Alexander Tschulakow

Effects of a single intravitreal injection of aflibercept and ranibizumab on glomeruli of monkeys.

Purpose It is known that endothelial cells in the kidney are also strongly VEGF-dependent. Whether intravitreal drugs can be detected within the glomeruli or affect VEGF in glomerular podocytes is not known. Therefore, the aim of this pilot study was to investigate the effects of a single intravitreal injection of aflibercept and ranibizumab on glomeruli of monkeys. Methods The kidneys of eight cynomolgus monkeys, which were intravitreally injected either with 2 mg of aflibercept or with 0.5 mg of ranibizumab, were investigated one and seven days after injection. Two animals served as controls. The distribution of aflibercept, ranibizumab and VEGF was evaluated using anti-Fc- or anti-F(ab)-fragment and anti-VEGF antibodies respectively. The ratio of stained area/nuclei was calculated using a semi-quantitative computer assisted method. Glomerular endothelial cell fenestration was quantified in electron microscopy using a systematic uniform random sampling protocol and estimating the ratio of fenestrae per µm. Results Compared to the controls, the anti-VEGF stained area/nuclei ratio of the ranibizumab-treated animals showed no significant changes whereas the stained areas of the aflibercept-treated monkeys showed a significant decrease post-treatment. Immune reactivity (IR) against aflibercept or ranibizumab was detected in aflibercept- or ranibizumab treated animals respectively. The number of fenestrations of the glomerular endothelial cells has shown no significant differences except one day after aflibercept injection in which the number was increased. Conclusion Surprisingly, both drugs could be detected within the capillaries of the glomeruli. After a single intravitreal injection of aflibercept, VEGF IR in the podocytes was significantly reduced compared to controls. Ranibizumab injection had no significant effect on the glomerulis VEGF level. Whether this is caused by aflibercepts higher affinity to VEGF or because it is used in a higher stoichiometric concentration compared to ranibizumab remains to be investigated.

Establishment of a novel retinoblastoma (Rb) nude mouse model by intravitreal injection of human Rb Y79 cells – Comparison of in vivo analysis versus histological follow up

ABSTRACT Retinoblastoma (Rb) is the most frequent primary intraocular tumour in children and, if left untreated, can cause death. Preclinical animal models that mimic molecular, genetic, and cellular features of cancers are essential for studying cancer and searching for promising diagnosis and treatment modalities. There are several models described for Rb, but none of them fully meet our requirements. The aim of this study was to create a novel xenograft-nude mouse-model with broad application possibilities, which closely resembles the clinical observations of Rb patients and which could be used to investigate the development and spread of the tumour by using scanning laser ophthalmoscopy/optical coherence tomography (SLO/OCT) as well as histology methods. We injected human retinoblastoma Y79 cells intravitreally in both eyes of immune-deficient nude mice. The incidences of retinoblastoma as well as growth velocity were analysed 3, 6, 9 and 12 weeks after cell injection in vivo by SLO/OCT as well as ex vivo by electron microscopy (EM) and hematoxylin/eosin (HE) staining. Moreover, internal organs were histologically screened for potentially occurring metastases. Three weeks post-injection, animals developed a retinoblastoma, and after five weeks tumour growth resulted in swelling of the eyes in individual animals, showing a similar phenotype to that of untreated Rb patients at advanced stages of tumour-development. After 12 weeks, 67.5% of all analysed eyes (29 of 42) contained a retinoblastoma. At early stages of Rb development, the SLO/OCT analysis correlated with the histology results. If the tumours were too large, only histological investigations were feasible. The ultrastructural characteristics of the xenograft-tumours were very similar to those described for patients tumours. In one mouse, brain metastases were observed. Our retinoblastoma mouse model closely resembles the human disease. SLO/OCT can be used for the detection of Rb at early stages of development and could be used for monitoring the success of future therapies. Summary: We present a novel retinoblastoma nude xenograft mouse model which closely resembles the human disease and allows broad application possibilities and a comparison of in vivo and histological analysis.

The anatomy of the foveola reinvestigated

Objective In the foveola of the eye, photoreceptors and Müller cells with a unique morphology have been described, but little is known about their 3D structure and orientation. Considering that there is an angle-dependent change in the foveolar photoreceptor response for the same light beam, known as the Stiles Crawford Effect of the first kind (SCE I), which is still not fully understood, a detailed analysis of the anatomy of the foveolar cells might help to clarify this phenomenon. Methods Serial semithin and ultrathin sections, and focused ion beam (FIB) tomography were prepared from 32 foveolae from monkeys (Macaca fascicularis) and humans. Foveolae were also analyzed under the electron microscope. Serial sections and FIB analysis were then used to construct 3D models of central Müller and photoreceptor cells. In addition, we measured the transmission of collimated light under the light microscope at different angles after it had passed through human foveae from flat mounted isolated retinae. Results In monkeys, outer segments of central foveolar cones are twice as long as those from parafoveal cones and do not run completely parallel to the incident light. Unique Müller cells are present in the central foveolae (area of 200 µm in diameter) of humans and monkeys. Light entering the fovea center, which is composed only of cones and Müller cells, at an angle of 0° causes a very bright spot after passing through this area. However, when the angle of the light beam is changed to 10°, less light is measured after transpasssing through the retina, the foveolar center becomes darker and the SCE-like phenomenon is directly visible. Measurements of the intensities of light transmission through the central foveola for the incident angles 0 and 10° resemble the relative luminance efficiency for narrow light bundles as a function of the location where the beam enters the pupil as reported by Stiles and Crawford. The effect persisted after carefully brushing away the outer segments. Conclusion We show that unique cones and Müller cells with light fibre-like properties are present in the center of the fovea. These unique Müller cells cause an angle dependent, SCE-like drop in the intensity of light guided through the foveola. Outer segments from the foveolar cones of monkeys are not straight.

Ultrastructural alterations in the retinal pigment epithelium and photoreceptors of a Stargardt patient and three Stargardt mouse models: indication for the central role of RPE melanin in oxidative stress

Background Stargardt disease (SD) is characterized by the accumulation of the age-pigment lipofuscin in the retinal pigment epithelium (RPE) and subsequent neuroretinal degeneration. The disease leads to vision loss early in life. Here, we investigate age-dependent ultrastructural changes in three SD mouse models: albino Abca4-/- and pigmented Abca4-/- and Abca4-/-.Rdh8-/- mice. Since we found indications for oxidative stress primarily in albino SD mice, we tested RPE melanin for its antioxidative capabilities. Methods SD mouse eyes were investigated by light, fluorescence and electron microscopy and were compared to the respective albino and pigmented wild type mice and to a human donor SD eye. To confirm the role of RPE melanin in scavenging oxidative stress, melanin from S. officinalis as a standard and porcine RPE were tested for their capability to quench superoxide anions. Results Histological alterations indicative of oxidative stress and/or lysosomal dysfunction were present in albino Abca4-/- and Abca4-/-.Rdh8-/- mice. Retinal damage, such as inner segment rupture and pyknotic or free photoreceptor nuclei in the subretinal space and RPE vacuolization were exclusively found in albino Abca4-/- mice. Shortened and disorganized photoreceptor outer segments and dead RPE cells were found in albino Abca4-/- and Abca4-/-.Rdh8-/- mice, with earlier onset in albino Abca4-/- mice. Undegraded phagosomes and lipofuscin accumulation were present in the RPE of all three SD strains, but numbers were highest in Abca4-/-.Rdh8-/- mice. Lipofuscin morphology differed between SD strains: (melano-)lipofuscin granules in pigmented Abca4-/- mice had a homogenous electron density and sharp demarcations, while lipofuscin in albino Abca4-/- mice had a flocculent electron density and often lacked a surrounding membrane, indicating loss of lysosomal integrity. Young Abca4-/-.Rdh8-/- mice showed (melano-)lipofuscin granules with homogenous electron density, while in aged animals granules with flocculent electron density predominated. Both strains of pigmented SD mice had melanolipofuscin clusters as found in the human SD eye. Like melanin from S. officinalis, porcine RPE melanin can also quench superoxide anions. Discussion The presented pathologies in albino Abca4-/- and Abca4-/-.Rdh8-/- mice suggest oxidative stress and/or lysosomal dysfunction within the RPE. Since albino Abca4-/- mice have the earliest onset and severest damage and as absence of melanin and also melanin turnover with age are known to diminish RPEs anti-oxidative properties, we assume that RPE melanin plays a role in SD related damages. A lack of pathology in pigmented Abca4-/- mice due to lower stress levels as compared to the Abca4-/-.Rdh8-/- mice underlines this hypothesis. It is also supported by the finding that RPE melanin can quench superoxide anions. We therefore suppose that RPE melanin is important in retinal health and we discuss its role as an oxidative stress scavenger.

Data showing the shapes of cones and Müller cells within the fovea of monkeys reconstructed from serial sections and focused ion beam analysis

The data presented in this article are related to the research paper entitled “The anatomy of the foveola reinvestigated” (Tschulakow et al., 2018) [1]. Here we show the original aligned serial sections through the foveal centre of monkeys at different planes of section and 3 D models of central foveal cells.

The radioprotector ortho-phospho-L-tyrosine (pTyr) attenuates the side effects of fractionated irradiation in retinoblastoma mouse models but also decreases the local tumour control

BACKGROUND Radiotherapy (RT) is used to treat retinoblastoma (Rb), the most frequent ocular tumour in children. Besides eradicating the tumour, RT can cause severe side effects including secondary malignancies. This study aimed to define whether the radioprotector ortho-phospho-L-tyrosine (pTyr) prevents RT-induced side effects and affects local tumour control in a xenograft and a genetic orthotopic Rb mouse model. METHODS B6;129-Rb1tm3Tyj/J (Rb+/-) and Y79-Rb cell-xenografted nude mice were fractionated external beam irradiated (15 fractions of 5Gy 6MV photons during 3weeks) with or without pTyr pre-treatment (100mg/kg BW, 16h prior to each irradiation). One, three, six and nine months after RT, tumour control and RT toxicity were evaluated using in vivo imaging and histology. We also analysed pTyr dependant post irradiation cell survival and p53 activity in vitro. RESULTS In vitro pTyr pre-treatment showed no radioprotection on Y79 cells, but led to p53 stabilisation in unirradiated Y79 cells and to a facilitation of radiation-induced p21 up-regulation, confirming a modulation of p53 activity by pTyr. In both mouse models, secondary tumours were undetectable. In Rb+/- mice, pTyr significantly lowered RT-induced greying of the fur, retinal thickness reduction and photoreceptor loss. However, in the xenografted Rb model, pTyr considerably decreased RT-mediated tumour control, which was observed in 16 out of 22 control eyes but in none of the 24 pTyr treated eyes. CONCLUSIONS In Rb+/- mice pTyr significantly prevents RT-induced greying of the fur as well as retinal degeneration. However, since non-irradiated control mice were not used in our study, a formal possibility exists that the effect shown in the retina of Rb+/- mice may be due to ageing of the animals and/or actions of pTyr alone. Unfortunately, as tested in a xenograft model, pTyr treatment reduced the control of Rb tumours.