Nuclear Volume Research Articles

Actomyosin and vimentin cytoskeletal networks regulate nuclear shape, mechanics and chromatin organization

The regulation of nuclear state by the cytoskeleton is an important part of cellular function. Actomyosin stress fibres, microtubules and intermediate filaments have distinct and complementary roles in integrating the nucleus into its environment and influencing its mechanical state. However, the interconnectedness of cytoskeletal networks makes it difficult to dissect their individual effects on the nucleus. We use simple image analysis approaches to characterize nuclear state, estimating nuclear volume, Poisson’s ratio, apparent elastic modulus and chromatin condensation. By combining them with cytoskeletal quantification, we assess how cytoskeletal organization regulates nuclear state. We report for a number of cell types that nuclei display auxetic properties. Furthermore, stress fibres and intermediate filaments modulate the mechanical properties of the nucleus and also chromatin condensation. Conversely, nuclear volume and its gross morphology are regulated by intracellular outward pulling forces exerted by myosin. The modulation exerted by the cytoskeleton onto the nucleus results in changes that are of similar magnitude to those observed when the nucleus is altered intrinsically, inducing chromatin decondensation or cell differentiation. Our approach allows pinpointing the contribution of distinct cytoskeletal proteins to nuclear mechanical state in physio- and pathological conditions, furthering our understanding of a key aspect of cellular behaviour.

Computer simulations reveal mechanisms that organize nuclear dynein forces to separate centrosomes.

Centrosome separation along the surface of the nucleus at the onset of mitosis is critical for bipolar spindle assembly. Dynein anchored on the nuclear envelope is known to be important for centrosome separation, but it is unclear how nuclear dynein forces are organized in an anisotropic manner to promote the movement of centrosomes away from each other. Here we use computational simulations of Caenorhabditis elegans embryos to address this fundamental question, testing three potential mechanisms by which nuclear dynein may act. First, our analysis shows that expansion of the nuclear volume per se does not generate nuclear dynein-driven separation forces. Second, we find that steric interactions between microtubules and centrosomes contribute to robust onset of nuclear dynein-mediated centrosome separation. Third, we find that the initial position of centrosomes, between the male pronucleus and cell cortex at the embryo posterior, is a key determinant in organizing microtubule aster asymmetry to power nuclear dynein-dependent separation. Overall our work reveals that accurate initial centrosome position, together with steric interactions, ensures proper anisotropic organization of nuclear dynein forces to separate centrosomes, thus ensuring robust bipolar spindle assembly.

Effects of gamma rays on rat testis tissue according to the morphological parameters and immunohistochemistry: radioprotective role of silymarin.

ObjectiveTo determine the radioprotective effects of Silymarin in adult male Sprague-Dawley rats irradiated with γ-rays.MethodsThe present experimental study was performed in Tehran University of Medical Sciences, Tehran, Iran from December 2009 to March 2010. The study was performed on 40 rats, which were randomly and equally divided into four groups: 1) control group: neither received Silymarin nor irradiated with γ-rays; 2) γ-irradiation group: testis region exposed to 2Gy of γ-rays; 3) Silymarin & γ-irradiation: rats received 100 mg/kg of Silymarin 24hrs before exposure to 2Gy of γ-rays; 4) Silymarin & γ-irradiation: rats received 200 mg/kg of Silymarin 24hrs before exposure to 2Gy of γ-rays. After animal experiments and preparing the tissue sections, different histological and histomorphological parameters of seminiferous tubules and the biological characteristics of Leydig cells were evaluated applying quantitative assessment, Johnson scoring, and Leydig cell apoptosis assay by TUNEL method. The data were analyzed applying ANOVA and Tukey’s post hoc test, using SPSS software (V.19).ResultsIrradiation of 2 Gy γ-rays to the testis of the rats significantly affected the frequency of spermatogonia, primary spermatocyte, round spermatid, spermatozoa, seminiferous tube and lumen diameters, thickness of the epithelium, Leydig cell nuclear diameter and volume, epithelium height, and apoptotic cells (p<0.05). However, administration of Silymarin improved the mentioned parameters specifically in 200 mg/kg of dosage.ConclusionSilymarin could act as a potent radioprotector and it can be used in modulation as well as improvement to radiation therapy to prevent male reproductive function, specifically seminiferous tubules in an animal model; however, its molecular mechanism is still not clear and needs more molecular researches.

Application of Nuclear Volume Measurements to Comprehend the Cell Cycle in Root-Knot Nematode-Induced Giant Cells.

Root-knot nematodes induce galls that contain giant-feeding cells harboring multiple enlarged nuclei within the roots of host plants. It is recognized that the cell cycle plays an essential role in the set-up of a peculiar nuclear organization that seemingly steers nematode feeding site induction and development. Functional studies of a large set of cell cycle genes in transgenic lines of the model host Arabidopsis thaliana have contributed to better understand the role of the cell cycle components and their implication in the establishment of functional galls. Mitotic activity mainly occurs during the initial stages of gall development and is followed by an intense endoreduplication phase imperative to produce giant-feeding cells, essential to form vigorous galls. Transgenic lines overexpressing particular cell cycle genes can provoke severe nuclei phenotype changes mainly at later stages of feeding site development. This can result in chaotic nuclear phenotypes affecting their volume. These aberrant nuclear organizations are hampering gall development and nematode maturation. Herein we report on two nuclear volume assessment methods which provide information on the complex changes occurring in nuclei during giant cell development. Although we observed that the data obtained with AMIRA tend to be more detailed than Volumest (Image J), both approaches proved to be highly versatile, allowing to access 3D morphological changes in nuclei of complex tissues and organs. The protocol presented here is based on standard confocal optical sectioning and 3-D image analysis and can be applied to study any volume and shape of cellular organelles in various complex biological specimens. Our results suggest that an increase in giant cell nuclear volume is not solely linked to increasing ploidy levels, but might result from the accumulation of mitotic defects.

Study of the role of plant nuclear envelope and lamina-like components in nuclear and chromatin organisation using 3D imaging

The linker of nucleoskeleton and cytoskeleton (LINC) complex is an evolutionarily well-conserved protein bridge connecting the cytoplasmic and nuclear compartments across the nuclear membrane. While recent data supports its function in nuclear morphology and meiosis, its implication for chromatin organisation has been less studied in plants. The first aim of this work was to develop NucleusJ a simple and user-friendly ImageJ plugin dedicated to the characterisation of nuclear morphology and chromatin organisation in 3D. NucleusJ quantifies 15 parameters including shape and size of nuclei as well as intra-nuclear objects and their position within the nucleus. A step-by-step documentation is available for self-training, together with data sets of nuclei with different nuclear organisation. Several improvements are ongoing to release a new version of this plugin. In a second part of this work, 3D imaging methods have been used to investigate nuclear morphology and chromatin organisation in interphase nuclei of the plant model Arabidopsis thaliana in which heterochromatin domains cluster in conspicuous chromatin regions called chromocentres. Chromocentres form a repressive chromatin environment contributing to the transcriptional silencing of repeated sequences a general mechanism needed for genome stability. Quantitative measurements of 3D position of chromocentres in the nucleus indicate that most chromocentres are situated in close proximity to the periphery of the nucleus but that this distance can be altered according to nuclear volume or in specific mutants affecting the LINC complex. Finally, the LINC complex is proposed to contribute at the proper chromatin organisation and positioning since its alteration is associated with the release of transcriptional silencing as well as decompaction of heterochromatic sequences. The last part of this work takes advantage of available genomic sequences and RNA-seq data to explore the evolution of NE proteins in plants and propose a minimal requirement to built the simplest functional nuclear envelope. Altogether, work achieved in this thesis associate genetics, molecular biology, bioinformatics and imaging to better understand the contribution of the nuclear envelope in nuclear morphology and chromatin organisation and suggests the functional implication of the LINC complex in these processes.

A systematic genomic screen implicates nucleocytoplasmic transport and membrane growth in nuclear size control.

How cells control the overall size and growth of membrane-bound organelles is an important unanswered question of cell biology. Fission yeast cells maintain a nuclear size proportional to cellular size, resulting in a constant ratio between nuclear and cellular volumes (N/C ratio). We have conducted a genome-wide visual screen of a fission yeast gene deletion collection for viable mutants altered in their N/C ratio, and have found that defects in both nucleocytoplasmic mRNA transport and lipid synthesis alter the N/C ratio. Perturbing nuclear mRNA export results in accumulation of both mRNA and protein within the nucleus, and leads to an increase in the N/C ratio which is dependent on new membrane synthesis. Disruption of lipid synthesis dysregulates nuclear membrane growth and results in an enlarged N/C ratio. We propose that both properly regulated nucleocytoplasmic transport and nuclear membrane growth are central to the control of nuclear growth and size.

Adapting the French nuclear fleet to integrate variable renewable energies via the production of hydrogen: Towards massive production of low carbon hydrogen?

Producing low-carbon hydrogen at a competitive rate is becoming a new challenge with respect to efforts to reduce greenhouse gas emissions. We examine this issue in the French context, which is characterised by a high nuclear share and the target to increase variable renewables by 2050. The goal is to evaluate the extent to which excess nuclear power could contribute to producing low-carbon hydrogen.Our approach involves designing scenarios for nuclear and renewables, modelling and evaluating the potential nuclear hydrogen production volumes and costs, examining the latter through the scope of hydrogen market attractiveness and evaluating the potential of CO2 mitigation.This article shows that as renewable shares increase, along with the hydrogen market expected growth driven by mobility uses, opportunities are created for the nuclear operator. If nuclear capacities are maintained, nuclear hydrogen production could correspond to the demand by 2030. If not, possibilities could still exist by 2050.

Caloric restriction minimizes aging effects on the femoral medial condyle

This study aimed to analyze the effects of caloric restriction on aged femoral articular cartilage of Wistar rats. Three groups of eight animals each were considered: young (YC) and old (OC) control groups fed with a normal diet and old caloric restriction group (OCR) composed of 18-month-old animals fed with a 31% less caloric diet from 6-months of age. Articular cartilage was studied through morphometry and immunohistochemistry. Body mass was 12% less in the OCR group than in the OC group. The articular cartilage from OC rats show thinner medial condyles, fewer chondrocytes, smaller chondrocytes nuclear volume and, in both condyles, a predominance of collagen type II and less collagen density compared to both YC and OCR groups (p < .001). In contrast, OCR articular cartilage show thicker medial condyles, larger chondrocytes nuclear volume and increased collagen density compared to OC group (p < 0.001). We concluded that caloric restriction minimizes the effects of aging on medial condyles of the femoral articular cartilage.

Cytomorphologic parameters in monitoring cytogenotoxic effects of fertilizer in Allium cepa L.

The present investigation was done to assess cytomorphologic parameters as indicators of genotoxicity as it is a simpler method and could be suggested for rapid screening of the vast range of agrochemicals used all over the world. The excessive and indiscriminate use of agrochemicals is responsible for increasing the level of pollutants in the soil environment resulting in cellular and molecular damage to the plants. The cellular damage caused manifestation of the resulting oxidative stress due to pollutants which can go up to the level of DNA. The roots of Allium cepa were treated with 0.5mg Nml-1 concentration of ammonium nitrate fertilizer for 3, 6, 9 and 12h, and in mitotic preparation of their respective root, mitotic index, phase indices and the genotoxic markers viz. chromosomal aberrations and binucleate cells were observed and the data statistically analysed. A significant decrease in mitotic index and increase in abnormality percentage as compared to control was observed which increased with the treatment duration. Chromosomal aberrations like stickiness, fragmentation, precocious movement, bridges and disorientations were observed in varying frequencies. A cytomorphologic study revealed that the interphase cell volume of cells of treated roots and their respective interphase nuclear volume were reduced as compared to control. The ratio between nuclear and cytoplasmic volume has been reported to relate to cell integrity. Both these markers viz. cytomorphologic and genotoxic can be used for assessment of the toxicity of agrochemicals including fertilizer; in our study, they have revealed the cytogenotoxic behaviour of ammonium nitrate fertilizer.

Volume Transitions of Isolated Cell Nuclei Induced by Rapid Temperature Increase

Understanding the physical mechanisms governing nuclear mechanics is important as it can impact gene expression and development. However, how cell nuclei respond to external cues such as heat is not well understood. Here, we studied the material properties of isolated nuclei in suspension using an optical stretcher. We demonstrate that isolated nuclei regulate their volume in a highly temperature-sensitive manner. At constant temperature, isolated nuclei behaved like passive, elastic and incompressible objects, whose volume depended on the pH and ionic conditions. When the temperature was increased suddenly by even a few degrees Kelvin, nuclei displayed a repeatable and reversible temperature-induced volume transition, whose sign depended on the valency of the solvent. Such phenomenon is not observed for nuclei subjected to slow heating. The transition temperature could be shifted by adiabatic changes of the ambient temperature, and the magnitude of temperature-induced volume transition could be modulated by modifying the chromatin compaction state and remodeling processes. Our findings reveal that the cell nucleus can be viewed as a highly charged polymer gel with intriguing thermoresponsive properties, which might play a role in nuclear volume regulation and thermosensing in living cells.

Neuroprotection and visual function after optic neuritis.

This article discusses the advantages and pitfalls of testing neuroprotective treatment strategies in patients suffering from optic neuritis. Spectral domain optical coherence tomography now permits for automated segmentation of individual retinal layers. The peripapillary retinal nerve fibre layer (pRNFL) has been used in 13 of the 15 trials reviewed. Twelve trials also made use of electrophysiology. Overestimation of good visual recovery in the past has recently been recognized. Assessment of low contrast visual acuity and colour vision are now mainstream. The availability of highly accurate and robust trial outcome measures has facilitated research on this topic. A single long-term structural outcome measurement of the pRNFL is sufficient. For shorter term, assessments of the ganglion cell/inner plexiform layer and axonal birefringence are promising. Longitudinal blood levels of neurofilament proteins permit to recognize axonal loss at presentation and monitor changes longitudinally. Inner nuclear layer volume changes relate to inflammatory disease activity.Pitfalls are related to the timing of events. Hyperacute recruitment is needed for future trials. The onset of demyelination is not known, which complicates timing of electrophysiological recordings. Optic disc oedema precludes the use of the pRNFL from the affected eye as a baseline variable. The concomitant use of corticosteroids complicates interpretation of trial data.

Butterfly eyespot organiser: in vivo imaging of the prospective focal cells in pupal wing tissues

Butterfly wing eyespot patterns are determined in pupal tissues by organisers located at the centre of the prospective eyespots. Nevertheless, organiser cells have not been examined cytochemically in vivo, partly due to technical difficulties. Here, we directly observed organiser cells in pupal forewing epithelium via an in vivo confocal fluorescent imaging technique, using 1-h post-pupation pupae of the blue pansy butterfly, Junonia orithya. The prospective eyespot centre was indented from the plane of the ventral tissue surface. Three-dimensional reconstruction images revealed that the apical portion of “focal cells” at the bottom of the eyespot indentation contained many mitochondria. The mitochondrial portion was connected with a “cell body” containing a nucleus. Most focal cells had globular nuclei and were vertically elongated, but cells in the wing basal region had flattened nuclei and were tilted toward the distal direction. Epithelial cells in any wing region had cytoneme-like horizontal processes. From 1 h to 10 h post-pupation, nuclear volume increased, suggesting DNA synthesis during this period. Morphological differences among cells in different regions may suggest that organiser cells are developmentally ahead of cells in other regions and that position-dependent heterochronic development is a general mechanism for constructing colour patterns in butterfly wings.

DNA methylation is dispensable for changes in global chromatin architecture but required for chromocentre formation in early stem cell differentiation.

Epiblast stem cells (EpiSCs), which are pluripotent cells isolated from early post-implantation mouse embryos (E5.5), show both similarities and differences compared to mouse embryonic stem cells (mESCs), isolated earlier from the inner cell mass (ICM) of the E3.5 embryo. Previously, we have observed that while chromatin is very dispersed in E3.5 ICM, compact chromatin domains and chromocentres appear in E5.5 epiblasts after embryo implantation. Given that the observed chromatin re-organization in E5.5 epiblasts coincides with an increase in DNA methylation, in this study, we aimed to examine the role of DNA methylation in chromatin re-organization during the in vitro conversion of ESCs to EpiSCs. The requirement for DNA methylation was determined by converting both wild-type and DNA methylation-deficient ESCs to EpiSCs, followed by structural analysis with electron spectroscopic imaging (ESI). We show that the chromatin re-organization which occurs in vivo can be re-capitulated in vitro during the ESC to EpiSC conversion. Indeed, after 7days in EpiSC media, compact chromatin domains begin to appear throughout the nuclear volume, creating a chromatin organization similar to E5 epiblasts and embryo-derived EpiSCs. Our data demonstrate that DNA methylation is dispensable for this global chromatin re-organization but required for the compaction of pericentromeric chromatin into chromocentres.

The LINC complex contributes to heterochromatin organisation and transcriptional gene silencing in plants.

The linker of nucleoskeleton and cytoskeleton (LINC) complex is an evolutionarily well-conserved protein bridge connecting the cytoplasmic and nuclear compartments across the nuclear membrane. While recent data support its function in nuclear morphology and meiosis, its involvement in chromatin organisation has not been studied in plants. Here, 3D imaging methods have been used to investigate nuclear morphology and chromatin organisation in interphase nuclei of the model plant Arabidopsis thaliana in which heterochromatin clusters in conspicuous chromatin domains called chromocentres. Chromocentres form a repressive chromatin environment contributing to transcriptional silencing of repeated sequences, a general mechanism needed for genome stability. Quantitative measurements of the 3D position of chromocentres indicate their close proximity to the nuclear periphery but that their position varies with nuclear volume and can be altered in specific mutants affecting the LINC complex. Finally, we propose that the plant LINC complex contributes to proper heterochromatin organisation and positioning at the nuclear periphery, since its alteration is associated with the release of transcriptional silencing as well as decompaction of heterochromatic sequences.

Chinese Journal of Nuclear Medicine and Molecular Imaging Volume 37, Number 11, November 25, 2017

Chinese Journal of Nuclear Medicine and Molecular Imaging Volume 37, Number 11, November 25, 2017

Chinese Journal of Nuclear Medicine and Molecular Imaging Volume 37, Number 10, October 25, 2017

Chinese Journal of Nuclear Medicine and Molecular Imaging Volume 37, Number 10, October 25, 2017

Optical model with multiple band couplings using soft rotator structure

A new dispersive coupled-channel optical model (DCCOM) is derived that describes nucleon scattering on 238 U and 232 Th targets using a soft-rotator-model (SRM) description of the collective levels of the target nucleus. SRM Hamiltonian parameters are adjusted to the observed collective levels of the target nucleus. SRM nuclear wave functions (mixed in K quantum number) have been used to calculate coupling matrix elements of the generalized optical model. Five rotational bands are coupled: the ground-state band, β-, γ-, non-axial- bands, and a negative parity band. Such coupling scheme includes almost all levels below 1.2 MeV of excitation energy of targets. The “effective” deformations that define inter-band couplings are derived from SRM Hamiltonian parameters. Conservation of nuclear volume is enforced by introducing a monopolar deformed potential leading to additional couplings between rotational bands. The present DCCOM describes the total cross section differences between 238 U and 232 Th targets within experimental uncertainty from 50 keV up to 200 MeV of neutron incident energy. SRM couplings and volume conservation allow a precise calculation of the compound-nucleus (CN) formation cross sections, which is significantly different from the one calculated with rigid-rotor potentials with any number of coupled levels.

Abstract IA19: Mechanisms of alternative telomere recombination

Abstract Telomere length maintenance is a requisite feature of cellular immortalization and a hallmark of human cancer. While most human cancers express telomerase activity, approximately 10-15% employ a recombination-dependent telomere maintenance pathway known as Alternative Lengthening of Telomeres (ALT), an incompletely understood process that is characterized by multi-telomere clusters. We have recently shown that a DNA double-strand break (DSB) response at ALT telomeres triggers long-range movement and clustering between chromosome termini, resulting in homology-directed telomere synthesis (Cho et. al. Cell 2014). Damaged telomeres initiate increased random surveillance of nuclear volumes before displaying rapid directional movement and association with recipient telomeres over micron-range distances. This phenomenon required Rad51 and the Hop2-Mnd1 heterodimer, implicating a specialized homology searching mechanism that exhibits similarities to meiotic recombination in ALT dependent telomere maintenance. This presentation will describe unpublished data that defines signaling events that are responsible for homology directed telomere synthesis during ALT. Using a novel methodology to purify nascent telomeres, we have identified the basic requirements for break induced telomere synthesis, including the specific DNA polymerases and helicases involved. Data derived from this approach reveals mechanisms of homology directed DNA synthesis that substantially differ from either normal replicative DNA synthesis or from existing models of break induced replication that have been defined in yeast. These data reveal an ordered series of events necessary for ALT telomere synthesis and have implications for homology directed DNA repair at other genomic locations. This work was supported by NIH grants GM101149, CA138835, and CA17494, and funds from the Abramson Family Cancer Research Institute and Basser Research Center for BRCA Citation Format: Roger A. Greenberg. Mechanisms of alternative telomere recombination. [abstract]. In: Proceedings of the AACR Precision Medicine Series: Cancer Cell Cycle - Tumor Progression and Therapeutic Response; Feb 28-Mar 2, 2016; Orlando, FL. Philadelphia (PA): AACR; Mol Cancer Res 2016;14(11_Suppl):Abstract nr IA19.

A three-dimensional spheroidal cancer model based on PEG-fibrinogen hydrogel microspheres.

Three-dimensional (3D) invitro cancer models offer an attractive approach towards the investigation of tumorigenic phenomena and other cancer studies by providing dimensional context and higher degree of physiological relevance than that offered by conventional two-dimensional (2D) models. The multicellular tumor spheroid model, formed by cell aggregation, is considered to be the "gold standard" for 3D cancer models, due to its ease and simplicity of use. Although better than 2D models, tumor spheroids are unable to replicate key features of the native tumor microenvironment, particularly due to a lack of surrounding extracellular matrix components and heterogeneity in shape, size and aggregate forming tendencies. In order to address this issue, we have developed a 3D "tumor microsphere" model, formed by a dual-photoinitiator, aqueous-oil emulsion technique, for the encapsulation of cancer cells within PEG-fibrinogen hydrogel microspheres and for subsequent long-term 3D culture. In comparison to self-aggregated tumor spheroids, the tumor microspheres displayed a higher degree of size and shape homogeneity throughout long-term culture. In sharp contrast to cells in tumor spheroids, cells within tumor microspheres demonstrated significant loss in apico-basal polarity and cellular architecture, cellular and nuclear atypia, increased disorganization, elevated nuclear cytoplasmic ratio and nuclear volume density and reduction in cell-cell junction length, all of which are hallmarks of malignant transformation and tumorigenic progression. Additionally, the tumor microsphere model was extended for the 3D encapsulation and maintenance of a wide range of other cancer cell (metastatic and non-metastatic) types. Taken together, our results reinforce the importance of incorporating a biomimetic matrix in the cellular microenvironment of 3D tumor models and the influential effects of the matrix on the tumorigenic morphology of 3D cultured cells. The tumor microsphere system established in this study has the potential to be used in future investigations of 3D cancer cell-cell and cell-ECM interactions and in drug-testing applications.

Retinal glymphatic system: an explanation for transient retinal layer volume changes?

This scientific commentary refers to ‘Retinal inner nuclear layer volume reflects response to immunotherapy in multiple sclerosis’, by Knier et al. (doi:10.1093/brain/aww219) . A recent article in Brain by Gelfand et al. (2012) on the inner nuclear layer (INL) of the retina in multiple sclerosis triggered a lively exchange of Letters to the Editor. With the advent of optical coherence tomography (OCT), a forgotten autopsy observation was restored to the top of the research agenda in multiple sclerosis. Over 150 years ago, severe atrophy of the retinal nerve fibre layer (RNFL) and ganglion cell layer (GCL) was shown to be associated with only subtle changes in the INL whilst the outer retinal layers were completely preserved (Muller, 1857). Although Heinrich Muller was cautious in interpreting potential INL changes, he was more convinced about the existence of secondary (retrograde) axonal degeneration. ‘Die sogenannten Korner erschienen etwas kornig, doch war mir sehr zweifelhaft, ob dies als pathologische Veranderung anzusprechen sei, da man Aehnliches auch sonst zu Gesicht bekommt.’ This case was discussed on pages 92–98 and the citation taken from page 93 (Muller, 1857). For the contemporary translation it is important to refer back to the terminology of the time. There were three ‘Kornerschichten’ that correspond to the present day: ‘ganglion cell layer’, ‘inner nuclear layer’ and ‘outer nuclear layer’. Muller was not able to see the GCL and his comment is on the next nuclear layer, the INL: ‘The so called nuclei seemed more grainy, but I am rather doubtful if this should be interpreted as a (specific) pathological finding as one can see similar features in other situations’. Subsequent histological studies of the macaque monkey eye following experimental lesions to the afferent visual pathway provided evidence for retrograde trans-synaptic degeneration reaching the GCL with associated changes in the …