Destructive effect of intravitreal heat shock protein 27 application on retinal ganglion cells and neurofilament

Heat shock protein 27 (HSP27) is commonly involved in cellular stress. Increased levels of HSP27 as well as autoantibodies against this protein were previously detected in glaucoma patients. Moreover, systemic immunization with HSP27 induced glaucoma-like damage in rodents. Now, for the first time, the direct effects of an intravitreal HSP27 application were investigated. For this reason, HSP27 or phosphate buffered saline (PBS, controls) was applied intravitreally in rats ($\it n$ = 12/group). The intraocular pressure (IOP) as well as the electroretinogram recordings were comparable in HSP27 and control eyes 21 days after the injection. However, significantly fewer retinal ganglion cells (RGCs) and amacrine cells were observed in the HSP27 group via immunohistochemistry and western blot analysis. The number of bipolar cells, on the other hand, was similar in both groups. Interestingly, a stronger neurofilament degeneration was observed in HSP27 optic nerves, while no differences were noted regarding the myelination state. In summary, intravitreal HSP27 injection led to an IOP-independent glaucoma-like damage. A degeneration of RGCs as well as their axons and amacrine cells was noted. This suggests that high levels of extracellular HSP27 could have a direct damaging effect on RGCs

Proteomic analysis of retinal tissue in an S100B autoimmune glaucoma model

Glaucoma is a neurodegenerative disease that leads to damage of retinal ganglion cells and the optic nerve. Patients display altered antibody profiles and increased antibody titer, e.g., against S100B. To identify the meaning of these antibodies, animals were immunized with S100B. Retinal ganglion cell loss, optic nerve degeneration, and increased glial cell activity were noted. Here, we aimed to gain more insights into the pathophysiology from a proteomic point of view. Hence, rats were immunized with S100B, while controls received sodium chloride. After 7 and 14 days, retinae were analyzed through mass spectrometry and immunohistology. Using data-independent acquisition-based mass spectrometry, we identified more than 1700 proteins on a high confidence level for both study groups, respectively. Of these 1700, 43 proteins were significantly altered in retinae after 7 days and 67 proteins revealed significant alterations at 14 days. For example, $\alpha$2-macroglobulin was found significantly increased not only by mass spectrometry analysis, but also with immunohistological staining in S100B retinae at 7 and 14 days. All in all, the identified proteins are often associated with the immune system, such as heat shock protein 60. Once more, these data underline the important role of immunological factors in glaucoma pathogenesis

Reduced retinal degeneration in an oxidative stress organ culture model through an iNOS-inhibitor

In retinal organ cultures, $H_{2}O_{2}$ can be used to simulate oxidative stress, which plays a role in the development of several retinal diseases including glaucoma. We investigated whether processes underlying oxidative stress can be prevented in retinal organ cultures by an inducible nitric oxide synthase (iNOS)-inhibitor. To this end, porcine retinal explants were cultivated for four and eight days. Oxidative stress was induced via 300 $\mu$M $H_{2}O_{2}$ on day one for three hours. Treatment with the iNOS-inhibitor 1400 W was applied simultaneously, remaining for 72 h. Retinal ganglion cells (RGC), bipolar and amacrine cells, apoptosis, autophagy, and hypoxia were evaluated immunohistologically and by RT-qPCR. Additionally, RGC morphology was analyzed via transmission electron microscopy. $H_{2}O_{2}$-induced RGCs loss after four days was prevented by the iNOS-inhibitor. Additionally, electron microscopy revealed a preservation from oxidative stress in iNOS-inhibitor treated retinas at four and eight days. A late rescue of bipolar cells was seen in iNOS-inhibitor treated retinas after eight days. Hypoxic stress and apoptosis almost reached the control situation after iNOS-inhibitor treatment, especially after four days. In sum, the iNOS-inhibitor was able to prevent strong $H_{2}O$-induced degeneration in porcine retinas. Hence, this inhibitor seems to be a promising treatment option for retinal diseases

Minocycline reduces inflammatory response and cell death in a S100B retina degeneration model

$\bf Background:$ Previous studies noted that intravitreal injection of S100B triggered a glaucoma-like degeneration of retina and optic nerve as well as microglia activation after 14 days. The precise role of microglia in our intravitreal S100B model is still unclear. Hence, microglia were inhibited through minocycline. The aim is to investigate whether microglia have a significant influence on the degeneration process or whether they are only a side effect in the model studied here. $\bf Methods:$ Minocycline was applied daily in rats by intraperitoneal injection using two different concentrations (13.5 mg/kg body weight, 25 mg/kg body weight). One day after treatment start, S100B or PBS was intravitreally injected in one eye per rat. The naïve groups received no injections. This resulted in a total of five groups (naïve $\it n$ = 14, PBS $\it n$ = 14, S100B $\it n$ = 13, 13.5 mg/kg mino $\it n$ = 15, 25 mg/kg mino $\it n$ = 15). At day 14, electroretinogram measurements were performed, followed by immunofluorescence and label-free quantitative proteomics analysis. The focus of these investigations was on the survival of RGCs as well as their axons, the response of the microglia, and the identification of further pathological modes of action of S100B. $\bf Results:$ The best signal transmission was detected via ERG in the 13.5 mg/kg mino group. The inhibition of the microglia protected optic nerve neurofilaments and decreased the negative impact of S100B on RGCs. However, the minocycline treatment could not trigger complete protection of RGCs. Furthermore, in retina and optic nerve, the minocycline treatment reduced the number and activity of S100B-triggered microglia in a concentration-dependent manner. Proteomics analysis showed that S100B application led to numerous metabolic functions and cellular stress, mainly an increased inflammatory response, glycolysis, and mitochondrial dysfunction, which caused oxidative stress in the retina. Importantly, the protective capability of lower dose of minocycline was unraveled by suppressing the apoptotic, inflammatory, and the altered metabolic processes caused by S100B insult in the retina. $\bf Conclusion:$ Intravitreally injected S100B not only led to a pro-inflammatory microglial reaction, but also a mitochondrial and metabolic dysfunction. Also, these results suggest that an excessive microglial response may be a significant degenerative factor, but not the only trigger for increased cell death

Oxidative stress-induced retinal damage is prevented by mild hypothermia in an ex vivo model of cultivated porcine retinas

$\bf Background$ Hydrogen peroxide (H$_2$O$_2$) can be used in vitro to simulate oxidative stress. In retinal organ cultures, H$_2$O$_2$ induces strong neurodegeneration of the retina. It is known that oxidative stress plays a role in the development of several retinal diseases including glaucoma and ischemia. Thus, we investigated whether processes underlying oxidative stress can be prevented by hypothermia using an ex vivo organ culture model of porcine retinas. $\bf Methods$ Porcine retinal explants were cultivated for 5 and 8 days. Oxidative stress was induced via 300 $\mu$M H$_2$O$_2$ on day 1 for 3 hours. Hypothermia treatment at 30°C was applied simultaneously with H$_2$O$_2$, for 3 hours. Retinal ganglion cells (RGCs), apoptosis, bipolar and cholinergic amacrine cells, microglia and macroglia were evaluated immunohistologically. Apoptosis rate was additionally analysed via western blot. $\bf Results$ Reduced apoptosis rates through hypothermia led to a preservation of RGCs ($\it P$ < .001). Amacrine cells were rescued after hypothermia treatment ($\it P$ = .17), whereas bipolar cells were only protected partly. Additionally, at 8 days, microglial response due to oxidative stress was completely counteracted via hypothermia ($\it P$ < .001). $\bf Conclusions$ H2O2 induced strong degenerative processes in porcine retinas. The role of oxidative stress in the progression of retinal diseases makes this ex vivo organ culture model suitable to investigate new therapeutic approaches. In the present study, the damaging effect of H$_2$O$_2$ to several retinal cell types was counteracted or strongly alleviated through hypothermia treatment. Especially RGCs, which are affected in glaucoma disease, were protected due to a reduced apoptosis rate through hypothermia

Simultaneous complement response via lectin pathway in retina and optic nerve in an experimental autoimmune glaucoma model

Glaucoma is a multifactorial disease and especially mechanisms occurring independently from an elevated intraocular pressure (IOP) are still unknown. Likely, the immune system contributes to the glaucoma pathogenesis. Previously, IgG antibody depositions and retinal ganglion cell (RGC) loss were found in an IOP-independent autoimmune glaucoma model. Therefore, we investigated the possible participation of the complement system in this model. Here, rats were immunized with bovine optic nerve homogenate antigen (ONA), while controls (Co) received sodium chloride ($\it n$ = 5–6/group). After 14 days, RGC density was quantified on flatmounts. No changes in the number of RGCs could be observed at this point in time. Longitudinal optic nerve sections were stained against the myelin basic protein (MBP). We could note few signs of degeneration processes. In order to detect distinct complement components, retinas and optic nerves were labeled with complement markers at 3, 7, 14, and 28 days and analyzed. Significantly more C3 and MAC depositions were found in retinas and optic nerves of the ONA group. These were already present at day 7, before RGC loss and demyelination occurred. Additionally, an upregulation of C3 protein was noted via Western Blot at this time. After 14 days, quantitative real-time PCR revealed significantly more $\it C3$ mRNA in the ONA retinas. An upregulation of the lectin pathway-associated mannose-serine-protease-2 (MASP2) was observed in the retinas as well as in the optic nerves of the ONA group after 7 days. Significantly more MASP2 in retinas could also be observed via Western Blot analyses at this point in time. No effect was noted in regard to C1q. Therefore, we assume that the immunization led to an activation of the complement system via the lectin pathway in retinas and optic nerves at an early stage in this glaucoma model. This activation seems to be an early response, which then triggers degeneration. These findings can help to develop novel therapy strategies for glaucoma patients

Anti-inflammatory cytokine and angiogenic factors levels in vitreous samples of diabetic retinopathy patients

Evaluation of cytokines in patients with diabetic retinopathy (DR) is important for the identification of future additive or alternative treatment options. Therefore, vitreous samples were obtained from patients with DR and patients with macular hole or macular pucker (control group) during 23-gauge-vitrectomy (n = 17/group). The levels of three pro-inflammatory (IL-1$\beta$, IL-6, IFN-$\gamma$) and pleiotropic cytokines (IL-2, IL-4, IL-13) as well as VEGF, VEGF-A, and PGF were measured using an enzyme linked immunosorbent assay (ELISA). IL-1$\beta$ (p = 0.02) and IFN-$\gamma$ (p = 0.04), two of the three tested pro-inflammatory cytokines, were elevated in the DR patients, while IL-6 (p = 0.51) level was comparable in both groups. Moreover, in DR samples, a trend towards an IL-13 down-regulation (p = 0.36) was observable. The IL-2 (p = 0.62) and IL-4 (p = 0.78) levels were comparable in both groups. All analyzed angiogenetic factors were up-regulated in DR patients (VEGF: p<0.001; VEGF-A: p = 0.002; PGF: p<0.001). The up-regulation of angiogenetic factors underline their importance in DR development. However, the interaction of the other cytokines showed an interesting pattern. Pro-inflammatory cytokines were also up-regulated, which could be evidence for inflammation processes in the diabetic retina. Furthermore, it seems that a counter response of immunomodulatory cytokines is in an initial process, but not strong enough to regulate the processes. Therefore, to support these anti-inflammatory mechanisms might be additive treatment option in the future

Laquinimod protects optic nerve and retina in an experimental autoimmune encephalomyelitis model

$\bf Background:$ The oral immunomodulatory agent laquinimod is currently evaluated for multiple sclerosis (MS) treatment. Phase II and III studies demonstrated a reduction of degenerative processes. In addition to anti-inflammatory effects, laquinimod might have neuroprotective properties, but its impact on the visual system, which is often affected by MS, is unknown. The aim of our study was to investigate potential protective effects of laquinimod on the optic nerve and retina in an experimental autoimmune encephalomyelitis (EAE) model. $\bf Methods:$ We induced EAE in C57/BL6 mice via MOG$_{35–55}$ immunization. Animals were divided into an untreated EAE group, three EAE groups receiving laquinimod (1, 5, or 25 mg/kg daily), starting the day post-immunization, and a nonimmunized control group. Thirty days post-immunization, scotopic electroretinograms were carried out, and mice were sacrificed for histopathology (HE, LFB), immunohistochemistry (MBP, Iba1, Tmem119, F4/80, GFAP, vimentin, Brn-3a, cleaved caspase 3) of the optic nerve and retina, and retinal qRT-PCR analyses ($\textit {Brn-3a, Iba1, Tmem119, AMWAP, CD68, GFAP}$). To evaluate the effect of a therapeutic approach, EAE animals were treated with 25 mg/kg aquinimod from day 16 when 60% of the animals had developed clinical signs of EAE. $\bf Results:$ Laquinimod reduced neurological EAE symptoms and improved the neuronal electrical output of the inner nuclear layer compared to untreated EAE mice. Furthermore, cellular infiltration, especially recruited phagocytes, and demyelination in the optic nerve were reduced. Microglia were diminished in optic nerve and retina. Retinal macroglial signal was reduced under treatment, whereas in the optic nerve macroglia were not affected. Additionally, laquinimod preserved retinal ganglion cells and reduced apoptosis. A later treatment with laquinimod in a therapeutic approach led to a reduction of clinical signs and to an improved b-wave amplitude. However, no changes in cellular infiltration and demyelination of the optic nerves were observed. Also, the number of retinal ganglion cells remained unaltered. $\bf Conclusion:$ Fromour study, we deduce neuroprotective and anti-inflammatory effects of laquinimod on the optic nerve and retina in EAE mice, when animals were treated before any clinical signs were noted. Given the fact that the visual system is frequently affected by MS, the agent might be an interesting subject of further neuro-ophthalmic investigations