Impaired drainage through capillary-venous networks contributes to age-related white matter loss.

The gradual loss of cerebral white matter contributes to cognitive decline during aging. However, microvascular networks that support the metabolic demands of white matter remain poorly defined. We used in vivo deep multi-photon imaging to characterize microvascular networks that perfuse cortical layer 6 and corpus callosum, a highly studied region of white matter in the mouse brain. We show that these deep tissues are exclusively drained by sparse and wide-reaching venules, termed principal cortical venules, which mirror vascular architecture at the human cortical-U fiber interface. During aging, capillary networks draining into deep branches of principal cortical venules are selectively constricted, reduced in density, and diminished in pericyte numbers. This causes hypo-perfusion in deep tissues, and correlates with gliosis and demyelination, whereas superficial tissues become relatively hyper-perfused. Thus, age-related impairment of capillary-venular drainage is a key vascular deficit that contributes to the unique vulnerability of cerebral white matter during brain aging.

Endothelial structure contributes to heterogeneity in brain capillary diameter.

The high metabolic demand of brain tissue is supported by a constant supply of blood flow through dense microvascular networks. Capillaries are the smallest class of vessels in the brain and their lumens vary in diameter between ~2 and 5 µm. This diameter range plays a significant role in optimizing blood flow resistance, blood cell distribution, and oxygen extraction. The control of capillary diameter has largely been ascribed to pericyte contractility, but it remains unclear if the architecture of the endothelial wall also contributes to capillary diameter. Here, we use public, large-scale volume electron microscopy data from mouse cortex (MICrONS Explorer, Cortical mm3) to examine how endothelial cell number, endothelial cell thickness, and pericyte coverage relates to microvascular lumen size. We find that transitional vessels near the penetrating arteriole and ascending venule are composed of two to six interlocked endothelial cells, while the capillaries intervening these zones are composed of either one or two endothelial cells, with roughly equal proportions. The luminal area and diameter are on average slightly larger with capillary segments composed of two interlocked endothelial cells vs one endothelial cell. However, this difference is insufficient to explain the full range of capillary diameters seen in vivo. This suggests that both endothelial structure and other influences, including pericyte tone, contribute to the basal diameter and optimized perfusion of brain capillaries.

Endothelial structure contributes to heterogeneity in brain capillary diameter.

The high metabolic demand of brain tissue is supported by a constant supply of blood through dense microvascular networks. Capillaries are the smallest class of vessels and vary in diameter between ∼2 to 5 µm in the brain. This diameter range plays a significant role in the optimization of blood flow resistance, blood cell distribution, and oxygen extraction. The control of capillary diameter has largely been ascribed to pericyte contractility, but it remains unclear if endothelial wall architecture also contributes to capillary diameter heterogeneity. Here, we use public, large-scale volume electron microscopy data from mouse cortex (MICrONS Explorer, Cortical MM^3) to examine how endothelial cell number, endothelial cell thickness, and pericyte coverage relates to microvascular lumen size. We find that transitional vessels near the penetrating arteriole and ascending venule are composed of 2 to 5 interlocked endothelial cells, while the numerous capillary segments intervening these zones are composed of either 1 or 2 endothelial cells, with roughly equal proportions. The luminal area and diameter is on average slightly larger with capillary segments composed of 2 interlocked endothelial cells versus 1 endothelial cell. However, this difference is insufficient to explain the full range of capillary diameters seen in vivo. This suggests that both endothelial structure and other influences, such as pericyte tone, contribute to the basal diameter and optimized perfusion of brain capillaries.

Deep Imaging to Dissect Microvascular Contributions to White Matter Degeneration in Rodent Models of Dementia.

The increasing socio-economic burden of Alzheimer disease (AD) and AD-related dementias has created a pressing need to define targets for therapeutic intervention. Deficits in cerebral blood flow and neurovascular function have emerged as early contributors to disease progression. However, the cause, progression, and consequence of small vessel disease in AD/AD-related dementias remains poorly understood, making therapeutic targets difficult to pinpoint. Animal models that recapitulate features of AD/AD-related dementias may provide mechanistic insight because microvascular pathology can be studied as it develops in vivo. Recent advances in in vivo optical and ultrasound-based imaging of the rodent brain facilitate this goal by providing access to deeper brain structures, including white matter and hippocampus, which are more vulnerable to injury during cerebrovascular disease. Here, we highlight these novel imaging approaches and discuss their potential for improving our understanding of vascular contributions to AD/AD-related dementias.

Pericyte remodeling is deficient in the aged brain and contributes to impaired capillary flow and structure.

Deterioration of brain capillary flow and architecture is a hallmark of aging and dementia. It remains unclear how loss of brain pericytes in these conditions contributes to capillary dysfunction. Here, we conduct cause-and-effect studies by optically ablating pericytes in adult and aged mice in vivo. Focal pericyte loss induces capillary dilation without blood-brain barrier disruption. These abnormal dilations are exacerbated in the aged brain, and result in increased flow heterogeneity in capillary networks. A subset of affected capillaries experience reduced perfusion due to flow steal. Some capillaries stall in flow and regress, leading to loss of capillary connectivity. Remodeling of neighboring pericytes restores endothelial coverage and vascular tone within days. Pericyte remodeling is slower in the aged brain, resulting in regions of persistent capillary dilation. These findings link pericyte loss to disruption of capillary flow and structure. They also identify pericyte remodeling as a therapeutic target to preserve capillary flow dynamics.

Multiphoton-Guided Creation of Complex Organ-Specific Microvasculature.

Engineering functional human tissues in vitro is currently limited by difficulty replicating the small caliber, complex connectivity, cellularity, and 3D curvature of the native microvasculature. Multiphoton ablation has emerged as a promising technique for fabrication of microvascular structures with high resolution and full 3D control, but cellularization and perfusion of complex capillary-scale structures has remained challenging. Here, multiphoton ablation combined with guided endothelial cell growth from pre-formed microvessels is used to successfully create perfusable and cellularized organ-specific microvascular structures at anatomic scale within collagen hydrogels. Fabrication and perfusion of model 3D pulmonary and renal microvascular beds is demonstrated, as is replication and perfusion of a brain microvascular unit derived from in vivo data. Successful endothelialization and blood perfusion of a kidney-specific microvascular structure is achieved, using laser-guided angiogenesis. Finally, proof-of-concept hierarchical blood vessels and complex multicellular models are created, using multistep patterning with multiphoton ablation techniques. These successes open new doors for the creation of engineered tissues and organ-on-a-chip devices.

Lipids status and copper in a single astrocyte of the rat model for amyotrophic lateral sclerosis: Correlative synchrotron-based X-ray and infrared imaging.

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease, causing death of motor neurons controlling voluntary muscles. The pathological mechanisms of the disease are only partially understood. The hSOD1-G93A ALS rat model is characterized by an overexpression of human mutated SOD1, causing increased vulnerability by forming intracellular protein aggregates, inducing excitotoxicity, affecting oxidative balance and disturbing axonal transport. In this study we followed the bio-macromolecular organic composition and compartmentalization together with trace metal distribution in situ in single astrocytes from the ALS rat model and compared them to the control astrocytes from nontransgenic littermates by simultaneous use of two synchrotron radiation-based methods: Fourier transform infrared microspectroscopy (SR-FTIR) and hard X-ray fluorescence microscopy (XRF). We show that ALS cells contained more Cu, which colocalized with total lipids, increased carbonyl groups and oxidized lipids, thus implying direct involvement of Cu in oxidative stress of lipidic components without direct connection to protein aggregation in situ.

In vivo Cell Tracking Using Non-invasive Imaging of Iron Oxide-Based Particles with Particular Relevance for Stem Cell-Based Treatments of Neurological and Cardiac Disease.

Stem cell-based therapeutics is a rapidly developing field associated with a number of clinical challenges. One such challenge lies in the implementation of methods to track stem cells and stem cell-derived cells in experimental animal models and in the living patient. Here, we provide an overview of cell tracking in the context of cardiac and neurological disease, focusing on the use of iron oxide-based particles (IOPs) visualized in vivo using magnetic resonance imaging (MRI). We discuss the types of IOPs available for such tracking, their advantages and limitations, approaches for labeling cells with IOPs, biological interactions and effects of IOPs at the molecular and cellular levels, and MRI-based and associated approaches for in vivo and histological visualization. We conclude with reviews of the literature on IOP-based cell tracking in cardiac and neurological disease, covering both preclinical and clinical studies.

Synchrotron radiation-based FTIR spectro-microscopy of the brainstem of the hSOD1 G93A rat model of amyotrophic lateral sclerosis.

Pathological mechanisms in amyotrophic lateral sclerosis (ALS), a fatal neurodegenerative disease, are still poorly understood. One subset of familial ALS cases is caused by mutations in the metallo-enzyme copper-zinc superoxide dismutase (SOD1), increasing the susceptibility of the SOD1 protein to form insoluble intracellular aggregates. Here, we employed synchrotron radiation-based Fourier transform infrared spectroscopy and microscopy to investigate brainstem cross-sections from the transgenic hSOD1 G93A rat model of ALS that overexpresses human-mutated SOD1. We compared the biomacromolecular organic composition in brainstem tissue cross-sections of ALS rats and their non-transgenic littermates (NTg). We demonstrate that the proteins and especially their antiparallel ß-sheet structure significantly differed in all three regions: the facial nucleus (FN), the gigantocellular reticular nucleus (GRN) and the trigeminal motor nucleus (TMN) in the brainstem tissue of ALS rats. The protein levels varied between different brainstem areas, with the highest concentration observed in the region of the FN in the brainstem tissue of NTg animals. Furthermore, the concentration of lipids and esters was significantly decreased in the TMN and FN of ALS animals. A similar pattern was detected for choline and phosphate assigned to nucleic acids with the highest concentrations in the FN of NTg animals. The spectroscopic analysis showed significant differences in phosphates, amide and lipid structure in the FN of NTg animals in comparison with the same area of ALS rats. These results show that the hG93A SOD1 mutation causes metabolic cellular changes and point to a link between bioorganic composition and hallmarks of protein aggregation.

Multimodal Synchrotron Radiation Microscopy of Intact Astrocytes from the hSOD1 G93A Rat Model of Amyotrophic Lateral Sclerosis.

Amyotrophic lateral sclerosis (ALS), a fatal neurodegenerative disease, is the most common adult onset neurodegenerative disorder affecting motor neurons. Disruptions in metal ion homeostasis have been described in association with ALS, but the pathological mechanisms are still poorly understood. One of the familial ALS cases is caused by mutations in the metallo-enzyme copper-zinc superoxide dismutase (SOD1). In this study, we employed orthogonal cellular synchrotron radiation based spectro-microscopies to investigate the astrocytes of an ALS animal model: the rat hSOD1 G93A that overexpresses human mutated SOD1, which is known to increase the susceptibility of the SOD1 protein to form insoluble intracellular aggregates. Specifically, we applied soft X-ray transmission tomography and hard X-ray fluorescence microscopy in situ, Fourier transform infrared spectro-microscopy to detect and analyze aggregates, as well as to determine the alterations in the cellular ultrastructure and the elemental and the organic composition of ALS model astrocytes with respect to the control astrocytes isolated from nontransgenic littermates (NTg). The present study demonstrates that large aggregates in the form of multivesicular inclusions form exclusively in the ALS model astrocytes and not in the NTg counterpart. Furthermore, the number of mitochondria, the cellular copper concentration, and the amount of antiparallel ß-sheet structures were significantly changed within the cells of the ALS model as well as the lipid localization and composition. Also, our data indicate that choline was decreased in the ALS model astrocytes, which could explain their higher sensitivity to oxidative stress that we observed. These results show that the hG93A SOD1 mutation causes metabolic and ultrastructural cellular changes and point to a link between an increased copper concentration and aggregation: the most probable that the aggregation of G93A hSOD1 may perturb its binding to Cu, thus directly or indirectly affecting Cu homeostasis.

Imaging of glial cell morphology, SOD1 distribution and elemental composition in the brainstem and hippocampus of the ALS hSOD1G93A rat.

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disorder affecting motor and cognitive domains of the CNS. Mutations in the Cu,Zn-superoxide dismutase (SOD1) cause 20% of familial ALS and provoke formation of intracellular aggregates and copper and zinc unbinding, leading to glial activation and neurodegeneration. Therefore, we investigated glial cell morphology, intracellular SOD1 distribution, and elemental composition in the brainstem and hippocampus of the hSOD1G93A transgenic rat model of ALS. Immunostaining for astrocytes, microglia and SOD1 revealed glial proliferation and progressive tissue accumulation of SOD1 in both brain regions of ALS rats starting already at the presymptomatic stage. Glial cell morphology analysis in the brainstem of ALS rats revealed astrocyte activation occurring before disease symptoms onset, followed by activation of microglia. Hippocampal ALS astrocytes exhibited an identical reactive profile, while microglial morphology was unchanged. Additionally, ALS brainstem astrocytes demonstrated progressive SOD1 accumulation in the cell body and processes, while microglial SOD1 levels were reduced and its distribution limited to distal cell processes. In the hippocampus both glial cell types exhibited SOD1 accumulation in the cell body. X-ray fluorescence imaging revealed decreased P and increased Ca, Cl, K, Ni, Cu and Zn in the brainstem, and higher levels of Cl, Ni and Cu, but lower levels of Zn in the hippocampus of symptomatic ALS rats. These results bring new insights into the glial response during disease development and progression in motor as well as in non-motor CNS structures, and indicate disturbed tissue elemental homeostasis as a prominent hallmark of disease pathology.

In vivo EPR pharmacokinetic evaluation of the redox status and the blood brain barrier permeability in the SOD1G93A ALS rat model.

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disorder affecting the motor pathways of the central nervous system. Although a number of pathophysiological mechanisms have been described in the disease, post mortem and animal model studies indicate blood-brain barrier (BBB) disruption and elevated production of reactive oxygen species as major contributors to disease pathology. In this study, the BBB permeability and the brain tissue redox status of the SOD1G93A ALS rat model in the presymptomatic (preALS) and symptomatic (ALS) stages of the disease were investigated by in vivo EPR spectroscopy using three aminoxyl radicals with different cell membrane and BBB permeabilities, Tempol, 3-carbamoyl proxyl (3CP), and 3-carboxy proxyl (3CxP). Additionally, the redox status of the two brain regions previously implicated in disease pathology, brainstem and hippocampus, was investigated by spectrophotometric biochemical assays. The EPR results indicated that among the three spin probes, 3CP is the most suitable for reporting the intracellular redox status changes, as Tempol was reduced in vivo within minutes (t1/2 =2.0±0.5min), thus preventing reliable kinetic modeling, whereas 3CxP reduction kinetics gave divergent conclusions, most probably due to its membrane impermeability. It was observed that the reduction kinetics of 3CP in vivo, in the head of preALS and ALS SOD1G93A rats was altered compared to the controls. Pharmacokinetic modeling of 3CP reduction in vivo, revealed elevated tissue distribution and tissue reduction rate constants indicating an altered brain tissue redox status, and possibly BBB disruption in these animals. The preALS and ALS brain tissue homogenates also showed increased nitrilation, superoxide production, lipid peroxidation and manganese superoxide dismutase activity, and a decreased copper-zinc superoxide dismutase activity. The present study highlights in vivo EPR spectroscopy as a reliable tool for the investigation of changes in BBB permeability and for the unprecedented in vivo monitoring of the brain tissue redox status, as early markers of ALS.

The extracellular matrix glycoprotein tenascin-C and matrix metalloproteinases modify cerebellar structural plasticity by exposure to an enriched environment.

The importance of the extracellular matrix (ECM) glycoprotein tenascin-C (TnC) and the ECM degrading enzymes, matrix metalloproteinases (MMPs) -2 and -9, in cerebellar histogenesis is well established. This study aimed to examine whether there is a functional relationship between these molecules in regulating structural plasticity of the lateral deep cerebellar nucleus. To this end, starting from postnatal day 21, TnC- or MMP-9-deficient mice were exposed to an enriched environment (EE). We show that 8 weeks of exposure to EE leads to reduced lectin-based staining of perineuronal nets (PNNs), reduction in the size of GABAergic and increase in the number and size of glutamatergic synaptic terminals in wild-type mice. Conversely, TnC-deficient mice showed reduced staining of PNNs compared to wild-type mice maintained under standard conditions, and exposure to EE did not further reduce, but even slightly increased PNN staining. EE did not affect the densities of the two types of synaptic terminals in TnC-deficient mice, while the size of inhibitory, but not excitatory synaptic terminals was increased. In the time frame of 4-8 weeks, MMP-9, but not MMP-2, was observed to influence PNN remodeling and cerebellar synaptic plasticity as revealed by measurement of MMP-9 activity and colocalization with PNNs and synaptic markers. These findings were supported by observations on MMP-9-deficient mice. The present study suggests that TnC contributes to the regulation of structural plasticity in the cerebellum and that interactions between TnC and MMP-9 are likely to be important for these processes to occur.

Iron-sulfur cluster damage by the superoxide radical in neural tissues of the SOD1(G93A) ALS rat model.

Extensive clinical investigations, in hand with biochemical and biophysical research, have associated brain iron accumulation with the pathogenesis of the amyotrophic lateral sclerosis (ALS) disease. The origin of iron is still not identified, but it is proposed that it forms redox active complexes that can participate in the Fenton reaction generating the toxic hydroxyl radical. In this paper, the state of iron in the neural tissues isolated from SOD1(G93A) transgenic rats was investigated using low temperature EPR spectroscopy and is compared with that of nontransgenic (NTg) littermates. The results showed that iron in neural tissues is present as high- and low-spin, heme and non-heme iron. It appears that the SOD1(G93A) rat neural tissues were most likely exposed in vivo to higher amounts of reactive oxygen species when compared to the corresponding NTg tissues, as they showed increased oxidized [3Fe-4S](1+) cluster content relative to [4Fe-4S](1+). Also, the activity of cytochrome c oxidase (CcO) was found to be reduced in these tissues, which may be associated with the observed uncoupling of heme a3 Fe and CuB in the O2-reduction site of the enzyme. Furthermore, the SOD1(G93A) rat spinal cords and brainstems contained more manganese, presumably from MnSOD, than those of NTg rats. The addition of potassium superoxide to all neural tissues ex vivo, led to the [4Fe-4S]→[3Fe-4S] cluster conversion and concurrent release of Fe. These results suggest that the superoxide anion may be the cause of the observed oxidative damage to SOD1(G93A) rat neural tissues and that the iron-sulfur clusters may be the source of poorly liganded redox active iron implicated in ALS pathogenesis. Low temperature EPR spectroscopy appears to be a valuable tool in assessing the role of metals in neurodegenerative diseases.

Fasting induced cytoplasmic Fto expression in some neurons of rat hypothalamus.

Fat mass and obesity associated protein (Fto) is a nucleic acid demethylase, with a preference for thymine or uracil, according to the recent structural data. This fact suggests that methylated single-stranded RNA, rather than DNA, may be the primary Fto substrate. Fto is abundantly expressed in all hypothalamic sites governing feeding behavior. Considering that selective modulation of Fto levels in the hypothalamus can influence food intake, we set out to investigate the effect of 48 h fasting on the Fto expression in lateral hypothalamic area, paraventricular, ventromedial and arcuate nucleus, the regulatory centres of energy homeostasis. We have demonstrated that 48 h fasting causes not only an increase in the overall hypothalamic levels of both Fto mRNA and protein, but also alters Fto intracellular distribution. This switch happens in some neurons of paraventricular and ventromedial nucleus, as well as lateral hypothalamic area, resulting in the majority of the enzyme being localized outside the cell nuclei. Interestingly, the change in the Fto intracellular localization was not observed in neurons of arcuate nucleus, suggesting that fasting did not universally affect Fto in all of the hypothalmic sites involved in energy homeostasis regulation. Both Fto mRNA and catechol-O-methyltransferaze mRNA were upregulated in the identical time-dependent manner in fasting animals. This fact, combined with the knowledge of the Fto substrate preference, may provide further insight into monoamine metabolism in the state of disturbed energy homeostasis.

Cellular markers of neuroinflammation and neurogenesis after ischemic brain injury in the long-term survival rat model.

MRI was employed to follow the neurodegenerative foci and the localization of inflammatory cells by magnetically labeled CD4+ or CD8+ lymphocytes in the ischemia/reperfusion long-lived rats (9 and 13 months after 10 min of cardiac arrest). MRI of ischemic rats showed: (1) blood-brain barrier (BBB) leakage in the area of the dorsal hippocampus and brainstem-hindbrain level in basal cerebellum, (2) unlike anti-CD8 magnetic antibodies anti-CD4 ultra small paramagnetic iron oxide particles (USPIO) antibodies revealed hypointense areas in the brainstem-interbrain region and caudoputamen not found in animals that were not injected with USPIO antibodies, and (3) dilation in the retrosplenial area. Immunocytochemistry revealed microglial activation in the hippocampus and striatum, with indications of activation in thalamic lateral dorsal nuclei and the subventricular zone. In the CA1 and CA3 regions, it was noted that OX42- and ED1-positive granules appear in neuronal somata. Immunostaining of lymphocytes with TCR confirmed the T-cell presence in ischemic brain parenchyma of the hippocampus and striatum. The above observations thus point to a persistent dysfunction of BBB that in long-term may still lead to infiltration of T cells that are predominantly of helper (CD4+) type. Such inflammatory processes are backed by microglial activity even up to 1 year after ischemia/reperfusion. Moreover, in these animals an augmented expression of neurogenesis markers and neuroblast migration was also revealed in the subventricular zone. Thus, a balance of degenerative processes and inflammatory surveillance with neurogenesis could determine the long-term outcome of global ischemia survival or the previously proposed formation of amyloid plaques and Alzheimer's-type dementia.