Changes in vitreal protein profile and retina mRNAs in Reeler mice: NGF, IL33 and Müller cell activation

A possible link between Nerve Growth Factor (NGF) and Reelin might take place during impaired retinal development occurring in the Reelin deficient mouse model (Reeler). To better characterize NGF and retina impairments at the Reeler retina, vitreous and retina were investigated by means of protein expression and glial cell activation. Reeler (n = 9; RELN-/-) and WT (n = 9; wild-type RELN+/+, B6C3Fe) mice were analyzed at 14, 21 and 28 postnatal days (p). Retinas and vitreous were subjected to confocal analysis and protein array, followed by conventional analysis. A significant increase of NGF, IL33 and TIMP1, a trend to a decrease of IL12 and IL6, as well as a significant decrease of NT3 were detected in Reeler vitreous, particularly at p28 (p<0.05). MIP3β mRNA was decreased while IL33mRNA was significantly upregulated in Reeler retina. Increased number of GFAP+ and Nestin+ cells as well as upregulation of Glutamine Synthetase and Nestin mRNAs were observed in Reeler retinas (p<0.05). These findings extend our previous studies on Reeler retina showing a selective Müller cell activation. NGF and IL33 release into vitreous would suggest a local activation of Müller cells, in addition to retinal ganglion and accessory cells. Overall, the data from this experimental study would strength the potential neuroprotective role played by activated Muller cells through NGF release.


Introduction
The absence of Reelin-a glycoprotein crucial for physiological retinogenesis-has been recently associated with changes in both Nerve Growth Factor (NGF) protein and mRNA in the retina [1][2][3]. NGF and Reelin have been reported to take actively part in neurogenesis and retinogenesis [4][5][6]. NGF has been hypothesized to interact with Reelin by modulating neuronal migration, neurodevelopment and other physiological processes in the central nervous system and retina [1,2,7]. NGF activities encompass cell proliferation, cytoskeletal reorganization, migration, differentiation, survival and/or apoptosis [4,8]. In the retina, NGF modulates retinal cell development, differentiation and functional activity, and promotes survival/recovery of PLOS  Retinal Ganglion Cells (RGCs), photoreceptors and optic axons after experimental injuries as well as normal functional and anatomical development of visual acuity and binocularity [4,[8][9][10].
The neurodegenerative process occurring in Reeler retina evolves through a series of changes at different cell types (neural, vascular and glial cells) and comprises several overlapping/interrelated molecular pathways [5]. Glial cell activation (astrocytes, Müller cells and resident microglia) represents a crucial step for protecting neurones from degeneration [7]. Müller cells work in concert with other glial cells and neurones to guarantee optimal development of retinal structure [9][10][11][12]. An open question regards the glia cell activation upon Reelin deficiency and the possibility for NGF to maintain retinal homeostasis via glial cell activation, as observed in other neuronal degenerating tissues [13,14].
Therefore, the aim of this study was to look for some proinflammatory/profibrogenic mediator changes in the vitreous and retina as well as glial cell activation in the retina of Reeler mice.

Animals and ethical approval
Eighteen (18) animals were used for the study, including 9 Reeler (RELN -/-; 9-11 gr bodyweight) and 9 WT (RELN +/+ ; B6C3Fe; 12-14 gr body-weight) mice (Charles River, Calco, Como). Experimental procedures were approved by the Ethical Committee of Tor Vergata University (Rome, Italy), according with ethical standards stated in the Declaration of Helsinki and the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research. All the steps in the procedure were in compliance with the directive 2010/63/EU guidelines, under the authorization provided by the Italian Ministry of Health. All efforts were made to minimize suffering.
All analytic and molecular grade reagents were purchased from Carlo Erba (Milan, Italy), Euroclone (Milan, Italy) and Sigma (Milan, Italy), otherwise specified in the text. Daily produced MilliQ RNAse-free water was provided for biochemical and molecular purposes (Direct Q5; Millipore Corp., Billerica, MA).

Experimental procedure: Vitreous and retina
At postnatal day (p) p14, p21 and p28, mice were anaesthetized by intraperitoneal injection of 2 mg/mL ketamine (0.2 mL/10 gr body-weight; Ketavet, Gellini Pharmaceutics, Italy) and 0.23 mg/mL medetomidine (0.24 mL/10 gr body-weight; Domitor, Orion Corp., Espoo, Finland) mixture. Sampling was carried out under a dissecting microscope (SMZ645; Nikon, Tokyo, Japan) equipped with cold-light optic fibers (PL2000 photonic; Axon, Vienna, Austria), as previously reported with slight modifications [2]. A corneal incision was produced and lens, retina and vitreous were collected in a microvial with separating membrane. Centrifugation (13.000rpm/15min) was performed to detach vitreous from retina and lens. Vitreous (left/ right eyes) and retina (right eye) were appropriately stored for biochemical and molecular studies. Other retinas (left eye) were used for immunofluorescent analysis.

Protein array
A customised chip-based array was used to quantify inflammatory/profibrogenic factors in vitreous and retinal lysates, between a list of potential candidates (G-series arrays; Ray Biotech, Norcross, CA). Each glass-slide comprised 14 identical sub-arrays containing 50 factors (antibody spots in duplicate) retrospectively selected by literature search [15][16][17][18]. Reeler and WT samples were processed simultaneously. Briefly, normalized protein extracts (50μg total protein; 70μL per well) were diluted in appropriate buffer and hybridized in sub-arrays. Washing, detection and labelling steps were performed according to the manufacturer's recommendation. Spin-dried slides were scanned in a GenePix 4100A Microarray platform (Molecular Devices LLC, Sunnyvale, Silicon-Valley, CA). Capturing conditions and image digital acquisitions were done as previously reported [19]. Images were uniformly adjusted for size, brightness, contrast and chip-to-chip comparisons by the software and provided as 8-bit Tiff format (Axon GenePix Pro 6.0.1.25 software; Molecular Devices). Inter-assay normalization was guaranteed by the presence of multiple internal controls for each sub-array. The sensitivity range was 3.8-56 pg/mL, as provided by the manufacturer. Microarray data are available in the ArrayExpress database (http://www.ebi.ac.uk/arrayexpress) under accession number E-MTAB-7622.

Total RNA extraction, cDNA synthesis and real-time PCR analysis
Total RNA was extracted from retinas according to the TRIfast procedure (EuroClone) and rehydrated in 10μL fresh RNAse free MilliQ water, before treating with RNase-Free DNaseI (2U/μL; AM-1907; Turbo DNA free kit; Ambion Ltd., Huntingdon, Cambridgeshire, UK). Quantity and purity (>1.8; A280 program, Nanodrop) as well as sign of RNA degradation (1% agarose gel analysis) were checked. cDNAs were generated from normalised templates (1μg RNA) (ImProm-II Reverse Transcription System; Promega Corp., Madison, USA) in a One Cycler programmable thermocycler (PeqLab Biotech, Erlangen, Germany) and amplified using the SYBR Green PCR core reagent kit (Applied Biosystems, Foster City, CA) in Eco Real Time PCR thermocycler equipped for 48-well plate (Illumina, MA, USA). Specific primers and amplification procedure are summarised in Table 1. Samples were amplified in duplicate and in parallel with negative controls (either without template or with mRNA as template). Real Cycle numbers (Cn) were recorded and normalized for referring genes run in parallel (nCq = Cq target -Cq referring ). The Cq averages were calculated from these replicates and showed as expression ratios (log2-scale) of a normalized target gene REST analysis [20].

Data management and statistical analysis
Graphics were assembled by using the Prism5software (GraphPad software Inc., La Jolla, CA, USA), while statistical analysis was carried out with the StatView II software for PC (Abacus Concepts. Inc., Barkley, CA, USA). A p<0.05 value was considered statistically significant.
For chip-based array, the Fluorescent Intensity (FI) values were obtained by subtracting the background signal (GenePix Pro 6.0.1.25 software-Molecular Devices). Single FI values were entered into a Microsoft Excel database (Microsoft, Redmond, WA, USA) and duplicate spots outside the 10% coefficient of variability were refused from the statistical analysis. FDR value of 0.01 was set. FI averages (means±SD) were calculated from replicates (2 spots) of not-pooled samples. The following cut-offs were used: Fold Changes � or � 2 (FC; herein defining a given candidate factor with respect to control) and pValues � 0.001 for multiple testing (p = 0.05/50 targets; two tails T-test followed by Bonferroni Correction).

A specific protein profile characterizes vitreous at p14, p21 and p28
To characterize the pro-inflammatory/fibrogenic profile, both vitreous and retinal extracts were analyzed for protein array (see above). The Volcano plots in Fig 1A highlight  Significant results are listed in Table 2.

Reactive müller cells populate Reeler retinas at p28
Serial sections were immunostained for GFAP, Nestin and CD45 (leukocyte common antigen expressed by T/B lymphocytes, granulocytes, monocytes/macrophages and retinal microglia) specific antibodies [21]. Vimentin and GS were not investigated by immunofluorescence. An increased immunoreactivity for GFAP (red) was observed in Reeler retinas (Fig 3B) with respect to WT (Fig 3A), as merged on a blue nuclear staining. Increased GFAP immunoreactivity was mainly localized at the Ganglion Cellular Layer (GCL), although a slight immunostaining was also detected at the inner nuclear layer (INL; see arrows). A co-expression of GFAP (red) and Nestin (green) was observed in Reeler retinas, as pointed by arrow in the white frame (Fig 3B), with respect to WT retina (A). This GFAP-Nestin co-expression in Reeler vs. WT retinas is better shown in related magnifications (Fig 3D vs. 3C). A positive correlation between GFAP and Nestin was provided by Kendall analysis (Fig 3E) Fig 3F. Either alone or in combination with GFAP, not significant changes were

Discussion
Herein, we show for the first-time changes in vitreous proteins and the presence of reactive Müller cells in the retinas of Reeler mice. The results are below discussed. Our previous studies highlighted the overexpression of NGF and NGF receptors (mRNA/ protein) in Reeler retina at p14, p21 and particularly p28, postulating a potential compensatory NGF activity in Reelin deficiency [1,2]. Albeit Reelin deficient retina does not show any crucial inflammatory state, the presence of several cell-to-cell and cell-to-mediator events cannot be excluded during the neurodegenerative process that might include the expression and/or modulation of gene and related products [1,3]. Therefore, to better characterize retinal microenvironment and provide further information on NGF interplays in Reelin-impaired retinogenesis, possible mediator changes were investigated in vitreous and retina from Reeler and WT mice, at the same time-points (p14-p21-p28).
First, increased expression for NGF, IL33 and TIMP1 proteins as well as a trend to an increase for MMP9 and TNFR-I/II were quantified in p28 Reeler vitreous. Vitreous represents a precious tool to support retinal disorder management (personalized medicine), being a reservoir of active mediators and an indicator of the underneath suffering retina, especially as vitreal reflux [16,22,23]. Vitreous is a normal clear jelly-form ocular fluid composed of collagens, sulphated proteoglycans and hyaluronan [24]. Widely, pathological vitreous is filled of inflammatory/angiogenic factors (cytokines, chemokines, growth factors and enzymes of extracellular matrix), depending on the grade of retinal inflammation/degeneration conditions or even microcirculation / systemic influences [25,26]. The detection of changes in NGF, IL33 and TIMP1 as well as MMP9 and soluble TNFR-I/II proteins in Reeler vitreous suggest the presence of a "low" proinflammatory/profibrogenic profile, in line with previous studies [27]. This Reeler-associated protein profile might be consistent with the degenerative condition due to neuron-to-neuron impairments, morphological changes and reduced retinal cell migration (rod bipolar cells) [5,28]. The overexpression of NGF in Reeler vitreous at p21 and p28 is in line with our previous studies on Reeler retina and other systems [1,2,9]. With respect to MMP9 and TIMP1 changes in Reeler vitreous, a possible explanation might lay in a steady MMP9/TIMP1 ratio known to be crucial for the extracellular matrix stability and particularly during development and synaptic plasticity [29]. Furthermore, the increased TIMP1 levels in Reeler vitreous might be an attempt to counteract the lack of Reelin by inhibiting the MMPs that are involved in the cleavage of Reelin glycoprotein, as observed in other experimental models [30]. Although needing further investigations, the increased MMP7 and MMP13 levels might participate in tissue remodeling and protective effects [30]. Although not significant, the overexpression of TNF-RI/II might be consistent with the recent evidence that a soluble counterpart exists for a number of signaling receptors (for review, see [31]). Soluble receptors participate to cell apoptosis, survival, proliferation and differentiation, and particularly the "receptor-shedding" mechanism seems to modulate or inhibit ligand activity/function by preventing the interaction with proper cellular targets [32].
The consistent overexpression of vitreal IL33 at p28 appears of great interest. A possible explanation might be found in the pathogenic IL33 route during the impaired retinogenesis occurring in Reelin-deficient cells/retina. Liu and coworkers associated IL33 human expression with retinal lesions occurring in Age related Macular Disease as well as with the loss of photoreceptor and retinal ganglion cells in the experimental model [33]. In other systems, the constant "bright light exposure"-a condition that can trigger photoreceptor lost-was associated with IL33 overexpression and explained as an attempt to balance the minor cell damage and glial cell activation [34,35].
Pathological vitreous retains most of retina-derived products, representing an indirect source of retina microenvironment information in vitreoretinal practice [25]. The majority of vitreous-detectable soluble proteins originate from vitreous itself (hyalocytes) or the surrounding tissues (ciliary body and retina), while albumin might be also of plasma source [26]. Therefore, a molecular approach was performed on retinal total RNA extracts to validate some protein changes quantified in the vitreous. While the biomolecular expression of NGF in the retina was previously investigated, the herein observed IL33mRNA overexpression in retinal extracts corroborated the biochemical data on vitreous, suggesting a potential IL33 driven activity on Reeler retinal cells. The significant downregulation of MIP3α and MIP3β mRNAs in Reeler retinas can sustain the absence of inflammation and local macrophage recruitment [1,2,36]. The observation of increased IL33mRNA expression in the retina might provide explanation for increased IL33 in the vitreous and suggest in situ glial cell (Müller cell) activation. In rodent and human retina, IL33 (IL1 family) is locally expressed (epithelial, endothelial, glial and Müller cells), modulates immune cells (T-helper, macrophages, eosinophils and mast cells), recruits innate cells (neutrophils, macrophages, dendritic cells and eosinophils), induces hematopoietic stem / progenitor cell mobilization and even triggers the Th2-cytokine pattern [37][38][39][40]. Of interest, Müller cells (i.) are crucial in maintaining laminar structure, neuronal survival, metabolic homeostasis and retinal regeneration; (ii.) participate in reactive gliosis in response to injury and interesting (iii.) synthesize/release IL33 in vivo under stressing conditions [35,41].
To verify the presence of reactive Müller cells, we checked for GFAP and GS mRNAs in retinal extracts as well as GFAP and Nestin immunoreactivity in retina sections, all known as Müller cell markers in aged, damaged, injured and stressed retinas [42]. Merely, GS and Nestin represent well-known marker for Müller cell identification in situ while GFAP represents a marker of activated Müller cells [43]. A significant increase of GS mRNA expression and immunoreactivity (cell number and protein accumulation in Müller cells) occurred in Reeler retinas. Since GFAP is a marker of both astrocytes and activated Müller cell, the observation of increased GFAP-positive and GFAP/Nestin-positive cells would suggest the presence of astrocytes and activated Müller cells [44]. The absence of GFAP/CD45-immunoreactivity would exclude the microglia involvement [45]. Newly generated GFAP-positive glial cells (gliogenesis), as well as their local differentiation into astrocytes, have been previously reported in Reeler mice and this increase might reflect the "misorganization" of retinal layers [5,28]. According to literature, gliosis and reactive Müller cells occur in response to injury and/or upon cytokine as well as growth factor stimulation (VEGF, NGF, . . .) [41]. On the other hand, both astrocytes and Müller cells are source of NGF and in turn can utilize NGF by means of trkA NGF expression, as reported in human retinal diseases and experimental models [43,46]. Therefore, the increased NGF expression described in Reeler retina and the observation of increased NGF level in Reeler vitreous might be view as a direct product of astrocytes/Müller cell activation, in addition to the neuronal task [1,2,47]. Corroborating data indicate that Müller cells are active players in retinal injury and chronic inflammatory/autoimmune mediated retinal disorders [48]. Long-term gliosis releases a plethora of inflammatory cytokines that lead to secondary injury, exacerbating the inflammatory reaction [47]. Local reactive gliosis might be view as an attempt to protect neuronal tissue from further damage [49].

Conclusions
Taken together, the finding herein reported reinforce the observation that NGF might be a compensatory effector of Reelin deficiency during postnatal brain and retinal development. NGF appears to function as an intrinsic determinant of cell migration, acting as a compensative regulator of Reelin expression [50,51]. Cumulating, the NGF and IL33 overexpression in Reeler vitreous and the Müller cell activation in the retina would strength the presence of a protective mechanism to prevent damage and/or promote tissue repair. According to literature, the proposed (neuro)-protective attempt might be not enough due to the activation of other proinflammatory target genes (cytokines, chemokines, growth and neurotoxic factors) [2,[9][10]18,25,33,41,51]. On the other hand, the overexpression of NGF, IL33, TIMP1, MMP9 and sTNFR-I/II factors to counteract/delay failures in retinogenesis and the potential NGF modulation of Müller cell activation represent interesting points that deserve further investigation for alternative strategies not restricted to reelin deficiency.