicker implicit time, and (D) flicker amplitude. The Diabetic retinopathy (DR) group showed significantly lower values compared to the CON group, indicating the induction of DR. *p<0.05, CON, n=9 rats, 18 eyes; DR, n=26 rats, 52 eyes.

Subsequently, ERG was measured at 14 weeks to observe the therapeutic effect after subconjunctival injection of DMSO. ERG results at 14 weeks, one week after two subconjunctival injections, were as follows: the average flash b-wave amplitudes for the CON, DMSO 0, DMSO 98, DMSO 50, DMSO 10 groups were 123.99±28.52 μV, 68.01±26.34 μV, 97.76±51 μV, 155.85±59.44 μV, and 107.77±39.63 μV, respectively (Figure 4A and A’). The value for the CON group was significantly higher than that for the DMSO 0 group (p<0.05). Notably, the amplitude value for the DMSO 50 group was like that of the CON group, and no significant difference was found between these groups.

Figure 4.

Figure 4

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Comparative analysis of flicker and flash b-wave amplitudes in ERG across groups. (A) Flash B wave stimulus ERG results at week 14; the DMSO 50 group exhibited significantly greater amplitude than the DMSO 0 group. (A)’ This graph images are representative pictures of the flash B wave amplitude of each group. (B) Flicker stimulus ERG results at week 14; the DMSO 50 group and the DMSO 10 group showed significantly greater amplitude than the DMSO 0 group. (B)’ This graph images are representative pictures of the flicker wave amplitude of each group. *p<0.05. CON, n=9 rats, 18 eyes; DMSO 0, n=8 rats, 16 eyes; DMSO 98, n=6 rats; DMSO 50, n=6 rats; DMSO 10, n=6 rats. ERG: Electroretinogram; CON: normal control group; DMSO 0: DR group with PBS injection; DMSO 98: diabetic retinopathy (DR) group with subconjunctival 98% DMSO injection; DMSO 50: DR group with subconjunctival 50% DMSO injection; DMSO 10: DR group with sub-conjunctival 10% DMSO injection.

The average flicker amplitudes for the CON, DMSO 0, DMSO 98, DMSO 50, DMSO 10 groups were 121.05±18.86 μV, 53.81±25.22 μV, 91.37±42.15 μV, 126.63±39.55 μV, and 103.9±38.09 μV, respectively (Figure 4B and B’). Similar to the b-wave, significant differences were observed between the CON and DMSO 0 groups in the flicker response. Notably, the DMSO 50 and DMSO 10 groups exhibited significantly higher amplitude values compared to the DMSO 0 group, suggesting an improvement in retinal function in these groups.

When the amplitude decreases, the retinal function deteriorates. The retinal function of the CON group was recorded as more than twice that of the DMSO 0 group, indicating a decline in retinal cell function in the DMSO 0 group. In contrast, the DMSO injection groups showed an improvement in retinal function compared to the DMSO 0 group, since the DMSO 50 group exhibited enhancements in both B amplitude and flicker amplitude, reaching levels like those of the CON group.

Histological evaluation of the retinal layer. The rats were euthanized, and their eyes were harvested at week 16 to evaluate the tissues using H&E staining. This was 15 weeks after the induction of diabetes. In the DMSO 0 group, proliferative vitreous membranes around the retina were observed, along with changes in the morphology of NFL+GCL cells, such as cell necrosis and re-modeling, and a reduction in the number of nuclei in other retinal cells. These changes were not observed in the CON group or other DMSO groups (Figure 5A). The total retinal thickness was measured and the results are presented in Table I.

Figure 5.

Figure 5

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Evaluation of retinal histology using H&E staining. (A) At 4 weeks post-initial subconjunctival injections, which occurred 15 weeks after diabetes induction, the all retinal layers of each group were assessed. (B) Measurements of total retinal thickness revealed that retinas in the DMSO groups were significantly thicker than those in the DMSO 0 group and were comparable to the CON group. (C) When measuring the thickness of the INL (white arrow), it was found that the DMSO 0 group had the thinnest INL, significantly differing from the CON, DMSO 50, and DMSO 10 groups, which showed no statistically significant differences among themselves. (D) ONL (red arrow) thickness measurements indicated a significant difference between the DMSO 0 group and other DMSO groups. (E) PRL (black arrow) in the DMSO 0 group was significantly thinner compared to the CON and DMSO 50 groups, with no significant differences noted between the CON, DMSO 50, and DMSO 10 groups. The slides were examined under a magnification of ×200. *p<0.05. CON, n=9 rats, 18 eyes; DMSO 0, n=8 rats, 16 eyes; DMSO 98, n=6 rats; DMSO 50, n=6 rats; DMSO 10, n=6 rats. H&E: Hematoxylin and eosin; NFL: nerve fiber layer; RGC: retinal ganglion cell layer; IPL: inner plexiform layer; INL: inner nuclear layer; OPL: outer plexiform layer; ONL: outer nuclear layer; PRL: photoreceptor layer; CON: normal control group; DMSO 0: diabetic retinopathy (DR) group with PBS injection; DMSO 98: DR group with subconjunctival 98% DMSO injection; DMSO 50: DR group with subconjunctival 50% DMSO injection; DMSO 10: DR group with subconjunctival 10% DMSO injection. Bar=100 μm.

Table I. Comparison of total retinal thickness, inner nuclear layer, outer nuclear layer, and photoreceptor layer between the CON group and the DMSO groups.

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Each value is represented as Mean±SD, and the unit is μm. Statistical significance is indicated in the graph in Figure 5.

There was a significant difference between the CON and DMSO 0 group in total retina thickness (Figure 5B), with the DMSO 0 group showing the thinnest retina. The DMSO 50 and DMSO10 groups showed significantly thicker retina than the DMSO 0 group, but DMSO 98 did not differ a significantly from the DMSO 0 group. The remaining three layers were significantly thicker in the DMSO 50 and DMSO 10 groups compared to the DMSO 0 group, but not in the DMSO 98 group. This means histologically, NPDR was observed in DMSO 0 group, while in the CON group and DMSO groups, there was no decrease in tissue thickness or cellular damage. Among the DMSO groups, both DMSO 50 and DMSO 10 showed no retinal damage compared to the DMSO 0 group (Figure 5C-E).

Tissue images from the CON group and DMSO 0 group were analyzed. In the CON group, Muller cell nuclei were clearly visible, and the distinction between the outer and inner retinal layers was well-defined (Figure 6A). In the DMSO 0 group, signs of Muller cell atrophy and loss of cell nuclei were evident (Figure 6B). Additionally, fibrous membrane proliferation was observed above the NFL layer, accompanied by hemorrhagic spots (Figure 6C). In another image from the DMSO 0 group, fibrous membrane proliferation was noted above the NFL layer (Figure 6D).

Figure 6.

Figure 6

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Tissue images of the CON group and the DMSO 0 group. (A) In the CON group, the nuclei of Muller cells are clearly observed (black arrow), and the connectivity between the outer retinal layer and the inner retinal layer is distinct. (B) In the DMSO 0 group, Muller cell atrophy and loss of cell nuclei are observed (black arrow). (C) In the DMSO 0 group, there is a fibrous membrane proliferating above the NFL layer (red arrow), with hemorrhagic spots (black arrow). (D) In the DMSO 0 group, a fibrous membrane is proliferating above the NFL layer (red arrow). Bar=100 μm.

Compared with the nondiabetic CON group, GFAP immunoreactivity to was increased in the STZ-induced diabetic rats in the DMSO 0 group. As shown, GFAP immuno-reactivity is higher in the DMSO 0 group than in the DMSO 50 group (Figure 7).

Figure 7.

Figure 7

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GFAP immunohistochemical staining of the rat retina in CON, DMSO 0, and DMSO 50 groups. Sections of a control (A), DMSO 0 (B), and the 50% DMSO treatment group (C) retina were stained with anti-GFAP antibodies using 3.3’-diaminobenzidine (DAB: brown) as the chromogen. Nuclei were counterstained with hematoxylin (blue). Bar=100 μm.

Evaluation of cataract formation with slit lamp bio-microscopy. After intraperitoneal injection of STZ, DR was induced, and cataracts rapidly developed. In the DMSO 0 group, the severity of cataracts was the most pronounced, and the lens capsule was not uniform; inflammation was observed with lens material leaking into the anterior chamber. The DMSO 50 group showed a lower formation of cataracts compared to the DR control group, and this significant difference (p<0.05) was confirmed using slit-lamp bio-microscopy (Table II and Figure 8). Regarding cataract formation, DMSO 50 appeared to have the most delayed progression among the four groups. There was a significant difference between the DMSO 0 group and DMSO 50; however, the differences between DMSO 98 and DMSO 10 compared to the DMSO 0 group were non-significant. This indicates that while cataract progression was less in these groups compared to the DMSO 0 group, the results were not statistically significant.

Table II. Cataract staging.

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Figure 8.

Figure 8

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Comparative analysis of cataract formation staging at week 16. (A) DMSO 50 has a significantly different incidence rate of cataracts compared to DMSO 0. (B) The representative images of retro-illumination of each group using slit-lamp bio-microscopy. *p<0.05. CON, n=9 rats, 18 eyes; DMSO 0, n=8 rats, 16 eyes; DMSO 98, n=6 rats; DMSO 50, n=6 rats; DMSO 10, n=6 rats. ERG: Electroretinogram; CON: normal control group; DMSO 0: diabetic retinopathy (DR) group with PBS injection; DMSO 98: DR group with subconjunctival 98% DMSO injection; DMSO 50: DR group with subconjunctival 50% DMSO injection; DMSO 10: DR group with subconjunctival 10% DMSO injection.

Evaluation of systemic effects of subconjunctival injection. The comparison of serum enzymes in normal blood glucose rats before and after subconjunctival injection of DMSO was performed using the Wilcoxon signed-rank test. The analysis revealed that the p-values for each blood enzyme test exceeded 0.05, indicating that there was no statistically significant difference between the pre- and post-injection values. All blood test values before and after the injection remained within the normal range. This indicates that the subconjunctival injection does not produce any systemic effects (Figure 9).

Figure 9.

Figure 9

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Comparison of serum enzymes before and after subconjunctival injection of DMSO in normal blood glucose rats using the Wilcoxon signed-rank test. The experiment was conducted on three rats. Blood was collected from the tail vein before the injection, and like the experimental scheme, blood collection was performed after two weekly subconjunctival injections of 98% DMSO. Each enzyme was measured in specific units: ALP, ALT, and AMY are reported in U/l, while TBIL, BUN, CRE, and GLU are measured in mg/dl. TP, ALB, and GLOB are recorded in g/dl. The results of each blood enzyme test showed that the p-value was greater than 0.05, confirming that there was no statistically significant difference between the values before and after the injections. This suggests that the subconjunctival injection has no systemic effects.

Discussion

In this study, we verified the therapeutic effects of subconjunctival injections of DMSO in DR. We assessed the therapeutic efficacy through ERG, slit lamp bio-microscopy, and retinal tissue evaluation. Among them, the DMSO 50 group showed the highest effect in improving retinal function.

This experiment showed that DMSO alone had a preventive effect on cataract formation and retinal function decline in DR. Previous studies suggested that DMSO application to ocular tissues could have therapeutic effects on conditions like retinitis pigmentosa and macular degeneration (22). According to the reports, the progression of cataracts in the DMSO 50 group was slower (20,35). Similarly in our study, there was a significant difference in cataract progression stages between the DMSO 50 group and the DMSO 0 group.

Current treatments for DR include retinal laser photocoagulation to prevent abnormal vascular leakage, and the use of anti-VEGF antibodies or steroids injected into the vitreous or subretinal space to stabilize retinal vascular walls and reduce inflammation (1,4,9,10,36). However, laser treatment has side effects, such as damage of retinal tissues and intraocular hemorrhage, and does not work for every patient. The safety and efficacy of DR treatments have been questioned, highlighting the need for new therapeutic agents for DR (37).

DMSO is well-documented for its ability to enhance permeability through biological membranes, such as skin and cells, making it a valuable agent in pharmaceutical applications. It modifies the structure of the stratum corneum and disrupts lipid bilayers, thereby facilitating the transdermal delivery of drugs. This characteristic is utilized in drug delivery systems to improve the penetration and efficacy of therapeutic compounds (16,38,39).

DMSO also has shown potential anti-inflammatory effects in vitro, such as inhibiting LPS-induced pro-inflammatory cytokines in J774 macrophages and reducing the inflammatory response in Caco-2 cells (17,18,40,41). In vivo, DMSO reduced swelling in an arthritis mouse model and inhibited NF-kB activation in sepsis (42,43). Additionally, DMSO has been reported to enhance the effects of drugs for conditions like interstitial cystitis and rheumatoid arthritis (42,44,45). Additionally, the application of 50% DMSO as an eye drop and subsequent evaluation of cataract formation and toxicity revealed no significant abnormalities in vision aside from allergic reactions. N-acetyl carnosine, lanosterol, or glutathione have been mixed with DMSO to create eye drop formulations used for cataract prevention (35). Based on evidence from various studies, we selected 10% and 50% DMSO concentrations to explore their anti-inflammatory effects. The 10% DMSO concentration was chosen in line with the findings of Ahn et al., which demonstrated its anti-inflammatory properties in vitro (46). The 50% concentration was selected because it is FDA-approved for the treatment of interstitial cystitis in the United States, enabling us to assess its potential anti-inflammatory effects when administered subconjunctivally. Moreover, a higher concentration and an increased number of injections were chosen because the administration route was subconjunctival, rather a direct intravitreal or retinal injection. As observed in the previous study, DMSO acted to alleviate inflammation. Steroids are commonly used to manage inflammation in DR patients, but for patients who cannot use steroids, the potential application of DMSO merits consideration. While steroids act intranuclearly to suppress the transcription and translation of inflammatory mediators, DMSO targets oxidative stress caused by inflammation and regulates inflammatory cytokines, suggesting its potential as an alternative therapeutic agent. Although there have been claims regarding its low toxicity (47,48). In these studies, various concentrations, and routes of DMSO administration were experimented. The retinal function decreased after injecting DMSO into the vitreous body, and returned to normal within 24 h. In our study, DMSO 50 showed the most significant functional improvement. Also, the ERG evaluations for DMSO 98 and DMSO 10 showed improved function compared to the DMSO 0 group, but no statistically significant differences were found. This suggests that DMSO did not exhibit retinal toxicity in our research unlike previous studies (47,49), which is corroborated by ERG measurements taken one-week post-injection. The subconjunctival application likely minimized any adverse effects on the eye.

Traditionally, intravitreal administration has been the preferred method for delivering treatments for age-related macular degeneration and DR, but this approach has been accompanied by side effects, such as increased intraocular pressure and intraocular hemorrhage, necessitating the search for effective and less invasive methods (11). Additionally, in rats, because of the lens size and vitreous cavity volume (50 μl), excessive volume of intravitreal injection may cause intraocular structure damage. Therefore, in our experiment, the DMSO was injected subconjunctivally and this injection is also a widely used and effective clinical method that allows drugs to reach the posterior segment of the eye. The conjunctival epithelial barrier is penetrated through injection, minimizing adverse systemic or local effects (50,51).

Significant differences were observed in the BW and BG changes over time between the CON group and the DMSO 0 group, while no significant weight change was noted between the DR-induced group and the groups receiving subconjunctival DMSO injections. This indicates that subconjunctival administration of DMSO did not have systemic effects, and diabetes was the only factor contributing to these changes, suggesting that the concentration-specific treatment of DMSO could be the only evaluation factor. Additionally, as shown in Figure 9, further subconjunctival injection experiments conducted on normoglycemic rats allow us to rule out any systemic toxic effects of DMSO, such as its impact on blood glucose levels.

Prior to subconjunctival injections, retinal function tests were conducted on both normal control subjects and those with induced diabetes to verify the occurrence of DR. In this study, the b-wave originates predominantly from the depolarizing activity of bipolar cells, with a secondary contribution from Müller cells. Prior to the experiment, all rats underwent a dark adaptation period of 30 min, followed by exposure to a strong stimulus. This procedure allowed for the observation of a combined rod-cone response, indicating that cones also play a role in generating the b-wave under these conditions. Additionally, the amplitude of flicker responses serves as an indicator of retinal photoreceptor activity in reaction to flashing light stimuli. A higher flicker amplitude is indicative of more robust photoreceptor function (52-54). According to the literature, following intraperitoneal injection of streptozotocin and the onset of DR, a decrease in b-wave amplitude is typically observed after one month. By the third month, the a-wave amplitude also shows a reduction, with retinal function declining by approximately 50% compared to age-matched normoglycemic control groups (55,56). Based on these findings, our study utilized, ERG at week 6 to examine DR induction, where no significant difference in amplitude between the normal and diabetes groups was observed. However, a significant decrease in ERG amplitude in the diabetes group at week 10 confirmed DR induction. According to retinal potential evaluations in the rat diabetes model, starting with changes in the c-wave, a progressive decrease in b-wave and flicker amplitude occurred over time following diabetes induction. In our experiment, at 14 weeks, the DMSO 0 group showed relatively lower b-wave and flicker amplitude values, whereas those treated with subconjunctival DMSO exhibited a smaller decrease in amplitude. Notably, the DMSO 50 group demonstrated significantly higher values compared to the DMSO 0, indicating that retinal function did not significantly decline and remained closer to that of the CON group, suggesting high therapeutic potential among the treated groups. We initially planned to extend the ERG measurements to 15 and 16 weeks to further assess photopic, scotopic, and oscillatory responses under anesthesia. However, in a previous preliminary experiment, we encountered a high mortality rate during anesthesia and found that many subjects did not meet the euthanasia criteria, which indicate euthanasia if body weight decreases by more than 30%. Due to these challenges, we were unable to extend the experimental period beyond 14 weeks.

According to previous research, changes in the pre-proliferative stage of DR in tissues are observed in about half of the rats 6 months after diabetes induction (55). Our experimental group was evaluated 4 months after STZ injection, and thus, no retinal proliferation was observed. Histological changes in DR include retinal folds, new vessel formation in the optic disk, intraocular hemorrhage, anterior chamber angle, intraretinal neovascularization, mild focal retinal infarcts, and retinal gliosis of the internal plexiform layer (57). In most studies, tissue thinning has been reported following the induction of DR (58). However, there are studies indicating an increase in tissue thickness, which result in the loss of nerve cells, a consequence of intracellular edema formation (59,60). This has been investigated using H&E staining and in vivo imaging such as optical coherence tomography. It has been reported that the ONL, OPL, INL, IPL, and GCL are affected (52,61-63). In DMSO 0, we observed retinal folds, fibrin deposition within the retina, and hemorrhages. Furthermore, a comparative analysis of the retinas between the CON group and the DMSO 0 group revealed notable atrophy of Muller cells accompanied by the loss of their nuclei. These findings are consistent with previous studies reporting neuronal cell death leading to the degeneration of ganglion cells, amacrine cells, Muller cells, and photoreceptors in diabetic mice (63-66). A significant difference was observed in the overall tissue thickness decrease, and thinning of the NFL-GCL, INL, and ONL when compared to the CON group. The DMSO 50 group showed the least tissue damage among the groups, with a significant difference in tissue thickness compared to the DR control group, suggesting less histological damage occurring in DR. IHC staining was additionally performed to prove the difference in retinal inflammation between the experimental groups. Most neurologic diseases are associated with increased brain-blood barrier permeability, and thus the markers thought to indicate neuronal damage may indicate barrier dysfunction. In cases of central nervous system injury, inflammation typically originates from the activation of glial cells, resulting in the production and release of inflammatory mediators. Similarly, in diabetic retinopathy, neurodegeneration and glial activation are evident in the disease’s early stages, occurring prior to the onset of microangiopathy (67). During the above damage process, GFAP, an inflammation marker protein, is released outside the cells (68-71). In our study, we also showed that GFAP, a glial marker, increased in the diabetic retina. Our findings showed that 50% of DMSO can limit the increase in GFAP expression in the diabetic retina, suggesting that DMSO may inhibit glial activation and proliferation. However, our experiment has the limitation of not seeing the effect of DMSO at different concentrations in non-diabetic rats.

There are some limitations in this research; it evaluated the impact of different DMSO concentrations on retinal physiology and thickness, but the assessment was conducted over a short period post-DR induction. Due to the high mortality rate caused by diabetes, ERG imaging, which requires anesthesia, was not always performed. Additionally, due to limitations of our facilities and environment, we only conducted ERG experiments at flash waves and flicker waves, focusing on the rod-cone response and the effect of DMSO on improving retinal function while we were unable to examine the scotopic response and oscillatory potentials. The purpose of this study was to evaluate the clinical function of the retina through ERG, and thus it did not elucidate molecular pathways. Due to the severe cataract formation following diabetes induction the use of optical equipment like optical coherence tomography was challenging. However, despite these limitations, we were able to obtain meaningful results through IHC staining of retinal tissues, which showed reduced retinal inflammation. Future clinical trials in large animals, naturally occurring DR models, and Type-2 diabetes models prevalent in humans are necessary.

Conclusion

In conclusion, subconjunctival injection of DMSO was effective in DR. The potential applicability of 50% DMSO in the treatment of DR was supported, and the effectiveness of the subconjunctival injection route was confirmed.

Funding

This study was supported by “Regional Innovation Strategy (RIS)” through the National Research Foundation of Korea (NRF) funded by the Ministry of Education (MOE) (2021 RIS-001). This work was also supported by the Basic Research Lab Program (2022R1A4A1025557) funded by the Ministry of Science and ICT and the Korean Fund for Regenerative Medicine (KFRM) grant (the Ministry of Science and ICT, the Ministry of Health & Welfare) No. 22A0101L1-11, National Research Foundation of Korea (NRF) grant funded by the Korea government (MSIT) (RS-2024-00344226).

Conflicts of Interest

The Authors declare no conflicts of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript; or in the decision to publish the results.

Authors’ Contributions

Conceptualization and design, J.H. and K.P.; investigation, J.H., J.S.J., N.K., S.E.P., M.K., H.Y., and J.Y.; data curation, J.H.; writing-original draft preparation: J.H.; writing-review and editing, J.H., H.M.W. and K.P.; supervision, project administration and funding acquisition, K.P.; All Authors have read and agreed to the published version of the manuscript.

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