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In vivo bioluminescence imaging of neurogenesis – the role of the blood brain barrier in an experimental model of Parkinson's disease

Bioluminescence imaging in transgenic mice expressing firefly luciferase in Doublecortin+ (Dcx) neuroblasts might serve as a powerful tool to study the role of neurogenesis in models of brain injury and neurodegeneration using non‐invasive, longitudinal in vivo imaging. Therefore, we aimed to use BLI in B6(Cg)‐Tyrc‐2J/J Dcx‐Luc (Doublecortin‐Luciferase, Dcx‐Luc) mice to investigate its suitability to assess neurogenesis in a unilateral injection model of Parkinson's disease. We further aimed to assess the blood brain barrier leakage associated with the intranigral 6‐OHDA injection to evaluate its impact on substrate delivery and bioluminescence signal intensity. Two weeks after lesion, we observed an increase in bioluminescence signal in the ipsilateral hippocampal region in both, 6‐OHDA and vehicle injected Dcx‐Luc mice. At the same time, no corresponding increase in Dcx+ neuroblast numbers could be observed in the dentate gyrus of C57Bl6 mice. Blood brain barrier leakage was observed in the hippocampal region and in the degenerating substantia nigra of C57Bl6 mice in vivo using T1 weighted Magnetic Resonance Imaging with Gadovist® and ex vivo using Evans Blue Fluorescence Reflectance Imaging and mouse Immunoglobulin G staining. Our data suggests a BLI signal dependency on blood brain barrier permeability, underlining a major pitfall of substrate/tracer dependent imaging in invasive disease models.

Introduction
Adult neurogenesis, the life-long process in which new nerve cells are generated in the brain, is an intriguing target for restoring neurologic function after injury. It mainly takes place in two distinct regions: the dentate gyrus (DG) of the hippocampus generating new dentate granule cells, and the subventricular zone (SVZ) lining the lateral ventricles and generating new neurons for the olfactory bulb (Ming & Song, 2011). During neurogenesis, neural stem cells give rise to neuroblasts expressing the marker Doublecortin (Dcx) until their final differentiation into mature neurons.
Numbers of Dcx + cells were shown to correlate with the rate of neurogenesis (Couillard-Despres et al., 2005), allowing the use of Dcx as a marker to study modulation of neurogenesis in disease models and to test pharmacological interventions targeting neurogenesis.
Altered rates of neurogenesis were observed in several neurologic disease models, for example, increased neurogenesis following cerebral ischemia and traumatic brain injury (Ruan et al., 2014). In neurodegenerative diseases such as Alzheimer's, Parkinson's and Huntington's disease (AD, PD, HD, respectively), several reports describe alterations in neurogenesis, varying from increased or unchanged (Fricke et al., 2016) neurogenic rates, to decreased rates (Ruan et al., 2014).
To study the role of neurogenesis in brain injury and neurodegeneration, approaches using non-invasive, longitudinal in vivo imaging are particularly attractive. Approaches enabling the study of adult neurogenesis non-invasively include positron emission tomography (PET) with the tracer 3 0 -deoxy-3 0 -[ 18 F]fluorothymidine ([ 18 F]FLT), Magnetic Resonance Imaging (MRI) with in situ iron oxide particle labelling or genetic MRI reporter genes, and optical imaging (bioluminescence and fluorescence) using viral vectors or transgenic animals for reporter gene delivery and expression (Couillard-Despres et al., 2011;Fricke et al., 2016).
Compared to other imaging approaches, bioluminescence imaging (BLI) in transgenic mice expressing firefly luciferase (Luc) in Dcx positive neuroblasts might serve as a powerful tool to assess neurogenesis in a specific, sensitive and at the same time cost-and timeeffective way.
However, as BLI is dependent on reproducible substrate delivery to the brain, standardised imaging protocols (Aswendt et al., 2013) and conditions with unimpaired blood brain barrier (BBB) are of crucial importance. Consisting of endothelial cells, connected by tight junctions and surrounded by astrocytes and pericytes, the BBB restricts the passage of molecules from the blood stream into the interstitial space of the brain (Abbott et al., 2010). Besides being actively transported, substances enter the brain by passive diffusion, which is highly dependent on the size and hydrophilicity of the given substance. In the bioluminescence reaction, the substrate D-Luciferin is oxidised in an ATP-dependent manner by the firefly luciferase enzyme, leading to light emission. This light emission is linearly correlated with the amount of Luciferase if the substrate is present in excess (de Wet et al., 1987). A biodistribution study with [ 123 I]iodo-D-Luciferin demonstrated that only a small fraction of D-Luciferin reaches the brain, thereby limiting the BLI signal that is generated in the brain (Lee et al., 2003) and making it vulnerable to differences in substrate distribution due to increased BBB permeability.
This study aimed to use BLI in B6(Cg)-Tyrc-2J/J Dcx-Luc (Dcx-Luc) mice to investigate its suitability to assess neurogenesis in a unilateral injection model of Parkinson's disease (PD). We further aimed to assess the BBB leakage that is associated with intranigral 6-OHDA lesion in order to evaluate its impact on substrate delivery to the brain and the resulting BLI signal.

Animal experiments
All animal experiments were performed in accordance with the German laws for animal protection and were approved by the local bureau for animal care (LANUV, Landesamt f€ ur Natur, Umwelt und Verbraucherschutz Nordrhein-Westfalen). Experiments have been performed and reported in compliance with the ARRIVE guidelines. C57Bl6 (Janvier, Saint-Berthevin, France) and B6(Cg)-Tyrc-2J/J Dcx-Luc mice (Dcx-Luc; provided by S. Couillard-Despr es, Paracelsus Medical University, Salzburg, Austria) were housed at constant temperature (23°C) and relative humidity (40%), under a 12 h light/12 h dark schedule. Mice were given ad libitum access to food and water.
Image data analysis of SPECT/CT data was performed using the Inveon TM Research Workplace software package (Siemens Healthcare, Erlangen, Germany). Volumes of interest (VOIs) of equal size and orientation were applied to the right striatum and the cerebellum. A 50% threshold of the VOI maximum was applied to the VOI for right striatum, and the resulting VOI50% right was mirrored to the left brain hemisphere in order to quantify the left striatum (VOI50% left ). Tracer uptake was quantified as mean specific tracer uptake [(mean uptake striatum50% left/rightmean uptake cerebellum)/mean uptake cerebellum] and the specific uptake ratio left/right was calculated. A value below 0.2 was taken as a criterion for successful degeneration. Representative images in Fig. S2 show SPECT/CT images.

Bioluminescence imaging
BLI was performed before lesion and either weekly from week two till week seven post lesion (n 6-OHDA = 5; n vehicle = 3; data presented in Fig. 1) or at week 2 post lesion (n 6-OHDA = 7; n vehicle = 7; data presented in Fig. 6) using the IVIS Spectrum Imaging System and Living Image 4.0 software (PerkinElmer, Waltham, MA, USA). The day before measurement, depilatory cream (Pilca) was used to remove the fur on the head under isoflurane anaesthesia. Mice were injected i. p. with 300 mg/kg D-luciferin in phosphate buffered saline (PBS) without calcium and magnesium (PAA Laboratories). 3 min post D-luciferin injection, mice were anaesthetised with 2.5% isoflurane in 100% oxygen and placed in the imaging system before 14 acquisitions of 60 s were recorded between 6 and 20 min p. i.
[field of view (FOV): B, subject height: 1.5 cm, binning: 4, f/stop: 1]. Grayscale photographic images and bioluminescence colour images were superimposed. Regions of interest were drawn for the right and left OB, SVZ and DG to determine the signal intensity (Average Radiance [p/s/cm 2 /sr]) and are shown in Fig. S1. The time frame with the maximal brain bioluminescence signal was chosen for quantification.

Magnetic resonance imaging (MRI)
MRI was performed in C57Bl6 mice injected with 6-OHDA (n = 20), vehicle (n = 19), or healthy animals (n = 5) at various time points, as summarised in Table S1. In Dcx-Luc animals (n 6-OHDA = 7, n vehicle = 7), MRI images were acquired at 14 days post lesion. To assess BBB leakage with MRI, mice were anaesthetised and catheters were placed as described for the SPECT protocol. A T2 FSE 2D sequence (repetition time/echo time 5253/ 88.5 ms, 24 slices, slice thickness 0.8 mm, spacing between slices 0.9 mm) and a T1 SE 2D sequence (repetition time/echo time 500/ 12.4 ms, 20 slices, slice thickness 0.8 mm, spacing between slices 0.9 mm) were acquired in a nanoScan PET/MRI scanner equipped with an MH20 coil (Mediso Medical Imaging Systems). Gadovist â (Bayer Vital GmbH, Leverkusen, Germany) was injected via the catheter (50 lmol/kg) and a post Gd T1 sequence was acquired. MRI data were analysed using the in-house built software MEDgical.

Evans blue application and imaging
Following the final MRI scan, C57Bl6 (n 6-OHDA = 20, n vehicle = 19, n Healthy = 5) and Dcx-Luc mice (n 6-OHDA = 7, n vehicle = 7) were kept under isoflurane anaesthesia and 4 lL/g bodyweight of a 4% Evans Blue (EB; Sigma-Aldrich) solution in 0.9% NaCl were injected via the catheter. After a circulation time of 30 min, animals were transcardially perfused with 0.9% NaCl followed by 4% PFA and brains were explanted. Photographic images and fluorescence images of the whole brain as well as 2 mm thick coronal slices [sliced with a Stainless Steel Coronal Brain Matrix, Mouse, Slice Width 2 mm (Harvard Apparatus, Holliston, MA, USA)] were acquired from both sides. Photographic images were acquired using a Nikon SMZ800 stereomicroscope equipped with a Nikon SS-2Mv camera.
EB fluorescence was quantified using an IVIS Spectrum Imaging System and Living Image 4.0 software [Filter: excitation 675 nm, emission 720 nm; auto exposure, f/stop: 2, binning: 2, FOV: B, subject height: 5 mm (whole brain)/0 mm (brain slices)]. ROIs of equal size and shape were drawn for the left and right dentate gyrus and substantia nigra and radiant efficiency values ([p/s/cm 2 /sr]/[lW/cm 2 ]) were quantified.
Brain slices were post fixed in 4% PFA and processed for immunohistochemistry.

Microscopy
Microscopy was performed using a Nikon ECLIPSE Ni-E microscope (fluorescence stainings) and a Nikon ECLIPSE 90i microscope (immunoperoxidase stainings), both operated by the NIS-Elements AR software. Images were processed using the Fiji ImageJ distribution (Schindelin et al., 2012;Schneider et al., 2012). For immunoperoxidase stainings (TH and IgG), the whole brain slice was scanned using a 49 magnification. Contrast was enhanced using the 'Enhance Contrast' tool (0% saturated pixels, no histogram equalisation). For fluorescence Dcx stainings, the left and right dentate gyri were scanned in a 209 magnification. For quantification, a dentate gyrus ROI was generated based on the DAPI image and the left/right ratio of the Dcx stained area/DAPI stained area was calculated. Representative images show Z-Stacks in 209 magnification combined to a focused image using the extended depth of focus function. For Iba1 stainings, the whole brain slice was scanned using a 109 magnification and representative images of the injection tract region spanning the hippocampus were acquired as Zstacks in 109 magnification and combined to a focused image using the extended depth of focus function.

Statistical analysis
Statistical analysis was performed using Sigma Plot 13.0 (Systat Software Inc, San Jose, CA, USA) and GRAPH PAD PRISM 7 (Graph-Pad Software, La Jolla, CA, USA). Longitudinal BLI data were subjected to a two-way repeated measurements ANOVA with pairwise multiple comparison procedures (Holm-Sidak method) and mean (M) values are displayed. SPECT data, EB fluorescence ratios, and Dcx/DAPI left/right ratios were compared using a Mann-Whitney Rank Sum Test and median (Mdn) values are shown. For BLI data that was acquired at two time points, median values are shown and differences in time within a group were analysed using the Wilcoxon Signed Rank Test. A P-value below 0.05 was considered as statistically significant.

Results
In vivo imaging of neurogenesis following 6-OHDA lesion In order to induce degeneration of the nigrostriatal system, Dcx-Luc mice were unilaterally injected with 6-OHDA (n = 6) or vehicle (n = 4). One vehicle injected animal died during the pre injection scan and one 6-OHDA injected animal had to be sacrificed due to weight loss (≥ 20%) following lesion. In order to validate the successful lesion in 6-OHDA injected animals and the integrity of the nigrostriatal system in vehicle injected animals, we used [ 123 I]Ioflupane SPECT at week 8 post lesion (Fig. S2), followed by TH immunohistochemistry (data not shown). Striatal [ 123 I]Ioflupane uptake ratios in 6-OHDA compared to vehicle injected animals were significantly decreased (Mdn 6-OHDA = 0.10, n 6-OHDA = 5; Mdn vehicle = 0.93, n vehicle = 3, U = 0, P = 0.036, Mann-Whitney Rank Sum Test, Fig. S2b). Based on our previous experience, we defined a striatal [ 123 I]Ioflupane uptake ratio equal to or lower than 0.2 as a criterion for successful degeneration, leading to the exclusion of two animals from the analysis and a group size of n = 3 per group (Fig. S2c).
BLI measurements were performed before lesion and weekly between week 2 and 7 post lesion (Fig. 1a). In week 1 post lesion, a quantification of BLI signal was not possible due to scar tissue remaining from the surgery. Following injection, vehicle and 6-OHDA injected animals showed no significantly different hippocampus left/right ratios (F 1,41 = 0.114, P = 0.752, two-way repeated measurements ANOVA). Allowing for differences in group, a significantly increased hippocampus left/right BLI signal ratio (Fig. 1b, e) was observed at 2 weeks compared to the pre lesion condition [M w0 = 1.01, SD w0 = 0.03, M w2 = 1.24, SD w2 = 0.08, t 6 = 7.45, P < 0.001, two-way repeated measurements ANOVA with pairwise multiple comparison procedures (Holm-Sidak method)], while SVZ and OB signals were unchanged at all time points (Fig. 1b, c, d).

Potential sources of an increased Dcx-Luc BLI signal
We investigated the expression of Dcx in the dentate gyrus by IHC and the possibility of a facilitated substrate entry to the brain due to BBB leakage induced by our injection procedure by MRI and FRI. C57Bl6 mice received a unilateral injection of 6-OHDA (n = 21) or vehicle (n = 21) into the left substantia nigra and BBB leakage was imaged in vivo by Gadovist â enhanced T1 weighted MRI and ex vivo by FRI of EB extravasation at 2, 7, 14, or 28 days post injection. Two 6-OHDA and one vehicle injected animal died during image acquisition and did not enter the final data analysis. Additionally, five healthy C57Bl6 mice were included in the imaging and all brains were processed for IHC.
Numbers of Dcx + cells in the dentate gyrus of C57Bl6 mice following lesion Following injection of 6-OHDA or vehicle, no obvious difference in Dcx staining between the left (ipsilateral) and right (contralateral) dentate gyrus was observed (Fig. 2a). Quantification of Dcx stained area and DAPI stained area showed unchanged left/right ratio of Dcx/DAPI stained area between 6-OHDA and vehicle ( Fig. 2b; Mann-Whitney Rank Sum Test). On day 7, a significantly increased left/right ratio was observed in the 6-OHDA injected group compared to the healthy control group. In addition, a significantly increased ratio was observed in the vehicle injected group compared to healthy controls on day 14 and 28 (Mdn 6-OHDA, d7 = 1.72, U = 2.0, P = 0.032; Mdn Vehicle, d14 = 1.36, U = 2.0, P = 0.032; Mdn Vehicle, d28 = 1.42, U = 2.0, P = 0.032; all tested against Mdn Healthy = 0.82; Mann-Whitney Rank Sum Test). Taken together, the observed alterations in hippocampal Dcx expression following injection do not follow the same temporal pattern as the BLI signal.
The intranigral injection procedure leads to blood brain barrier damage in the dentate gyrus region To investigate whether a facilitated substrate entry could be the basis of the observed BLI signal increase, BBB leakage was assessed in a semi quantitative way by ex vivo FRI with EB ( Fig. 3a-c). In addition, Gd enhanced T1w MRI and immunostaining for IgG were performed to validate the FRI findings (Fig. 3a). When comparing white light and fluorescence images of EB distribution, we observed a clearer signal using the FRI approach, giving a better image of the regional distribution and concentration of EB and the possibility for quantification (Fig. S3). In the healthy control group, no EB fluorescence signal or Gd contrast enhancement was observed. In the vehicle injected group, EB and Gd extravasation was observed along the injection tract, also spanning the hippocampus region. Besides in the injection tract, additional EB and Gd extravasation were observed in the 6-OHDA lesioned SN. At all studied time points, hippocampus left/right FRI signal ratios were not differing significantly between 6-OHDA and vehicle injected group (Fig. 3b). However, the signal ratio in the 6-OHDA injected group was at all time points significantly higher compared to the healthy control group (Mdn Control = 1.05, n = 5; Mdn 6-OHDA, d2 = 1.37, n = 5, U = 0.0, P = 0.008; Mdn 6-OHDA, d7 = 1.32, n = 5, U = 0.0, P = 0.008; Mdn 6-OHDA, d14 = 1.26, n = 5, U = 2.0, P = 0.032; Mdn 6-OHDA, d28 = 1.40, n = 5, U = 0.0, P = 0.008). Additionally, the hippocampus left/right FRI signal ratio in the vehicle group was significantly increased on day 2 (Mdn Vehicle, d2 = 1.54, n = 4, U = 1.0, P = 0.032) and day 28 (Mdn Vehicle, d28 = 1.25, n = 5, U = 0.0, P = 0.008) compared to the healthy control. In conclusion, an increased BBB permeability was observed in the hippocampal region and along the injection tract in both, 6-OHDA and vehicle injected animals.

Intra-individual time course of BBB leakage
In order to investigate the intra-individual time course of BBB opening following intranigral injection of vehicle and 6-OHDA, four animals per group were subjected to repeated MRI measurements on day 2, 14, and 28 p. i. and EB FRI on day 28 (Fig. 4). The observed time course of Gd extravasation in the vehicle group ranges from no visible extravasation (Vehicle 1), over a declining signal in the hippocampal region (Vehicle 2 & 3) to a rather stable signal in the hippocampus (Vehicle 4). In the 6-OHDA lesioned group, a similar distribution is visible, ranging from a persistent BBB damage in the SN and/or injection tract region (6-OHDA 1, 3 & 4) to a declining BBB damage in the SN (6-OHDA 2). For all animals, the last T1w MRI shows a good spatial relation with the EB FRI images obtained on the same day (Fig. 4, FRI A-C). Despite the significant differences observed in the group comparison, the intra-individual pattern and timing of BBB disruption is highly variable.

Immune cell activation in areas of blood brain barrier leakage
In order to evaluate the role of the immune cells in the observed blood brain barrier leakage following dopaminergic neurodegeneration and mechanic injury, we performed immunohistochemistry for ionised calcium-binding adapter molecule 1 (Iba1), a marker for microglia/macrophages, at 14 and 28 days post injection of vehicle or 6-OHDA (Fig. 5). An increase in density in Iba1 + immune cells and a morphological change towards an activated phenotype was observed in areas of BBB leakage around the injection tract, spanning the area of the hippocampus, and the degenerating substantia nigra at all studied time points. At 2 weeks post lesion, more (activated) microglia were observed. Iba1 + cells with an activated phenotype were not observed on the contralateral side of the brain.

Imaging of blood brain barrier leakage and BLI in Dcx-Luc mice
In order to demonstrate in the same animal that the observed increase in BLI signal is in fact related to an increase in BBB permeability, we injected Dcx-Luc mice with 6-OHDA (n = 9) or vehicle (n = 7) into the substantia nigra and acquired BLI before and 2 weeks post injection, followed by BBB assessment using Gadovist â -enhanced T1w MRI and FRI with EB (summarised in Fig. 6a). Two animals were sacrificed due to weight loss (≥ 20%) following 6-OHDA lesion. As immunohistochemistry for TH revealed that the 6-OHDA lesion did not lead to a complete degeneration of left substantia nigra in five out of seven animals, we only included the data obtained for the vehicle injected group. An increased BLI signal left/right ratio was observed in the hippocampal region of vehicle injected animals at day 14 post injection compared to the pre lesion condition (Mdn Vehicle, d-1 = 0.98, Mdn Vehicle, d14 = 1.20, n = 7, W = 26, P = 0.0313; Wilcoxon Signed Rank Test; Fig. 6 b and e). A slight, but significant, increase in BLI signal left/right was also observed in the SVZ (Mdn Vehicle, d-1 = 0.99, Mdn Vehicle, d14 = 1.09, n = 7, W = 26, P = 0.0313, Wilcoxon Signed Rank Test, Fig. 6d), compared to the pre lesion condition. No difference was observed for the olfactory bulb (Fig. 6c). Using Gadovist âenhanced T1w MRI, BBB leakage along the injection tract was only observed in two out of seven vehicle injected animals. Quantification of the ex vivo FRI measurements following EB injection did not show a significant difference between vehicle injected and healthy control (C57Bl6 from the previous experiment shown in Fig. 3) animals with respect to Hippocampus or SN left/right signal ratios (Hippocampus: Mdn Vehicle = 0.99, n Vehicle = 7, Mdn Healthy = 1.05, n Healthy = 5; SN: Mdn Vehicle = 1.07, n Vehicle = 7, Mdn Healthy = 1.02, n Healthy = 5; Mann-Whitney Rank Sum Test; Fig. 6 f-h). Using immunohistochemistry for IgG, we detected BBB leakage around the injection tract in all vehicle injected mice.
In conclusion, we observed a clear increase in BLI left/right ratio in the hippocampal region in vehicle injected animals at 2 weeks post lesion, which is accompanied by a weak increase in BBB permeability detectable by IgG immunohistochemistry, but not by contrast enhanced T1w MRI or ex vivo FRI following EB administration.

Discussion
Following both, intranigral 6-OHDA and vehicle injection in Dcx-Luc mice, we observed a significantly increased BLI signal originating from the ipsilateral hippocampal region at 2 weeks post lesion, which slowly decreased in the following weeks. This temporal pattern was not recapitulated by the Dcx expression in the ipsilateral dentate gyrus of C57Bl6 mice post 6-OHDA or vehicle injection. Using in vivo Gd enhanced T1w MRI, ex vivo FRI with EB and IgG immunostaining, an increased BBB permeability was observed in the hippocampal region and along the injection tract in both, 6-OHDA and vehicle injected animals between day 2 and 28. Additionally, a persistent BBB leakage was observed in the 6-OHDA lesioned SN at all studied time points. In the intra-individual comparison, the observed pattern, severity, and timing of BBB disruption was highly variable. In a second set of Dcx-Luc mice, the increased BLI signal at week two post vehicle injection could be reproduced, even though the BBB leakage observed in these mice was rather weak, as it could only be detected by IgG IHC.
Expression of the neurogenesis marker Dcx in the dentate gyrus is unchanged following intranigral 6-OHDA application Even though we found an increased BLI signal in the hippocampal area of Dcx-Luc mice following intranigral injection of 6-OHDA or vehicle, this increase could not be confirmed in the immunohistochemical stainings for Dcx in C57Bl6 mice, suggesting that neither the injection procedure nor the nigrostriatal lesion has an effect on hippocampal neurogenesis with regards to total Dcx expression. In contrast, several studies reported a reduction of neurogenesis in the dentate gyrus in experimental models of PD as well as in PD patients, which was linked to dopamine depletion following dopaminergic neurodegeneration. In PD patients, a reduced density of progenitor cells in the DG was reported (H€ oglinger et al., 2004). A reduced cell proliferation was also described following intranigral 6-OHDA lesion in rats (Suzuki et al., 2010) and mice (Chiu et al., 2015), and in MPTP (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine)- lesioned mice (H€ oglinger et al., 2004). The opposite effect, a transient increase in progenitor cell division, was observed in the DG of MPTP-treated animals at 14 days post treatment, but not at 4 or 30 days post treatment (Park & Enikolopov, 2010). Another study reported an increase in new-born Type 2b cells following MPTP treatment, while the absolute number of Type 2b cells was unchanged (Lesemann et al., 2012). The controversial results obtained underline that the use of different markers, marker combinations, and models might result in a different experimental outcome. The increased BLI signal we observed in both, 6-OHDA and vehicle injected animals does not seem to be related to an actual increase in hippocampal neurogenesis. In addition to Dcx + cells in the hippocampus, Dcx + cells in the SVZ drive the overall intensity of the observed BLI signal in Dcx-Luc mice. In our second set of Dcx-Luc mice, we observed a very slight increase in BLI left/right signal ratios in the SVZ. This increase might be due to slight scatter from the strong signal in the hippocampal area. We observed previously (Fricke et al., 2016), that neurogenesis in the SVZ is unchanged following intranigral 6-OHDA lesion, indicating that also the neurogenesis in the SVZ is most likely not the source of the increased BLI signal. Hence, the observed alterations in BLI probably result from a different source.

BBB leakage induced by mechanic injury and dopaminergic neurodegeneration
The source for an increased BLI signal in spite of an unchanged Dcx expression could, at least in part, be an increased substrate availability due to a regional damage of the BBB. We observed a leakage of Gadovist â , EB and IgG along the injection tract, also spanning the hippocampus in both, 6-OHDA and vehicle injected C57Bl6 animals. This is in line with the finding of FITC-labelled albumin leakage around the injection tract at 10 days post 6-OHDA injection in rats (Carvey et al., 2005). Besides BBB damage due to the injection procedure, we also observed a persistent BBB damage in the 6-OHDA lesioned SN in these animals. In agreement with our results, FITC-labelled albumin leakage was reported in the ipsilateral SN and at the primary 6-OHDA injection site 10 days following striatal 6-OHDA injection in rats (Carvey et al., 2005). In the same study, MFB injections of 6-OHDA resulted in a BBB leakage in SN and striatum at 10 and 34 days post injection (Carvey et al., 2005), indicating that degeneration of dopaminergic cell bodies in the SN and dopaminergic fibres in the striatum induces a long-lasting BBB damage, which was already described by Cooper et al. (Cooper et al., 1982). In contrast to our results in C57Bl6 mice, we did not observe this increased BBB permeability in vehicle injected Dcx-Luc animals using in vivo Gd enhanced T1w MRI and ex vivo FRI following EB injection. In the immunohistochemical staining for IgG, a moderate extravasation was observed, showing that the degree of BBB impairment can vary between animals and experiments.
In addition to an increased BBB permeability, facilitated light penetration through the hole that is drilled into the skull during the stereotactic injection should be considered as another factor increasing the BLI signal at time points early after injection, likely also impacting the signal we observed at 2 weeks post lesion.

Blood brain barrier leakage is associated with microglia/ macrophage activation
We previously observed increased numbers and activation of microglia cells close to the injection tract and in the degenerating substantia nigra in our animal model (Fricke et al., 2016), indicating that immune cells might play a role in BBB opening in this model. In agreement with previous results, we observed increased density of Iba1 + microglia/macrophages in regions of IgG extravasation in the area of the injection tract.
The blood brain barrier as a limiting factor for substrate availability The intact BBB restricts diffusion of D-Luciferin into the brain and plays a role in eliminating D-Luciferin from the brain by active transport processes via drug efflux transporter ABCG2 (ATP-binding cassette subfamily G member 2) (Bakhsheshian et al., 2013). Thereby it notably restricts the availability of substrate in the brain. When increasing amounts of D-Luciferin (15, 150, 300, 750 mg/kg) were applied in healthy Dcx-Luc mice, no saturating photon emission level could be reached (Aswendt et al., 2013), highlighting that D-Luciferin is not present in excess under normal experimental conditions. This observation also holds true for tracer substances in nuclear imaging that possess limited BBB mobility. A biodistribution study of [ 125 I]iodo-D-Luciferin and 14 C-D-Luciferin in mice confirmed the very limited brain uptake in mice (< 1% ID/g 5 min post i. p. injection) (Lee et al., 2003;Berger et al., 2008).
A study using an Experimental Autoimmune Encephalomyelitis (EAE) model in Dcx-Luc mice already described an increase in BLI signal unrelated to neurogenesis, which followed the same temporal pattern as the opening of the BBB (Ayzenberg et al., 2015). Nevertheless, more research is needed to determine the impact of BBB alterations on substrate/tracer availability in different models and diseases.
The necessity of BBB assessment in substrate/tracer dependent brain imaging To allow for quantification in substrate or tracer dependent brain imaging in conditions that potentially increase BBB permeability, the BBB should be assessed. Various methods to assess BBB leakage exist, including in vivo MRI, ex vivo FRI with different tracer substances, and histological stainings. Ex vivo fluorescence imaging of EB leakage was previously used in a rat model of stroke (Jaffer et al., 2013) and in an approach to open the BBB using low frequency focused ultra-sound (Shen et al., 2014). It was described as more sensitive than the conventional EB approach using white light images of EB distribution or ultraviolet spectrophotometry (Jaffer et al., 2013). Using the same approach, we could confirm EB FRI as a relatively sensitive, fast and cost-efficient method. Nevertheless, EB FRI is less sensitive than immunohistochemistry for IgG extravasation, is only semi-quantitative, and lacks the option for longitudinal imaging.

Limitations of bioluminescence imaging in the brain
Bioluminescence imaging in the brain can be utilised for various purposes, but it also confronts us with a range of different challenges for its proper quantification and interpretation. Its 2D nature and the absorption and scattering of light travelling through tissue make it difficult to define proper regions of interest. In addition, surgical manipulations to the brain and skull like intracranial injection associated with local thinning of the skull after creating a hole for injection might result in a facilitated light penetration leading to an increase in detected BLI signal. Furthermore, increased substrate delivery to the brain due to a disrupted BBB might play a role in several models, including stereotactic injections, neurodegeneration, ischemic events, or tumour development.

Conclusions
An increased BLI signal in the neurogenic regions close to the injection tract was observed in both, 6-OHDA and vehicle injected animals, at 2 weeks post lesion, which was not recapitulated in Dcx + cell numbers in the dentate gyrus. In addition, a BBB damage associated with the injection procedure and the ongoing neurodegenerative processes was observed in C57Bl6 mice. In a second set of vehicle injected Dcx-Luc mice, a weaker BBB leakage was observed. These data suggest, that the measured BLI signals could result from increased substrate availability due to BBB damage and facilitated light penetration through the impacted skull, rather than a difference in neurogenesis. Our study underlines the importance of BBB assessment in in vivo imaging studies based on tracer substances and enzyme substrates with a limited BBB permeability. Furthermore, the variability of BBB damage observed in invasive intracranial injection models has to be taken into account in future studies.

Supporting Information
Additional supporting information can be found in the online version of this article: Fig. S1. BLI and FRI regions of interest. Fig. S2. Validation of striatal deafferentation in Dcx-Luc mice following 6-OHDA lesion. Fig. S3. Fluorescence imaging of EB signal gives a more reliable readout than conventional EB white light images. Table S1. Summary of the experimental setup of the BBB leakage assessment.


s2fieldsofstudy: Biology Medicine

text:
Relationship between initiation time of adjuvant chemotherapy and survival in ovarian cancer patients: a dose-response meta-analysis of cohort studies

Although several studies have previously investigated the association between the initiation time of adjuvant chemotherapy and survival in ovarian cancer, inconsistencies remain about the issue. We searched PubMed and Web of Science through the May 24, 2017 to identify cohort studies that investigated the aforementioned topic. Fourteen studies with 59,569 ovarian cancer patients were included in this meta-analysis. We conducted meta-analyses comparing the longest and shortest initiation time of adjuvant chemotherapy and dose-response analyses to estimate summary hazards ratios (HRs) and 95% confidence intervals (CIs). A random-effects model was used to estimate HRs with 95% CIs. When comparing the longest with the shortest category of initiation time of adjuvant chemotherapy, the summary HR was 1.18 (95% CI: 1.06–1.32; I 2 = 17.6; n = 7) for overall survival. Additionally, significant dose-response association for overall survival was observed for each week delay (HR = 1.04; 95% CI: 1.00–1.09; I 2 = 9.05; n = 5). Notably, these findings were robust in prospective designed cohort studies as well as studies with advanced stage (FIGO III-IV) patients. No evidence of publication bias was observed. In conclusion, prolonged initiation time of adjuvant chemotherapy is associated with a decreased overall survival rate of ovarian cancer, especially in patients with advanced stage ovarian cancer.

adjuvant chemotherapy in ovarian cancer is unclear. However, without quantitative findings and subgroup and sensitivity analyses might limit the interpretation of their results. To the best of our knowledge, there has been no quantitative assessment and dose-response analysis of published findings on this topic. Notably, several important studies 13,19,20 with large sample sizes were published after their study. Herein, to update the evidence as well as further clarify the relationship between initiation time of adjuvant chemotherapy and the prognosis of ovarian cancer patients, we carried out this dose-response meta-analysis of epidemiological studies.

Result
Search results and characteristics of included studies. The literature search strategy yielded 5,277 studies for eligibility from the databases of PubMed and Web of Science. After carefully screening each title and abstract, 26 studies were included for full reviews of original articles. Of these, 11 were excluded because of the inclusion and exclusion criteria. Finally, 15 studies 10-13, 15, 19-28 were included in the present meta-analysis (Fig. 1). The detail characteristics of these 15 studies are demonstrated in the Table S1 in the supplement. A total of 59,569 ovarian cancer cases were included. Quality assessment. The methodological quality of all studies is depicted in the Table S2 in the supplement. The major differences among these studies were comparability and outcome, especially in adjustments for potential confounders and duration of follow-up. Only five of 15 studies adjusted for two defined confounders (FIGO stage and residual disease) and received full scores. Additionally, ten studies followed their ovarian cancer patients more than twenty-four months.
Longest compared with shortest category of initiation time of adjuvant chemotherapy. Six observational studies provided the data on the longest compared with the shortest initiation time of adjuvant chemotherapy. The summary HR of OS was 1.16 (95% CI: 1.04-1.29) with moderate heterogeneity (I 2 = 67%, P = 21.2) (Fig. 2). There was no evidence of publication bias, both quantitatively (P = 0.88 for Begg and P = 0.16 for Egger) and qualitatively, on visual inspection of the funnel plot.
Several post hoc subgroup analyses stratified by study characteristics and adjustment for potential confounders were carried out (Table 1). In five prospective studies, longer initiation time of adjuvant chemotherapy was associated with a significant 22% risk decrease in OS of ovarian cancer. Subgroup analysis on geographic location, number of cases, FIGO stage, and chemotherapy, yielded similar results in European studies, studies with more than 600 cases, studies with FIGO III-IV stage patients, and studies with platinum based plus Taxane chemotherapy. Additionally, when stratified by whether there had been an adjustment for potential confounders, similar results were observed in studies with adjustment for FIGO and residual disease. Notably, there was no evidence of significant heterogeneity between subgroup analyses in meta-regression analysis. In a sensitivity analysis, we sequentially removed one study at a time and reanalyzed the data. The seven study-specific HRs ranged from a low of 1.14 (95% CI: 1.02-1.27, I 2 = 7.05%, P = 0.22) after omitting the study by Seagle et al. 25 to a high of 1.25 (95% CI: 1.16-1.34, I 2 = 5.48%, P = 0.36) after omitting the study by Feng et al. 13 .

Dose-response analysis of initiation time of adjuvant chemotherapy. Four observational studies
provided the sufficient data on dose-response analysis of initiation time of adjuvant chemotherapy. The summary HR was 1.04 (95% CI: 1.00-1.09) with moderate heterogeneity (I 2 = 55.8%, P = 0.06) (Fig. 3). There was no evidence of publication bias, both quantitatively (P = 0.62 for Begg and P = 0.65 for Egger) and qualitatively, on visual inspection of the funnel plot.
Although positive associations were observed in the majority of these subgroup analyses, not all of them showed statistical significance ( Table 2). Similar to the results of the longest versus the shortest analysis, there was no evidence of significant heterogeneity between subgroup analyses in meta-regression analysis. In a sensitivity analysis, we sequentially removed one study at a time and reanalyzed the data. The 4 study-specific HRs ranged from a low of 1.01 (95% CI: 1.00-1.02, I 2 = 2.75%, P = 0.43) after omitting the study by Heo et al. 19 to a high of 1.06 (95% CI: 1.01-1.12, I 2 = 4.12%, P = 0.25) after omitting the study by Seagle et al. 25 .

Discussion
The present meta-analysis comprehensively and systematically summarized the evidence from 15 cohort studies and found that early initiation of chemotherapy will improve the OS rates of patients with ovarian cancer. Relative OS decreases by 4% for each week in delay of initiating adjuvant chemotherapy. This finding was consistent in patients with FIGO III-IV stage. In the clinical practice, initiation time of adjuvant chemotherapy may need to be carefully considered by physicians when discussing adjuvant chemotherapy with ovarian cancer patients after surgery.
Currently, primary tumor resection surgery followed by platinum-taxane chemotherapy has been the most common and well-accepted treatment method for ovarian cancer patients 6 while the optional initiation time of adjuvant chemotherapy has remained unclear. A recent systematic literature review carried out by Alexander et al. 18 evaluated the association between initiation time of adjuvant chemotherapy and survival in six priority cancers. However, limited evidence of ovarian cancer was provided which might be attributed to a lack of quantitative assessments, dose-responses, subgroups and sensitivity analyses in their study. Additionally, since the relatively earlier search date of their study (April 2014), in the recent two years several studies with large sample sizes have been published. For example, Tewari et al. 20 carried out a post-trial ad hoc analysis on the basis of 1,718 ovarian cancer patients from a phase III randomized, double-blind, placebo-controlled trial. They suggested that initiation time of adjuvant chemotherapy was predictive of overall survival. Notably, when initiation time of adjuvant chemotherapy exceeded 25 days, the complete resection group (i.e., stage IV) encountered an increased risk of death. Additionally, two recent retrospective cohort studies 13,19 from Asia published their results, although one of them is a meeting abstract 19 . Several potential biological theoretical rationales may explain the benefits of initiating adjuvant chemotherapy without delay after curative surgery. Gunduz et al. 9 suggested that resection of the tumor could increase the tumor growth which may result from a conversion of non-cycling cells in G 0 phase into proliferation. Similarly, surgery has been shown to temporarily modulate the function of the immune system; for instance, antitumor effects of interleukin-2 and lymphokine-activated killer cells will be significantly reduced by the performance of a laparotomy 29 . Additionally, in animal models, peri-operative chemotherapy demonstrated a significant advantage in preventing systemic relapse and a short time span to chemotherapy resulted in the most Figure 2. Forest plots of the relationship between the interval from surgery to chemotherapy and overall survival of patients with ovarian cancer (longest compared with shortest interval). Squares indicate studyspecific risk estimates (size of the square reflects the study-specific statistical weight); horizontal lines indicate 95% CIs; diamonds indicate the summary hazards ratio with its 95% CI. HR: hazards ratio. effective control of metastases 7,8 . After primary surgery, a large proportion of tumor cells will grow rapidly, and thus, the earlier chemotherapy is started, the greater the chance that the chemotherapy treatment will control the residual disease.
To the best of our knowledge, this is the first meta-analysis comprehensively and quantitatively evaluating the effect of initiation time of adjuvant chemotherapy in ovarian cancer patients. On the basis of 59,569 cases from 15 cohort studies, we have sufficient statistical evidence to detect the aforementioned association and further reinforce earlier results of previous meta-analysis. Additionally, numerous subgroup analyses were carried out to increase the robustness as well as to explore the heterogeneity. Notably, we first provided evidence that advanced ovarian cancer patients (FIGO III-IV) should consider shorter initiation time of adjuvant chemotherapy to improve their survival. Our meta-analysis also had several limitations. Firstly, we included both prospective and retrospective cohort studies in the present meta-analysis. However, compared to prospective cohort studies, retrospective cohort studies might be limited by recall bias. Although we found no evidence of differences between these two study designs, the point estimate of prospective cohort studies was slightly stronger than retrospective cohort studies (1.22 versus 1.08). Secondly, administration of chemotherapy could be delayed by various factors including purpose of patients, age, performance status, and peri-operative co-morbidity in clinical practice that might impact the outcomes. Although positive findings were observed in subgroup analyses stratified by whether there was an adjustment for potential confounders (FIGO, histology, and residual disease), not all of them showed significance which might be partly attributed to the limited included studies. Therefore, we could not rule out the possibility that there might exist other potential confounders that not be adjusted by most of the studies affecting the results. Thus, further analyses adjusting or stratification for more confounders are needed. Thirdly, since there are no specific clinical manifestations and effective screening methods, over 75% ovarian cancer patients are diagnosed in advanced stages 3 . For example, although six studies mentioned that they included patients in all stages of the disease, the majority of these cases were in the advanced stages of the disease. The findings of the present meta-analysis should be interpreted with caution in patients with early stage ovarian cancer. Additionally,  . Squares indicate study-specific risk estimates (size of the square reflects the study-specific statistical weight); horizontal lines indicate 95% CIs; diamond indicates the summary hazards ratio with its 95% CI. HR: hazards ratio. small-study bias, such as publication bias, might be an issue in meta-analyses, however, we failed to detect statistical evidence in the present study. Finally, although dose-response analyses were carried out in the present study, considering the limited available data, we failed to evaluate whether there was evidence for a nonlinear aforementioned association.
In conclusion, the present meta-analysis suggests that early initiation of adjuvant chemotherapy after surgery will improve the overall survival rates of ovarian cancer patients, especially in patients with advanced stage cancer. Future prospective clinical trials randomizing patients to different time intervals are warranted to confirm our findings as well as to further clarify the definitive relevance of the time between surgery and chemotherapy. Consequently, longer initiation time of adjuvant chemotherapy should be advised as little as possible in clinical practice when treating ovarian cancer patients after surgery, especially for these patients with advanced stage cancer.

Materials and Methods
Search strategy. Two investigators (YiLiu and Tiening Zhang) carried out a systematic literature search of PubMed and Web of Science databases and extracted data up to May 24, 2017 for all relevant epidemiological studies without restriction. The language restriction was imposed on English and following keywords were employed in the search: (ovary OR ovarian) AND (cancer OR neoplasm OR tumor OR carcinoma) AND (time OR interval) AND (survival OR mortality). We followed standard criteria for conducting and reporting this meta-analysis 30 . Additionally, we also included an unpublished study 19 and searched the reference sections of the studies that were included in our study to identify publications for further potentially relevant articles 31-34 . Study selection criteria. Titles, abstracts, and articles for eligibility using the following criteria was reviewed by two investigators (YiLiu and Tiening Zhang) independently. The study was included if it accorded with following criteria (1) evaluated and clearly defined the time interval between primary surgery to inception of adjuvant chemotherapy as exposure, (2) reported ovarian cancer survival (progression-free or OS), (3) reported hazards ratios (HR) and 95% confidence interval (CIs) for aforementioned association. Studies were excluded if they: (1) were reviews without original data, ecological studies, editorials, or case reports and (2) failed to evaluate the aforementioned association as well as to report HR and 95% CI or the data necessary for calculating these risk estimates. Only data from the most recent comprehensive report were included when there were multiple publications from the same population. Any discrepancy was resolved by discussion with third investigator (Qijun Wu) 34-37 . Data extraction and quality assessmenth. From each study we extracted data on the first author, publication year, country, study design, numbers of cases within the cohorts, regimens of chemotherapy, tumor characteristics, time to chemotherapy (category/continuous), risk estimates and 95% CI and adjustment confounders. Extracted data were entered into a standardized Excel (Microsoft Corporation) file. We also sought supplementary appendixes of included studies [38][39][40] . Discrepancies were resolved by discussion. Data extraction was performed by YiLiu and confirmed independently by the other authors (Tiening Zhang and Qijun Wu) for accuracy.
Additionally, the methodological quality of included epidemiological studies was independently assessed by YiLiu using the Newcastle-Ottawa scale (NOS) and confirmed by the other investigator (Tiening Zhang) 41,42 . According to the NOS, The included studies were evaluated across three categories: selection, comparability, and exposure/outcome. Since quality scoring of these included studies might not only hide important information by combining disparate study features into a single score, but also introduce an arbitrary subjective element into the analysis, we described these studies instead of dividing them into categories of high or low quality utilizing a scoring system.

Statistical analysis.
For studies that reported the risk estimates stratified by residual disease and race, we used a previously described counting method proposed by Hamling et al. 43 to recalculate the HR and 95% CI. We used the main cut-off values mentioned in the manuscripts for studies that reported the risk estimates using different cut-off values. In dose-response analysis, for studies that reported the risk estimates for each day increment in initiation time of adjuvant chemotherapy, we used the methods first carried out by Danesh et al. 44 to transfer the information into per week increments.
Since these included studies differed both clinically and methodologically, we report the results from the DerSimonian and Laird random effects models over the fixed effects model 45 . If no heterogeneity exits in the pooled data, results of random and fixed effects models are the same, and if significant heterogeneity is present, a random effects model is more conservative 45 . Therefore, the random effects model, which considered both withinand between-study variation, was used to calculate summarized HR and 95% CIs. I 2 statistic was used to quantify the heterogeneity across studies. I 2 > 50% indicated significant heterogeneity. Subsequently, to explore the heterogeneity as well as check the influence of various factors on initiation time of adjuvant chemotherapy of ovarian cancer survival, we further carried out post hoc subgroup analyses stratified by study design (prospective versus retrospective), geographical location (Asia, North America, and Europe), number of cases (<600 versus ≥ 600), FIGO (International Federation of Gynecology and Obstetrics) stage (all, III-IV versus I-II), residual disease (yes versus no), chemotherapy (Platinum based, Platinum based plus Taxane versus N/A), and adjustment for potential confounders (FIGO, histology, and residual disease). Heterogeneity between subgroups was evaluated by meta-regression, which can be used to explore reasons for heterogeneity when trials, not patients, are pooled 46 . Sensitivity analyses were carried out to examine the influence of individual data sets on the overall estimate by deleting each study in turn. Small-study bias, such as publication bias, was assessed with visually inspecting a funnel plot for asymmetry as well as by the test developed by Egger et al. 47 and Begg and Mazumdar 48 . To avoid the results were driven by one large study or by a study with an extreme result, we carried out sensitivity analyses excluding one study at a time to explore whether All statistical analyses were performed with Stata 12.0 (StataCorp LP).


extfieldsofstudy: Biology Medicine

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The Role of HCMV and HIV-1 MicroRNAs: Processing, and Mechanisms of Action during Viral Infection

Viruses infect host cells releasing their genome (DNA or RNA) containing all information needed to replicate themselves. The viral genome takes control of the cells and helps the virus to evade the host immune system. Some viruses alter the functions of infected cells without killing them. In some cases infected cells lose control over normal cell proliferation and becomes cancerous. Viruses, such as HCMV and HIV-1, may leave their viral genome in the host cells for a certain period (latency) and begin to replicate when the cells are stressed causing diseases. HCMV and HIV-1 have developed multiple strategies to avoid recognition and elimination by the host’s immune system. These strategies rely on viral products that mimic specific components of the host cells to prevent immune recognition of virally infected cells. In addition to viral proteins, viruses encode short non-coding RNAs (vmiRNAs) that regulate both viral and host cellular transcripts to favor viral infection and actively curtail the host’s antiviral immune response. In this review, we will give an overview of the general functions of microRNAs generated by HCMV and HIV-1, their processing and interaction with the host’s immune system.

INTRODUCTION
MicroRNAs (miRNAs) are short (∼22 nucleotides) single-stranded non-coding RNA molecules that negatively regulate gene expression at post-transcriptional level. The miRNA maturation machinery involves several steps and multiple proteins in both nucleus and cytoplasm. miRNAs can be transcribed as part of independent primary transcripts (pri-miRNAs), which are mainly generated by RNA polymerase II (Lee et al., 2004;Ozsolak et al., 2008), and display all features commonly associated with Pol II-mediated transcription, such as histone marks, CpG islands, transcription factor binding sites (Cai et al., 2004). In the canonical miRNA biogenesis pathway, the Microprocessor complex, a multiprotein complex with Drosha and Di George Syndrome critical region gene 8 (DGCR8), cleaves the double-stranded pri-miRNA generating a hairpin-shaped RNA molecule (pre-miRNA) of about 70-100 bp (Denli et al., 2004;Han et al., 2006). This process can be modulated by different proteins (Suzuki et al., 2009;Trabucchi et al., 2009). Once generated, nuclear pre-miRNAs are exported to the cytoplasm by the exportin-5/Ran-GTP complex to be further processed by the RNase III enzyme Dicer (Grishok et al., 2001). The pre-miRNA processing is finely regulated and its inhibition affects miRNA-mediated differentiation in embryonic stem cells, embryonal carcinoma cells and certain primary tumors. An example is provided by Lin-28, an developing regulated RNA binding protein, which promotes cell proliferation and tumorigenesis of embryonic cells by affecting let-7 maturation (Viswanathan et al., 2008). The activity of Dicer can be either enhancing or inhibiting by A-to-I RNA editing mediated by ADARs enzymes which convert Adenosine to Inosine (Kawahara et al., 2008;Nishikura, 2010;Tomaselli et al., 2013). ADAR1 has been shown to directly bind Dicer, thus increasing the maximum rate of pre-microRNA cleavage by Dicer (Ota et al., 2013). In human adult brain, it has been estimated that approximately 20% of pri-miRNAs can undergo A-to-I RNA editing (Nishikura, 2010;Tomaselli et al., 2013) by affecting miRNAs maturation at different steps (Luciano et al., 2004;Chawla and Sokol, 2014;Tomaselli et al., 2015).
The result of Dicer cleavage is the formation of a double stranded RNA of 22 nt in length whose strand with the less stability is normally chosen as guide strand and transferred to the RNA-induced silencing complex (RISC) for the annealing of miRNAs to the target mRNA, whereas the other strand [the star ( * )-strand] is usually degraded. The RISC complex contains Dicer and many associated proteins such as Argonaute (Ago) protein family and the RNA-binding protein TRBP [human immunodeficiency virus transactivating response RNA (TAR) binding protein] (Chendrimada et al., 2005).
Mature miRNAs recognize their target mRNAs through 6-8 nucleotides (the seed region) at the 5 end of the miRNA. Complete complementarity between the miRNA and target mRNA sequence directs mRNA degradation, while absent of perfect complementarity will silence the gene target by preventing its translation (Lim et al., 2005). A given miRNA may have hundreds of different mRNA targets, and a given target mRNA might be regulated by several miRNAs. These evidences suggest that the biogenesis of miRNAs is extremely complex and regulated at different levels, thus highlighting the importance of these short RNA molecules in crucial cell programs including viral infection.
Viruses have developed multiple strategies to avoid recognition and elimination by the host's immune system. These strategies rely on viral products that mimic specific components of the host cells to prevent immune recognition of virally infected cells. In addition to viral proteins, viruses encode miRNAs (vmiRNA) that regulate both viral and host cellular transcripts during viral infection. The first vmiRNAs was identified in a cell line latently infected with Epstein-Barr virus (EBV) (Pfeffer et al., 2004), a member of the Herpesviridae, that in humans is associated with Burkitt's lymphoma, Hodgkin's disease and nasopharyngeal carcinoma (Raab-Traub, 2007). Currently, different members of Herpesviridae, Polyomaviridae and Adenoviridae families are known to express vmiRNAs (Skalsky and Cullen, 2010). To date, 172 viral-encoded mature miRNAs are listed in the miRBase collection 1 (Kozomara and Griffiths-Jones, 2014), the majority of which (93%) belongs to the three subfamilies (Alphaherpesvirinae, Betaherpesvirinae and Gammaherpesvirinae) of Herpesviridae family (Figure 1).
In general, virus and the host-cell's miRNAs use different mechanisms to interfere each other. Viruses can either block or impair the host cells miRNA pathway by interacting with key proteins (Lu and Cullen, 2004;Bennasser et al., 2006), synthesize and regulate their own miRNAs (Grundhoff and Sullivan, 2011;Harwig et al., 2014), exploit cellular miRNAs to complete their replication cycle (Luna et al., 2015). Conversely, host cells can target vmiRNAs with endogenous miRNAs (Lecellier et al., 2005;Delorme-Axford et al., 2013;Bai and Nicot, 2015). The complex interplay between viruses and host cells usually favors viral infection by either reducing immune recognition or promoting cell growth and lytic or latent infection. One of the best examples of virus-host-miRNA interplay important for human infection and diseases come from HCMV. In this review, we summarize the general functions of miRNAs generated by HCMV and their interactions with immune system and we discuss new finding regarding miRNA from HIV-1.

HCMV MicroRNAs
HCMV is a ubiquitous and highly specific herpesvirus that establishes lifelong latent infections, coexisting asymptomatically with its host in a healthy immune system, with periodic and spontaneous phases of reactivation, lytic replication and virus shedding (Stern-Ginossar et al., 2012). In immunocompromised individuals, such as transplant recipients, HIV-infected patients and individuals with an immunological immaturity, as the fetus in utero, HCMV can cause different clinical syndromes the severity of which depends on the degree of immunosuppression (Varani and Landini, 2011). Of note, during pregnancy HCMV primary infections can lead to mental retardation and severe neonatal pathologies.
To persist indefinitely within the host, HCMV has elaborated several strategies that act to subvert host cellular immune responses (Noriega et al., 2012). Many HCMV proteins and vmiRNAs are known to target cellular and viral transcripts to establish and maintain latency (Murphy et al., 2008). The complex interplay between virus and host is further enhanced by post-transcriptional events, such as RNA editing. Indeed, during HCMV infection, the expression of the short form of the RNA editing enzyme ADAR1 (ADAR1-p110) is enhanced and the host miR-376a precursor undergo editing at specific sites. The increased edited-miR-376a during HCMV infection, downregulates HLA-E transcript leading the infected cells more visible by NK cells (Nachmani et al., 2014).
To date, 15 stem-loop precursors and 26 mature HCMV miRNAs are deposited in the miRBase (Figure 2) 1 . HCMV miRNAs are differently expressed during latent and lytic infection. Of note, only a subset of vmiRNAs are produced during HCMV latency, most of which originate from the unique long (UL) region of the HCMV genome (Meshesha et al., 2016). Interestingly, during reactivation of the virus from latency all known vmiRNAs, including those that were absent during latency, restored their expression. An alternative expression of the two stands of miR-US29 was detected in the lytic and latent infection. Specifically, miR-US29-5p prevailed during lytic infection, whereas miR-US29-3p dominated viral latency, suggesting the presence of a specific mechanism that regulates expression of the two arms of the vmiRNA hairpins during the viral life cycle (Meshesha et al., 2016). It is possible that expression of vmiRNAs during latency is required to manipulate host-signaling pathways and make the latently infected cell ready for reactivation.
HCMV miRNAs are known to target several cellular genes to evade immune system, control cell cycle and vesicle trafficking ( Table 1). Since this subject has been recently reviewed by Piedade and Azevedo-Pereira (2016), in this review we will deepen the HCMV miRNAs with proved implications in viral evasion from innate and adaptive immune responses.
The first host cellular mRNA target reported for a HCMV vmiRNA encodes MICB, a stress-induced ligand for the NK cell activating receptor NKG2D critical for the NK cell killing of virus-infected and tumor cells. VmiR-UL112 specifically binds to MICB-3 untranslated regions (3 UTR) and downregulates MICB expression during viral infection, therefore, leading to decreased binding of NKG2D and reduced killing by NK cells (Stern-Ginossar et al., 2007). This miRNA-mediated MICB inhibition is not exclusive to HCMV, but conserved in the herpesviruses family. Other viral miRNAs encoded by EBV and HHV-8 (miR-BART2-5p and miR-k12-7, respectively), target MICB to escape NK cell recognition (Nachmani et al., 2009). These vmiRNAs exhibit poor sequence homology with HCMV miR-UL112 and target MICB at different binding sites. HCMV miR-UL112 attenuates NK cell activity also by targeting others transcripts, such as IL-32, type I IFN and the toll-like receptor 2 (TLR2) signaling (Landais et al., 2015).
In addition, the HCMV miR-UL148D, one of the most highly expressed vmiRNAs during latent infection, contribute to immune evasion by directly targeting the chemokine (C-C Motif) ligand 5 (CCL5), a chemokine known to attract immune cells to sites of inflammation and tissue damage (Kim et al., 2012). This downregulation was reverted by treatment with a miR-UL148D-specific inhibitor, supporting the role of this agent as Frontiers in Microbiology | www.frontiersin.org therapeutic tool against HCMV infection. VmiR-UL148D also targets the activating receptor type-1B (ACVR1B) in monocytes, resulting in a reduced secretion of IL-6 (Lau et al., 2016). More recently, vmiR-US5-1 and vmiR-UL112-3p have been shown to play important role in modulating NF-κB signaling at late time of infection by reducing the expression of the IKK complex and induce the release of proinflammatory cytokines (Hancock et al., 2017). The limited or delayed secretion of proinflammatory cytokines is thought to be one of the mechanisms of immune evasion exploited by vmiRNAs to limit the recruitment of immune cells and killing of infected cells.
Finally, the HCMV miR-US4-1 was shown to directly target the endoplasmic reticulum aminopeptidase 1 (ERAP1), a key peptidase that trims peptide precursors to their optimal length to bind MHC class I molecules (Kim et al., 2011). The reduced trimming due to HCMV-specific action resulted in an immunoevasion of HCMV-infected cells (Kim et al., 2011).
All these mechanisms focus on the possibility to enhance viral replication by hindering viral clearance by NK cells and T cells for the period of time necessary to virus to replicate.

HIV-1 miRNAs
HIV-1 infection cause progressive CD4 + T-cell loss making individuals susceptible to get infections and develop a wide range of immunological abnormalities until oncological complications. Although HIV infections are able to induce vigorous antiviral immune responses, HIV-1 replication is not fully controlled by the innate and adaptive immune system. Like many other viruses, HIV-1 has evolved a number of strategies to evade host immune responses, most notably by using viral accessory proteins and RNA.
Recent reports have demonstrated the existence of HIV-1derived miRNAs from coding and non-coding regions of the viral genome, which regulate both viral and host gene expression ( Table 1). Despite so, the presence of HIV-1 derived vmiRNAs has been highly controversial and further studies are necessary to better understand their potential role in viral infection and pathogenesis. The importance of viral and cell host miRNAs in the context of HIV-1 infection was first suggested by silencing of Drosha and Dicer leading to significant enhancement of HIV-1 replication (Triboulet et al., 2007). The first description of HIV-1-derived miRNAs came in 2004 from a group that identified a Nef-derived miRNA, named miR-N367 (Omoto et al., 2004). MiR-N367 reduces HIV-1 transcription by blocking Nef expression and long terminal repeat (LTR) transcription (Omoto et al., 2004;Omoto and Fujii, 2005). The HIV-1 transactivation RNA (TAR), which regulates viral translation, also encodes for a vmiRNA (called TAR-miR-5p and -3p) (Ouellet et al., 2008;Klase et al., 2009). TAR-miR has been shown to downregulate host genes (such as ERCC1 and IER3) important for apoptosis and cell survival, thus giving HIV-1-infected cells a survival advantage by preventing host cell death (Klase et al., 2009). Recent studies demonstrated that additional host substrates, including Caspase 8, Aiolos, Ikaros and Nucleophosmin (NPM)/B23, are modulated by TAR-miR (Ouellet et al., 2013). VmiR-H1, located in the LTR has been reported to downregulate the apoptosisantagonizing transcription factor (AATF) gene product and act as an antagonist of the anti-apoptotic effect mediated by TAR-miR. Additionally, hiv1-mir-H1 can downregulate the host miR149 expression recognized to target HIV-1 Vpr transcript (Kaul et al., 2009). Recently, Zhang et al. (2014) have reported the existence of a novel HIV-1-encoded vmiRNA called miR-H3 located in the region of the HIV-1 RNA genome that encodes for reverse transcriptase (RT). Overexpression of miR-H3 increases viral production and mutations within miR-H3 sequence significantly impair the viral replication of wild-type HIV-1 viruses by targeting HIV-1 5 -LTR (TATA box) . Two additional vmiRNAs (called as vmiR88 and vmiR99) were identified in overlapping regions of HIV LTR from viral infected human macrophages (Bernard et al., 2014). They were able to directly stimulate TNFα release by human macrophages through TLR8 activation by a mechanism that is partially dependent on vmiRNA sequence motifs. Of note, vmiR88 and vmiR99 were detected in sera of HIV infected individuals suggesting their role in stimulating recipient macrophages in vivo and contributing to chronic immune activation.

FUTURE OUTLOOK AND CONCLUSION
vmiRNAs have overall a protecting role for the viral infection. By interfering with host cellular proteins and mechanisms of immune evasion, they represent a potential target for future specific therapies. Studies on the complex interplay between viral miRNAs and host genes has been only recently started and further investigations are required to identify more efficient and less toxic therapeutic strategies. The identification of differentially expressed miRNAs during viral infection (for example in HIV-1 infection) may provide a new approach to control disease progression. Finally, understanding the mechanism of vmiRNA-mediated immune evasion will allow us to develop novel strategies to prevent and cure viral infections. Targeting vmiRNAs with molecules such as antagomirs may represent a novel therapeutic strategy to limit chronic immune activation and the progression of viral infection.

AUTHOR CONTRIBUTIONS
DF and AG analyzed the literature, wrote the manuscript, and approved the final version for publication. RR analyzed the literature, revised the manuscript and approved the final version for publication.


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Renoprotective Effects of Aldose Reductase Inhibitor Epalrestat against High Glucose-Induced Cellular Injury

Diabetic nephropathy (DN) is the leading cause of end stage renal disease worldwide. Increased glucose flux into the aldose reductase (AR) pathway during diabetes was reported to exert deleterious effects on the kidney. The objective of this study was to investigate the renoprotective effects of AR inhibition in high glucose milieu in vitro. Rat renal tubular (NRK-52E) cells were exposed to high glucose (30 mM) or normal glucose (5 mM) media for 24 to 48 hours with or without the AR inhibitor epalrestat (1 μM) and assessed for changes in Akt and ERK1/2 signaling, AR expression (using western blotting), and alterations in mitochondrial membrane potential (using JC-1 staining), cell viability (using MTT assay), and cell cycle. Exposure of NRK-52E cells to high glucose media caused acute activation of Akt and ERK pathways and depolarization of mitochondrial membrane at 24 hours. Prolonged high glucose exposure (for 48 hours) induced AR expression and G1 cell cycle arrest and decreased cell viability (84% compared to control) in NRK-52E cells. Coincubation of cells with epalrestat prevented the signaling changes and renal cell injury induced by high glucose. Thus, AR inhibition represents a potential therapeutic strategy to prevent DN.

Introduction
Diabetic nephropathy (DN) is the leading cause of end stage renal disease worldwide [1]. Chronic hyperglycemia causes progressive kidney damage in diabetic patients, which is characterized by three main findings: (1) persistent albuminuria (more than 300 mg/day or more than 200 g/min), (2) progressive and irreversible decline in glomerular filtration rate (GFR), and (3) increased arterial blood pressure [2]. In most DN cases, pathological changes are prominent in the glomeruli as well as the renal tubules of diabetes patients' kidneys even before the onset of microalbuminuria. In the glomeruli, the early adaptive changes are marked by glomerular hypertrophy and thickening of the glomerular basement membrane (GBM). As the disease progresses, the mesangial matrix expands, and the mesangial cells increase in size, leading to glomerulosclerosis and the development of nodular lesions also known as Kimmelstiel-Wilson nodules, a hallmark of DN. Similarly, the tubules also undergo early hypertrophy and thickening of the tubular basement membrane (TBM), which then progress to tubulointerstitial fibrosis and inflammation at later stages of the disease [3].
Growing body of evidences has shown that DN is induced by multiple factors such as impaired glucose signaling, increased blood pressure, imbalance in the cellular redox status, inflammation and altered protein folding, and turnover within the renal cells. Additionally, several signaling pathways are implicated in the development and progression of DN [4]. Akt, a 57 KDa serine/threonine kinase that regulates numerous cellular functions such as transcription, translation, cell proliferation, and survival, is thought to play a critical role in induced complications [5]. It has been shown that Akt increases the production of extracellular matrix (ECM) proteins such as collagen-I, collagen-IV, and laminin in diabetic kidney [6,7]. In addition, the activation of PI3K/Akt pathway in renal tubules has been shown to cause tubular hypertrophy via two mechanisms: (1) by a cell cycledependent mechanism and (2) by altering protein translation through phosphorylation of the protein translation regulator 4E-BP1 [8][9][10].

BioMed Research International
The extracellular signal-regulated kinase (ERK) pathway is another crucial signaling network involved in numerous cellular processes [11]. Under hyperglycemia, the Ras-Raf-MEK-ERK signal transduction cascade becomes activated and leads to the activation of renin-angiotensin-aldosteronesystem (RAAS), which is well known to cause renal injury [4,12]. Moreover, ERK has been shown to induce vascular endothelial growth factor (VEGF) and transforming growth factor-beta 1 (TGF-1), which causes albuminuria, increase in GFR, and kidney fibrosis [4,10,13]. Furthermore, several studies have showed crosstalk between ERK pathway and other pathways in DN such as advanced glycation end products (AGE) and protein kinase C (PKC) pathways [14]. Therefore, ERK activation is considered one of the significant signaling events in DN.
Aldose reductase (AR; EC 1.1.1.21) is a member of aldoketo reductase (AKR) superfamily [15], which constitutes more than 100 structurally related proteins [16]. AR is the rate-limiting enzyme in the polyol pathway, which is responsible for the reduction of glucose to sorbitol using NADPH as a cofactor. Subsequently, sorbitol is converted to fructose by sorbitol dehydrogenase using NAD+ as a cofactor. In normoglycemic conditions, glucose serves as a poor substrate for AR due to the high Km values of AR for glucose. Under hyperglycemic conditions, there is increase in flux of glucose through polyol pathway, which leads to imbalances in cofactors and products causing several deleterious effects such as osmotic stress, oxidative stress, increased AGE, and activation of PKC pathway [17].
Many studies have investigated the role of polyol pathway in the development of DN and evaluated the protective role of various aldose reductase inhibitors (ARIs) [18][19][20][21][22][23][24]. ARIs are thought to exert their beneficial effects by decreasing hyperfiltration, albuminuria, and accumulation of ECM proteins (antifibrotic effects) without affecting the blood glucose or HbA1c levels [25]. In addition, plethora of in vitro data supports the link between polyol pathway and PKC activity and TGF-1 and ECM protein production, which suggest the involvement of this pathway in fibrotic changes observed in DN [26][27][28][29]. Although several preclinical studies using ARIs have demonstrated nephroprotective effects against DN, the results from most clinical trials are not very encouraging [25]. There are several challenges to be faced when targeting AR enzyme including the difficulty of developing ARIs with high specificity towards ALR2 isoform and the use of optimum doses for AR inhibition. Moreover, the efficacy of ARIs depends on the levels of expression of AR in target tissues and the degree of involvement of polyol pathway in the development of diabetic complications, which varies significantly across diabetic patients [25,30].
In this study, we have investigated the effects of high glucose concentrations on rat renal proximal tubular cells cultured in vitro. The study is aimed to investigate the key molecular pathways that are activated in renal cells under high glucose conditions and to identify the cellular markers that mediate hyperglycemia-induced renal cell injury. We hypothesized that exposing renal tubular cells to high glucose concentrations would induce AR expression and, thus, inhibiting AR using epalrestat (a potent ARI) would confer protection against high glucose-induced renal cell injury.

Cell Culture. Normal rat kidney cells (NRK-52E; Health
Protection Agency, UK) were cultured in Dulbecco's modified Eagle medium (DMEM, Life Technologies, UK) supplemented with 10% fetal bovine serum (FBS), 1% L-glutamine, and 1% penicillin/streptomycin. The cells were maintained at 37 ∘ C in 5% CO 2 incubator. To obtain an in vitro model that represents the features of DN, cells were divided into 3 groups: (1) control, where cells were treated with DMEM medium containing 5 mM glucose and 1% FBS, (2) high glucose treated, where cells were treated with DMEM medium containing 30 mM glucose and 1% FBS, and (3) osmotic control, where cells were treated with DMEM medium containing 5 mM glucose and 25 mM mannitol with 1% FBS to simulate a milieu that is osmotically equivalent to the 30 mM high glucose media. The cells were exposed to the above-mentioned conditions for 24, 48, and 72 hours. To investigate the effects of AR inhibition on high glucose-induced stress in renal cells, NRK-52E cells were cotreated with epalrestat (Sigma Aldrich, Germany) at 1 M concentration and compared against vehicle-treated controls.

Assessment of Cell
Viability. NRK-52E cells were seeded on 96-well plates and treated according to the aforementioned protocol. At the end point, media were removed and replaced by 100 l of fresh serum-free media and 10 l of 5 mg/ml MTT (3-(4,5-dimethylthiazol-2-yl)-2,5diphenyltetrazolium bromide) reagent (Sigma Aldrich, Germany). The plates were incubated at 37 ∘ C for 3-4 hours. The purple formazan crystals formed were solubilized in 50 l of dimethylsulfoxide (DMSO), and the absorbance was measured at 570 nm using a microplate reader. Absorbance value from the control group was set as 100% and the values from treatment groups were expressed as percentage of control.
The densitometry values were normalized to actin (used as loading control) and expressed as percentage of control.

Assessment of Mitochondrial Membrane Potential (JC-1 Assay).
The high glucose-induced alterations in the mitochondrial membrane potential in NRK-52E cells were assessed using JC-1 (5,5 ,6,6 -tetrachloro-1,1 ,3,3 tetraethylbenzimidazolylcarbocyanine iodide; T-3168, Life Technologies, UK) as described previously [31]. Briefly, after 24 or 48 h high glucose treatment, cells were stained with JC-1 reagent (10 M) for 30 minutes at 37 ∘ C in dark, and the fluorescence was measured using a microplate reader (SpectraMax, USA) at excitation/emission (Ex/Em) 488 nm/529 nm to measure monomers and Ex/Em 488 nm/590 nm to measure J-aggregates. The ratio of fluorescent intensity of Jaggregates (red) to that of monomers (green) was calculated to determine changes in mitochondrial membrane potential. For fluorescence microscopy, cells were stained with JC-1 as described above and imaged under fluorescent microscope (Olympus, USA) with TRITC filter at 200x magnification.

Cell Cycle Analysis.
Following 24 or 48 h high glucose treatment, NRK-52E cells were fixed in 70% ethanol and kept at −20 ∘ C. On the day of analysis, cells were pelleted out by centrifugation at 1000 ×g for 5 minutes at 4 ∘ C and stained with Tali5 cell cycle solution (Life Technologies, UK) in dark for 30 minutes to achieve a cell density of 1 × 10 5 -5 × 10 6 cells/ml for analysis using Tali image-based cytometer (Life Technologies, UK).

Statistical Analysis.
All values were calculated as a percentage of control (5 mM glucose-treated group) and expressed as Mean ± SEM. One-way ANOVA test followed by Tukey's post hoc test was performed using GraphPad Prism 6 to detect statistical differences between groups. < 0.05 was considered statistically significant.

Effect of High Glucose on the Viability and Relative Mitochondrial Membrane Potential of Renal Proximal Tubular
Cells. NRK-52E cells were exposed to high glucose media (30 mM) for 24, 48, and 72 hours. 30 mM mannitol was used as osmotic control. At the endpoint, cell viability was assessed by MTT assay. It was observed that when using cells at low passage number (P2 to P6), there was a significant decrease in viability after 48 hours to 84%, which further declined to 68% after 72 h (Figure 1(a)). Intriguingly, the high glucoseinduced decrease in viability after 48 and 72 h exposure was not reproducible when using cells with passage numbers higher than P7 (data not shown). Thus, we chose to perform all experiments on NRK-52E cells between passage numbers P2 to P6. Alterations in the relative mitochondrial membrane potential (Δ m) of NRK-52E cells following exposure to high glucose concentrations were assessed using JC-1 staining. JC-1 is a cationic dye, which at high Δ m forms J-aggregate with red fluorescence, while at low Δ m it remains as monomers with green fluorescence. Therefore, the ratio of red to green fluorescence is indicative of the polarization status of the mitochondria membrane. NRK-52E cells exposed to 30 mM glucose for 24 h and stained with JC-1 showed increased green fluorescence, and the red to green fluorescence ratio was 81% as compared to control. Although the mitochondrial depolarization was sustained after 48 h of high glucose exposure, it was not statistically significant (Figure 1(b)). These findings indicate that acute high glucose exposure induces transient mitochondrial depolarization in renal tubular cells.

Effect of High Glucose on Akt and ERK Signaling Pathways in Renal Tubular
Cells. Activation of Akt and ERK signaling pathways was investigated in renal tubular cells following exposure to high glucose for 10 minutes, 30 minutes, 1 hour, 2 hours, and 4 hours. As shown in Figures 2 and 3, there was an acute and transient induction of p-Akt and p-ERK after 10 minutes of high glucose exposure, which then subsided at further time points tested.

Effect of High Glucose on Aldose Reductase (AR) Expression in Renal Tubular Cells.
To investigate if aldose reductase (AR) was induced in the model, AR expression was measured in NRK-52E cells subjected to high glucose treatment. Although the AR expression remained unchanged after 24 hours of high glucose treatment, it was significantly induced at 48 hours by about 1.5-fold as compared to control (Figure 4). A small induction in AR protein expression was also observed in cells exposed to high osmolar media (30 mM mannitol) at 48 hours.

Effect of Epalrestat on High Glucose-Induced Alterations in
Renal Cell Viability and Cell Cycle. To examine the protective effect of aldose reductase (AR) inhibition on high glucoseinduced renal cell injury, NRK-52E cells were exposed to high glucose in presence of 1 M epalrestat (an AR inhibitor) for 48 hours. At the endpoint, cell viability was measured using MTT assay. As observed earlier, with 48 hours' high glucose treatment, the cell viability declined to 84% ( < 0.05) to that of control. In contrast, coincubation of cells with epalrestat at 1 M was able to attenuate high glucose induced cell death (Figure 5(a)). In addition, when cells were exposed to high glucose medium for 48 h in presence of epalrestat, the percentage of cells increased in the G1 phase when compared to control but was not statistically significant ( Figure 5(b)). reversed the depolarization observed with 24 hours' high glucose treatment. Fluorescent cell images further confirmed this finding, that is, an intense green staining observed in high glucose-treated cells, whereas the cells coincubated with epalrestat show a mixed red and green staining pattern similar to control cells ( Figure 6).

Effect of Epalrestat on High Glucose-Induced Activation of Akt and ERK Pathways in Renal Tubular
Cells. NRK-52E cells were exposed to 10 minutes of high glucose in presence of epalrestat to investigate the effect of aldose reductase inhibition on high glucose-induced activation of Akt and ERK pathways. As seen in Figure 7, epalrestat cotreatment attenuated high glucose-mediated induction of Akt and ERK pathways, which suggests crosstalk between these two pathways and the polyol pathway in renal cells under high glucose conditions.

Discussion
Our studies indicate that exposure of NRK-52E cells to 30 mM glucose causes a progressive decline in cell viability, that is, 84% viability after 48 hours and 68% viability after 72 hours as compared to control (100%). Similarly, a study by Park et al. using primary rabbit renal proximal tubular cells observed inhibition of cell proliferation over 24, 48, and 72 hours following exposure to 25 mM glucose [32]. It has been shown that high glucose induces mitochondrial alterations and dysfunction, which ultimately leads to apoptosis. In the current study, the effect of high glucose on mitochondrial membrane potential was tested in NRK-52E cells using JC-1 dye. After 24 hours, there was a slight decrease (81% compared to control) in mitochondrial membrane potential, which was sustained at 48 h (86% compared to control) indicating that high glucose treatment induces mitochondrial membrane depolarization in renal tubular cells. Our finding is in line with other studies, which demonstrated the onset of mitochondrial dysfunction in human kidney cells (HK-2) following exposure to high glucose medium [33][34][35]. Therefore, these findings suggest that tubular cell death observed in the current model might be initiated by alterations in mitochondrial function.
PI3K/Akt pathway is a vital pathway that is implicated in numerous cellular processes [36]. Several studies have revealed impairments in the PI3K/Akt pathway during diabetes and signified its role in the development of diabetic complications [8,37,38]. For example, hyperglycemia has been shown to induce collagen-I synthesis in mesangial cells by stimulating cytokines such as TGF-and epidermal     Table 1.  growth factor (EGF) through PI3K/Akt dependent pathway [37,39]. In this study, exposing NRK-52E cells to 30 mM glucose caused acute activation of S473-phosphorylated Akt after 10 minutes of exposure, which then subsided at higher time points (30 minutes, 1 h, 2 h, and 4 h). This finding was consistent with other findings previously described in the literature where high glucose activates Akt pathway in renal cells [9,40]. ERK1/2 pathway is a major pathway that is actively involved in regulation of cellular functions. It has been shown that mitogen-activated protein kinase (MAPK) cascade was activated in glomerular mesangial cells exposed to high glucose concentrations as well as in diabetic rats' glomeruli [41]. Furthermore, inhibiting ERK 1/2 pathway in mesangial cells in vitro attenuated high glucose-induced TGF-1 expression and the induction of collagen-I and fibronectin mRNA [42,43]. In addition, in vitro studies using renal tubular cells showed that ERK1/2 pathway is implicated in high glucoseinduced TGF-1, fibronectin, and collagen-IV expression as well as tubular cell hypertrophy [44,45]. In the current study, high glucose treatment induced acute phosphorylation of ERK1/2 within 10 minutes of exposure in renal tubular cells. Taken together, these findings provide evidence for the role of ERK1/2 pathway in the development and progression of DN.
Findings from this study indicate that exposing tubular cells to high glucose concentrations in vitro induces low levels of cellular stress and injury, which is not unexpected given the complexity of mechanisms involved in diabetic complications and the inherent limitations of in vitro high glucose model to mimic diabetic setting. To establish an in vitro model of DN within a reasonable time frame, culturing cells at high glucose concentrations are commonly employed [32,46]. Alternatively, cells could be transfected with markers of DN such as angiotensin II and TGF-1 to aggravate DN in vitro. Additionally, albumin can be used in conjunction with high  glucose to mimic advanced stages of DN [47]. Serum (FBS) concentration is another crucial factor in the development of an in vitro DN model. In our model, serum deprivation has greatly affected the growth and morphology of NRK-52E cells, and consistent results were obtained when cells were exposed to 30 mM glucose media containing 1% FBS for 24 and 48 hours. It has been shown that activation of polyol pathway due to hyperglycemia in renal cells plays a pivotal role in the development of DN [48]. Therefore, it is hypothesized that inhibiting aldose reductase enzyme, the rate-limiting enzyme in the polyol pathway, can protect the cells against high glucose-induced cell injury in renal tubular cells [49]. Activation of polyol pathway in response to high glucose has been reported to cause a sevenfold increase in sorbitol production in renal proximal tubules [50]. Our results reveal that aldose reductase expression was increased by about 1.5fold with high glucose treatment as compared to control at 48 hours. Similar inductions of AR protein expression have been reported in studies using mesangial cells cultured under high glucose conditions [29,48].
Coincubation of NRK-52E cells with 1 M epalrestat (dose based on the literature [34] and our pilot studies) has prevented mitochondrial depolarization and loss of cell BioMed Research International 9 viability induced by high glucose. These findings suggest that AR inhibition can protect renal cells against high glucoseinduced mitochondrial alterations and improve cell survival. In addition, cell cycle analysis was performed to determine the effects of high glucose on cell cycle progression. Our results showed that incubation with high glucose for 48 h increased the proportion of cells in G1 phase as compared to control (59% versus 51%, resp.). Consistent with our findings, a similar G1 phase arrest has been observed in other studies using different types of renal tubular cells [8]. Together, these findings suggest that tubular renal cell hypertrophy occurs in cell cycle-dependent manner [8,32,51].
Although treatment with epalrestat did not block the effects of high glucose on cell cycle in our model, it ameliorated high glucose-mediated acute activation of Akt and ERK pathways. As mentioned earlier, both pathways are actively involved in DN and contribute to development of many of its pathologic features. Therefore, our results suggest that AR inhibition exerts a protective effect on kidney cells through attenuation of Akt and ERK dependent pathways. This finding is in line with another study, which showed that transfecting mesangial cells with AR gene augmented high glucose-mediated activation of Akt and ERK pathways in these cells [29].
In support to our findings, inhibiting aldose reductase has been shown to reverse high glucose-induced production of ECM proteins such as fibronectin [29]. Nevertheless, specific inhibition of aldose reductase with enzyme inhibitors is extremely challenging due to its extensive homology with other aldo-keto reductase family members and makes it hard to find specific inhibitors that are devoid of properties to block other aldo-keto enzymes involved in protective physiological detoxification of toxic aldehydes formed within cells.

Conclusions
In summary, in vitro exposure to high glucose acutely activates Akt and ERK pathways and induces pathologic changes such as decreases in cell viability and depolarization of mitochondrial membrane potential in renal tubular cells (Figure 8). These pathologic alterations in signaling pathways and cellular organelles initiated by hyperglycemia could potentially contribute to high glucose-induced renal injury. Most importantly, our studies demonstrate that polyol pathway plays a crucial role in development of renal tubular cell injury in a high glucose milieu, and inhibiting its ratelimiting enzyme aldose reductase (AR) protects renal cells against high glucose-mediated deleterious effects (Figure 8). Further studies in vitro and in vivo are required to ascertain their therapeutic potential of AR inhibitors to prevent and/or treat DN.

Disclosure
The sponsor had no involvement in the study design, data collection, analysis and interpretation of data, writing the manuscript, or in the decision to submit the article for publication.

Conflicts of Interest
The authors declare that there are no conflicts of interest regarding the publication of this paper.