A Cross-Species Neuroimaging Study of Sex Chromosome Dosage Effects on Human and Mouse Brain Anatomy.

All eutherian mammals show chromosomal sex determination with contrasting sex chromosome dosages (SCDs) between males (XY) and females (XX). Studies in transgenic mice and humans with sex chromosome trisomy (SCT) have revealed direct SCD effects on regional mammalian brain anatomy, but we lack a formal test for cross-species conservation of these effects. Here, we develop a harmonized framework for comparative structural neuroimaging and apply this to systematically profile SCD effects on regional brain anatomy in both humans and mice by contrasting groups with SCT (XXY and XYY) versus XY controls. Total brain size was substantially altered by SCT in humans (significantly decreased by XXY and increased by XYY), but not in mice. Robust and spatially convergent effects of XXY and XYY on regional brain volume were observed in humans, but not mice, when controlling for global volume differences. However, mice do show subtle effects of XXY and XYY on regional volume, although there is not a general spatial convergence in these effects within mice or between species. Notwithstanding this general lack of conservation in SCT effects, we detect several brain regions that show overlapping effects of XXY and XYY both within and between species (cerebellar, parietal, and orbitofrontal cortex), thereby nominating high priority targets for future translational dissection of SCD effects on the mammalian brain. Our study introduces a generalizable framework for comparative neuroimaging in humans and mice and applies this to achieve a cross-species comparison of SCD effects on the mammalian brain through the lens of SCT.SIGNIFICANCE STATEMENT Sex chromosome dosage (SCD) affects neuroanatomy and risk for psychopathology in humans. Performing mechanistic studies in the human brain is challenging but possible in mouse models. Here, we develop a framework for cross-species neuroimaging analysis and use this to show that an added X- or Y-chromosome significantly alters human brain anatomy but has muted effects in the mouse brain. However, we do find evidence for conserved cross-species impact of an added chromosome in the fronto-parietal cortices and cerebellum, which point to regions for future mechanistic dissection of sex chromosome dosage effects on brain development.

Neuroprotection in Cerebral Cortex Induced by the Pregnancy Hormone Estriol.

In multiple sclerosis (MS), demyelination occurs in the cerebral cortex, and cerebral cortex atrophy correlates with clinical disabilities. Treatments are needed in MS to induce remyelination. Pregnancy is protective in MS. Estriol is made by the fetoplacental unit, and maternal serum estriol levels temporally align with fetal myelination. Here, we determined the effect of estriol treatment on the cerebral cortex in the preclinical model of MS, experimental autoimmune encephalomyelitis (EAE). Estriol treatment initiated after disease onset decreased cerebral cortex atrophy. Neuropathology of the cerebral cortex showed increased cholesterol synthesis proteins in oligodendrocytes, more newly formed remyelinating oligodendrocytes, and increased myelin in estriol-treated EAE mice. Estriol treatment also decreased the loss of cortical layer V pyramidal neurons and their apical dendrites and preserved synapses. Together, estriol treatment after EAE onset reduced atrophy and was neuroprotective in the cerebral cortex.

Targeted deletion of PAC1 receptors in retinal neurons enhances neuron loss and axonopathy in a model of multiple sclerosis and optic neuritis.

Chronic inflammation drives synaptic loss in multiple sclerosis (MS) and is also commonly observed in other neurodegenerative diseases. Clinically approved treatments for MS provide symptomatic relief but fail to halt neurodegeneration and neurological decline. Studies in animal disease models have demonstrated that the neuropeptide pituitary adenylate cyclase-activating polypeptide (PACAP, ADCYAP1) exhibits anti-inflammatory, neuroprotective and regenerative properties. Anti-inflammatory actions appear to be mediated primarily by two receptors, VPAC1 and VPAC2, which also bind vasoactive intestinal peptide (VIP). Pharmacological experiments indicate that another receptor, PAC1 (ADCYAP1R1), which is highly selective for PACAP, provides protection to neurons, although genetic evidence and other mechanistic information is lacking. To determine if PAC1 receptors protect neurons in a cell-autonomous manner, we used adeno-associated virus (AAV2) to deliver Cre recombinase to the retina of mice harboring floxed PAC1 alleles. Mice were then subjected to chronic experimental autoimmune encephalomyelitis (EAE), a disease model that recapitulates major clinical and pathological features of MS and associated optic neuritis. Unexpectedly, deletion of PAC1 in naïve mice resulted in a deficit of retinal ganglionic neurons (RGNs) and their dendrites, suggesting a homeostatic role of PAC1. Moreover, deletion of PAC1 resulted in increased EAE-induced loss of a subpopulation of RGNs purported to be vulnerable in animal models of glaucoma. Increased axonal pathology and increased secondary presence of microglia/macrophages was also prominently seen in the optic nerve. These findings demonstrate that neuronal PAC1 receptors play a homeostatic role in protecting RGNs and directly protects neurons and their axons against neuroinflammatory challenge. SIGNIFICANCE STATEMENT: Chronic inflammation is a major component of neurodegenerative diseases and plays a central role in multiple sclerosis (MS). Current treatments for MS do not prevent neurodegeneration and/or neurological decline. The neuropeptide pituitary adenylate cyclase-activating polypeptide (PACAP) has been shown to have anti-inflammatory, neuroprotective and regenerative properties but the cell type- and receptor-specific mechanisms are not clear. To test whether the protective effects of PACAP are direct on the PAC1 receptor subtype on neurons, we delete PAC1 receptors from neurons and investigate neuropathologigical changes in an animal model of MS. The findings demonstrate that PAC1 receptors on neurons play a homeostatic role in maintaining neuron health and can directly protect neurons and their axons during neuroinflammatory disease.

Axonal damage in spinal cord is associated with gray matter atrophy in sensorimotor cortex in experimental autoimmune encephalomyelitis.

BACKGROUND: Gray matter (GM) atrophy in brain is one of the best predictors of long-term disability in multiple sclerosis (MS), and recent findings have revealed that localized GM atrophy is associated with clinical disabilities. GM atrophy associated with each disability mapped to a distinct brain region, revealing a disability-specific atlas (DSA) of GM loss. OBJECTIVE: To uncover the mechanisms underlying the development of localized GM atrophy. METHODS: We used voxel-based morphometry (VBM) to evaluate localized GM atrophy and Clear Lipid-exchanged Acrylamide-hybridized Rigid Imaging-compatible Tissue-hYdrogel (CLARITY) to evaluate specific pathologies in mice with experimental autoimmune encephalomyelitis (EAE). RESULTS: We observed extensive GM atrophy throughout the cerebral cortex, with additional foci in the thalamus and caudoputamen, in mice with EAE compared to normal controls. Next, we generated pathology-specific atlases (PSAs), voxelwise mappings of the correlation between specific pathologies and localized GM atrophy. Interestingly, axonal damage (end-bulbs and ovoids) in the spinal cord strongly correlated with GM atrophy in the sensorimotor cortex of the brain. CONCLUSION: The combination of VBM with CLARITY in EAE can localize GM atrophy in brain that is associated with a specific pathology in spinal cord, revealing a PSA of GM loss.

In vivo magnetic resonance images reveal neuroanatomical sex differences through the application of voxel-based morphometry in C57BL/6 mice.

Behaviorally relevant sex differences are often associated with structural differences in the brain and many diseases are sexually dimorphic in prevalence and progression. Characterizing sex differences is imperative to gaining a complete understanding of behavior and disease which will, in turn, allow for a balanced approach to scientific research and the development of therapies. In this study, we generated novel tissue probability maps (TPMs) based on 30 male and 30 female in vivo C57BL/6 mouse brain magnetic resonance images and used voxel-based morphometry (VBM) to analyze sex differences. Females displayed larger anterior hippocampus, basolateral amygdala, and lateral cerebellar cortex volumes, while males exhibited larger cerebral cortex, medial amygdala, and medial cerebellar cortex volumes. Atlas-based morphometry (ABM) revealed a statistically significant sex difference in cortical volume and no difference in whole cerebellar volume. This validated our VBM findings that showed a larger cerebral cortex in male mice and a pattern of dimorphism in the cerebellum where the lateral portion was larger in females and the medial portion was larger in males. These results are consonant with previous ex vivo studies examining sex differences, but also suggest further regions of interest.

Bringing CLARITY to gray matter atrophy.

Gray matter atrophy has been shown to be a strong correlate to clinical disability in multiple sclerosis (MS) and its most commonly used animal model, experimental autoimmune encephalomyelitis (EAE). However, the relationship between gray mater atrophy and the spinal cord pathology often observed in EAE has never been established. Here EAE was induced in Thy1.1-YFP mice and their brains imaged using in vivo magnetic resonance imaging (MRI). The brains and spinal cords were subsequently optically cleared using Clear Lipid-exchanged Acrylamide-hybridized Rigid Imaging-compatible Tissue-hYdrogel (CLARITY). Axons were followed 5mm longitudinally in three dimensions in intact spinal cords revealing that 61% of the axons exhibited a mean of 22 axonal ovoids and 8% of the axons terminating in axonal end bulbs. In the cerebral cortex, we observed a decrease in the mean number of layer V pyramidal neurons and a decrease in the mean length of the apical dendrites of the remaining neurons, compared to healthy controls. MRI analysis demonstrated decreased cortical volumes in EAE. Cross-modality correlations revealed a direct relationship between cortical volume loss and axonal end bulb number in the spinal cord, but not ovoid number. This is the first report of the use of CLARITY in an animal model of disease and the first report of the use of both CLARITY and MRI.

Overexpression of VIPR2 in mice results in microencephaly with paradoxical increased white matter volume.

Large scale studies in populations of European and Han Chinese ancestry found a series of rare gain-of-function microduplications in VIPR2, encoding VPAC2, a receptor that binds vasoactive intestinal peptide and pituitary adenylate cyclase-activating polypeptide with high affinity, that were associated with an up to 13-fold increased risk for schizophrenia. To address how VPAC2 receptor overactivity might affect brain development, we used a well-characterized Nestin-Cre mouse strain and a knock-in approach to overexpress human VPAC2 in the central nervous system. Mice that overexpressed VPAC2 were found to exhibit a significant reduction in brain weight. Magnetic resonance imaging analysis confirmed a decrease in brain size, a specific reduction in the hippocampus grey matter volume and a paradoxical increase in whole-brain white matter volume. Sex-specific changes in behavior such as impaired prepulse inhibition and contextual fear memory were observed in VPAC2 overexpressing mice. The data indicate that the VPAC2 receptor may play a critical role in brain morphogenesis and suggest that overactive VPAC2 signaling during development plays a mechanistic role in some forms of schizophrenia.

Estrogen receptor beta in astrocytes modulates cognitive function in mid-age female mice.

Menopause is associated with cognitive deficits and brain atrophy, but the brain region and cell-specific mechanisms are not fully understood. Here, we identify a sex hormone by age interaction whereby loss of ovarian hormones in female mice at midlife, but not young age, induced hippocampal-dependent cognitive impairment, dorsal hippocampal atrophy, and astrocyte and microglia activation with synaptic loss. Selective deletion of estrogen receptor beta (ERß) in astrocytes, but not neurons, in gonadally intact female mice induced the same brain effects. RNA sequencing and pathway analyses of gene expression in hippocampal astrocytes from midlife female astrocyte-ERß conditional knock out (cKO) mice revealed Gluconeogenesis I and Glycolysis I as the most differentially expressed pathways. Enolase 1 gene expression was increased in hippocampi from both astrocyte-ERß cKO female mice at midlife and from postmenopausal women. Gain of function studies showed that ERß ligand treatment of midlife female mice reversed dorsal hippocampal neuropathology.

Cortical atrophy in experimental autoimmune encephalomyelitis: in vivo imaging.

There are strong correlations between cortical atrophy observed by MRI and clinical disability and disease duration in multiple sclerosis (MS). The objective of this study was to evaluate the progression of cortical atrophy over time in vivo in experimental autoimmune encephalomyelitis (EAE), the most commonly used animal model for MS. Volumetric changes in brains of EAE mice and matched healthy controls were quantified by collecting high-resolution T2-weighted magnetic resonance images in vivo and labeling anatomical structures on the images. In vivo scanning permitted us to evaluate brain structure volumes in individual animals over time and we observed that though brain atrophy progressed differently in each individual animal, all mice with EAE demonstrated significant atrophy in whole brain, cerebral cortex, and whole cerebellum compared to normal controls. Furthermore, we found a strong correlation between cerebellar atrophy and cumulative disease score in mice with EAE. Ex vivo MRI showed a significant decrease in brain and cerebellar volume and a trend that did not reach significance in cerebral cortex volume in mice with EAE compared to controls. Cross modality correlations revealed a significant association between neuronal loss on neuropathology and in vivo atrophy of the cerebral cortex by neuroimaging. These results demonstrate that longitudinal in vivo imaging is more sensitive to changes that occur in neurodegenerative disease models than cross-sectional ex vivo imaging. This is the first report of progressive cortical atrophy in vivo in a mouse model of MS.

Estrogen treatment prevents gray matter atrophy in experimental autoimmune encephalomyelitis.

Gray matter atrophy is an important correlate to clinical disability in multiple sclerosis (MS), and many treatment trials include atrophy as an outcome measure. Atrophy has been shown to occur in experimental autoimmune encephalomyelitis (EAE), the most commonly used animal model of MS. The clinical severity of EAE is reduced in estrogen-reated mice, but it remains unknown whether estrogen treatment can reduce gray matter atrophy in EAE. In this study, mice with EAE were treated with either estrogen receptor (ER)-α ligand or ER-ß ligand, and diffusion tensor images (DTI) were collected and neuropathology was performed. DTI showed atrophy in the cerebellar gray matter of vehicle-treated EAE mice compared with healthy controls but not in ER-α or ER-ß ligand-treated EAE mice. Neuropathology demonstrated that Purkinje cell numbers were decreased in vehicle-treated EAE mice, whereas neither ER ligand-treated EAE groups showed a decrease. This is the first report of a neuroprotective therapy in EAE that unambiguously prevents gray matter atrophy while sparing a major neuronal cell type. Fractional anisotropy (FA) in the cerebellar white matter was decreased in vehicle- and ER-ß ligand-treated but not in ER-α ligand-treated EAE mice. Inflammatory cell infiltration was increased in vehicle- and ER-ß ligand-treated but not in ER-α ligand-treated EAE mice. Myelin staining was decreased in vehicle-treated EAE mice and was spared in both ER ligand-treated groups. This is consistent with decreased FA as a potential biomarker for inflammation rather than myelination or axonal damage in the cerebellum in EAE.

Chronic experimental autoimmune encephalomyelitis is an excellent model to study neuroaxonal degeneration in multiple sclerosis.

Animal models of multiple sclerosis (MS), specifically experimental autoimmune encephalomyelitis (EAE), have been used extensively to develop anti-inflammatory treatments. However, the similarity between MS and one particular EAE model does not end at inflammation. MS and chronic EAE induced in C57BL/6 mice using myelin oligodendrocyte glycoprotein (MOG) peptide 35-55 share many neuropathologies. Beyond both having white matter lesions in spinal cord, both also have widespread neuropathology in the cerebral cortex, hippocampus, thalamus, striatum, cerebellum, and retina/optic nerve. In this review, we compare neuropathologies in each of these structures in MS with chronic EAE in C57BL/6 mice, and find evidence that this EAE model is well suited to study neuroaxonal degeneration in MS.

Decreased neurofilament light chain levels in estriol-treated multiple sclerosis.

Estrogens have neuroprotective actions depending on estrogen type, dose, and timing in both preclinical models and in women during health and disease. Serum neurofilament light chain is a putative biomarker of neurodegeneration in multiple sclerosis, aging, and other neurodegenerative diseases. Here, oral treatment with an estrogen unique to pregnancy (estriol) using an 8 mg dose to induce a mid-pregnancy blood estriol level reduced serum neurofilament light chain in nonpregnant MS women at mean age of 37 years. This is consistent with estriol-mediated protection from neuro-axonal injury and supports the use of serum neurofilament light chain as a biomarker in MS.

Sex differences in brain atrophy in multiple sclerosis.

BACKGROUND: Women are more susceptible to multiple sclerosis (MS) than men by a ratio of approximately 3:1. However, being male is a risk factor for worse disability progression. Inflammatory genes have been linked to susceptibility, while neurodegeneration underlies disability progression. Thus, there appears to be a differential effect of sex on inflammation versus neurodegeneration. Further, gray matter (GM) atrophy is not uniform across the brain in MS, but instead shows regional variation. Here, we study sex differences in neurodegeneration by comparing regional GM atrophy in a cohort of men and women with MS versus their respective age- and sex-matched healthy controls. METHODS: Voxel-based morphometry (VBM), deep GM substructure volumetry, and cortical thinning were used to examine regional GM atrophy. RESULTS: VBM analysis showed deep GM atrophy in the thalamic area in both men and women with MS, whereas men had additional atrophy in the putamen as well as in localized cortical regions. Volumetry confirmed deep GM loss, while localized cortical thinning confirmed GM loss in the cerebral cortex. Further, MS males exhibited worse performance on the 9-hole peg test (9HPT) than MS females. We observed a strong correlation between thalamic volume and 9HPT performance in MS males, but not in MS females. CONCLUSION: More regional GM atrophy was observed in men with MS than women with MS, consistent with previous observations that male sex is a risk factor for worse disease progression.

Purkinje cell loss in experimental autoimmune encephalomyelitis.

Gray matter atrophy observed by brain MRI is an important correlate to clinical disability and disease duration in multiple sclerosis. The objective of this study was to link brain atrophy visualized by neuroimaging to its underlying neuropathology using the MS model, experimental autoimmune encephalomyelitis (EAE). Volumetric changes in brains of EAE mice, as well as matched healthy normal controls, were quantified by collecting post-mortem high-resolution T2-weighted magnetic resonance microscopy and actively stained magnetic resonance histology images. Anatomical delineations demonstrated a significant decrease in the volume of the whole cerebellum, cerebellar cortex, and molecular layer of the cerebellar cortex in EAE as compared to normal controls. The pro-apoptotic marker caspase-3 was detected in Purkinje cells and a significant decrease in Purkinje cell number was found in EAE. Cross modality and temporal correlations revealed a significant association between Purkinje cell loss on neuropathology and atrophy of the molecular layer of the cerebellar cortex by neuroimaging. These results demonstrate the power of using combined population atlasing and neuropathology approaches to discern novel insights underlying gray matter atrophy in animal models of neurodegenerative disease.

Ischemic axonal injury up-regulates MARK4 in cortical neurons and primes tau phosphorylation and aggregation.

Ischemic injury to white matter tracts is increasingly recognized to play a key role in age-related cognitive decline, vascular dementia, and Alzheimer's disease. Knowledge of the effects of ischemic axonal injury on cortical neurons is limited yet critical to identifying molecular pathways that link neurodegeneration and ischemia. Using a mouse model of subcortical white matter ischemic injury coupled with retrograde neuronal tracing, we employed magnetic affinity cell sorting with fluorescence-activated cell sorting to capture layer-specific cortical neurons and performed RNA-sequencing. With this approach, we identified a role for microtubule reorganization within stroke-injured neurons acting through the regulation of tau. We find that subcortical stroke-injured Layer 5 cortical neurons up-regulate the microtubule affinity-regulating kinase, Mark4, in response to axonal injury. Stroke-induced up-regulation of Mark4 is associated with selective remodeling of the apical dendrite after stroke and the phosphorylation of tau in vivo. In a cell-based tau biosensor assay, Mark4 promotes the aggregation of human tau in vitro. Increased expression of Mark4 after ischemic axonal injury in deep layer cortical neurons provides new evidence for synergism between axonal and neurodegenerative pathologies by priming of tau phosphorylation and aggregation.

Provenance in neuroimaging.

Provenance, the description of the history of a set of data, has grown more important with the proliferation of research consortia-related efforts in neuroimaging. Knowledge about the origin and history of an image is crucial for establishing data and results quality; detailed information about how it was processed, including the specific software routines and operating systems that were used, is necessary for proper interpretation, high fidelity replication and re-use. We have drafted a mechanism for describing provenance in a simple and easy to use environment, alleviating the burden of documentation from the user while still providing a rich description of an image's provenance. This combination of ease of use and highly descriptive metadata should greatly facilitate the collection of provenance and subsequent sharing of data.

Estriol-mediated neuroprotection in multiple sclerosis localized by voxel-based morphometry.

INTRODUCTION: Progressive gray matter (GM) atrophy is a hallmark of multiple sclerosis (MS). Cognitive impairment has been observed in 40%-70% of MS patients and has been linked to GM atrophy. In a phase 2 trial of estriol treatment in women with relapsing-remitting MS (RRMS), higher estriol levels correlated with greater improvement on the paced auditory serial addition test (PASAT) and imaging revealed sparing of localized GM in estriol-treated compared to placebo-treated patients. To better understand the significance of this GM sparing, the current study explored the relationships between the GM sparing and traditional MRI measures and clinical outcomes. METHODS: Sixty-two estriol- and forty-nine placebo-treated RRMS patients underwent clinical evaluations and brain MRI. Voxel-based morphometry (VBM) was used to evaluate voxelwise GM sparing from high-resolution T1-weighted scans. RESULTS: A region of treatment-induced sparing (TIS) was defined as the areas where GM was spared in estriol- as compared to placebo-treated groups, localized primarily within the frontal and parietal cortices. We observed that TIS volume was directly correlated with improvement on the PASAT. Next, a longitudinal cognitive disability-specific atlas (DSA) was defined by correlating voxelwise GM volumes with PASAT scores, that is, areas where less GM correlated with less improvement in PASAT scores. Finally, overlap between the TIS and the longitudinal cognitive DSA revealed a specific region of cortical GM that was preserved in estriol-treated subjects that was associated with better performance on the PASAT. CONCLUSIONS: Discovery of this region of overlap was biology driven, not based on an a priori structure of interest. It included the medial frontal cortex, an area previously implicated in problem solving and attention. These findings indicate that localized GM sparing during estriol treatment was associated with improvement in cognitive testing, suggesting a clinically relevant, disability-specific biomarker for clinical trials of candidate neuroprotective treatments in MS.

Brain atlases and neuroanatomic imaging.

Quantifying the effect of a genetic manipulation or disease is a complicated process in a population of animals. Probabilistic brain atlases can capture population variability and be used to quantify those variations in anatomy as measured by structural imaging. Minimum deformation atlases (MDAs), a subclass of probabilistic atlases, are intensity-based averages of a collection of scans in a common space unbiased by selection of a single target image. Here, we describe a method for generating an MDA from a set of magnetic resonance microscopy images. First, the images are segmented to remove any non-brain tissue and bias field corrected to remove field inhomogeneities. The corrected images are then linearly aligned to a representative scan, the geometric mean of all the transformations is calculated, and a minimum deformation target (MDT) is produced by averaging the volumes in this new space. The brains are then non-linearly aligned to the MDT to produce the MDA. Finally, the images are linearly aligned to the MDA using a full-affine transformation to spatially and intensity normalize them, removing global differences in size, shape, and position but retaining anatomically significant differences.

Bedside to bench to bedside research: Estrogen receptor beta ligand as a candidate neuroprotective treatment for multiple sclerosis.

Protective effects of pregnancy during MS have led to clinical trials of estriol, the pregnancy estrogen, in MS. Since estriol binds to estrogen receptor (ER) beta, ER beta ligand could represent a "next generation estriol" treatment. Here, ER beta ligand treatment was protective in EAE in both sexes and across genetic backgrounds. Neuroprotection was shown in spinal cord, sparing myelin and axons, and in brain, sparing neurons and synapses. Longitudinal in vivo MRIs showed decreased brain atrophy in cerebral cortex gray matter and cerebellum during EAE. Investigation of ER beta ligand as a neuroprotective treatment for MS is warranted.

Optical Clearing of the Mouse Central Nervous System Using Passive CLARITY.

Traditionally, tissue visualization has required that the tissue of interest be serially sectioned and imaged, subjecting each tissue section to unique non-linear deformations, dramatically hampering one's ability to evaluate cellular morphology, distribution and connectivity in the central nervous system (CNS). However, optical clearing techniques are changing the way tissues are visualized. These approaches permit one to probe deeply into intact organ preparations, providing tremendous insight into the structural organization of tissues in health and disease. Techniques such as Clear Lipid-exchanged Acrylamide-hybridized Rigid Imaging-compatible Tissue-hYdrogel (CLARITY) achieve this goal by providing a matrix that binds important biomolecules while permitting light-scattering lipids to freely diffuse out. Lipid removal, followed by refractive index matching, renders the tissue transparent and readily imaged in 3 dimensions (3D). Nevertheless, the electrophoretic tissue clearing (ETC) used in the original CLARITY protocol can be challenging to implement successfully and the use of a proprietary refraction index matching solution makes it expensive to use the technique routinely. This report demonstrates the implementation of a simple and inexpensive optical clearing protocol that combines passive CLARITY for improved tissue integrity and 2,2'-thiodiethanol (TDE), a previously described refractive index matching solution.