ABSTRACT
Synapse loss and altered plasticity are significant contributors to memory loss in aged individuals. Microglia, the innate immune cells of the brain, play critical roles in maintaining synapse function, including through a recently identified role in regulating the brain extracellular matrix. This study sought to determine the relationship between age, microglia, and extracellular matrix structure densities in the macaque retrosplenial cortex. Twenty-nine macaques ranging in age from young adult to aged were behaviorally characterized on 3 distinct memory tasks. Microglia, parvalbumin (PV)-expressing interneurons and extracellular matrix structures, known as perineuronal nets (PNNs), were immuno- and histochemically labeled. Our results indicate that microglia densities increase in the retrosplenial cortex of aged monkeys, while the proportion of PV neurons surrounded by PNNs decreases. Aged monkeys with more microglia had fewer PNN-associated PV neurons and displayed slower learning and poorer performance on an object recognition task. Stepwise regression models using age and the total density of aggrecan, a chondroitin sulfate proteoglycan of PNNs, better predicted memory performance than did age alone. Together, these findings indicate that elevated microglial activity in aged brains negatively impacts cognition in part through mechanisms that alter PNN assembly in memory-associated brain regions.
Subject(s)
Gyrus Cinguli , Microglia , Animals , Macaca mulatta/metabolism , Microglia/metabolism , Gyrus Cinguli/metabolism , Extracellular Matrix/metabolism , Parvalbumins/metabolism , Memory DisordersABSTRACT
Along with initiatives to understand the pathophysiology of stroke in detail and to identify neuroprotective targets, cell-stabilizing elements have gained increasing attention. Although cell culture experiments have indicated that tricellulin, α-catenin and microfibrillar-associated protein 5 (MFAP5) contribute to cellular integrity, these elements have not yet been investigated in the ischemic brain. Applying immunofluorescence labeling, this study explored tricellulin, MFAP5 and α-catenin in non-ischemic and ischemic brain areas of mice (24, 4 h of ischemia) and rats (4 h of ischemia), along with collagen IV and fibronectin as vascular and extracellular matrix constituents and microtubule-associated protein 2 (MAP2) and neurofilament light chain (NF-L) as cytoskeletal elements. Immunosignals of tricellulin and notably MFAP5 partially appeared in a fiber-like pattern, and α-catenin appeared more in a dotted pattern. Regional associations with vascular and extracellular constituents were found for tricellulin and α-catenin, particularly in ischemic areas. Due to ischemia, signals of tricellulin, MFAP5 and α-catenin decreased concomitantly with MAP2 and NF-L, whereby MFAP5 provided the most sensitive reaction. For the first time, this study demonstrated ischemia-related alterations in tricellulin, MFAP5 and α-catenin along with the vasculature, extracellular matrix and cytoskeleton. Confirmatory studies are needed, also exploring their role in cellular integrity and the potential for neuroprotective approaches in stroke.
Subject(s)
Brain Ischemia , Stroke , Animals , Mice , Rats , alpha Catenin , Brain Ischemia/metabolism , Cerebral Infarction , Cytoskeleton/metabolism , Ischemia , MARVEL Domain Containing 2 Protein , Stroke/metabolism , Intercellular Signaling Peptides and Proteins , Contractile ProteinsABSTRACT
The surfactant protein-G (SP-G) has recently been discovered in the brain and linked to fluid balance regulations. Stroke is characterized by impaired vessel integrity, promoting water influx and edema formation. The neurovascular unit concept (NVU) has been generated to cover not only ischemic affections of neurons or vessels but also other regionally associated cells. This study provides the first spatio-temporal characterization of SP-G and NVU elements after experimental stroke. Immunofluorescence labeling was applied to explore SP-G, vascular and cellular markers in mice (4, 24, and 72 h of ischemia), rats (24 h of ischemia), and sheep (two weeks of ischemia). Extravasated albumin indicated vascular damage within ischemic areas. Quantifications revealed decreasing SP-G signals in the ischemia-affected neocortex and subcortex. Inverse immunosignals of SP-G and vascular elements existed throughout all models. Despite local associations between SP-G and the vasculature, a definite co-localization was not seen. Along with a decreased SP-G-immunoreactivity in ischemic areas, signals originating from neurons, glial elements, and the extracellular matrix exhibited morphological alterations or changed intensities. Collectively, this study revealed regional alterations of SP-G, vascular, and non-vascular NVU elements after ischemia, and may thus stimulate the discussion about the role of SP-G during stroke.
Subject(s)
Brain Ischemia , Neocortex , Stroke , Animals , Cerebral Infarction , Mice , Rats , Sheep , Surface-Active AgentsABSTRACT
Targeting molecular alterations as an effective treatment for isocitrate dehydrogenase-wildtype glioblastoma (GBM) patients has not yet been established. Sterol-O-Acyl Transferase 1 (SOAT1), a key enzyme in the conversion of endoplasmic reticulum cholesterol to esters for storage in lipid droplets (LD), serves as a target for the orphan drug mitotane to treat adrenocortical carcinoma. Inhibition of SOAT1 also suppresses GBM growth. Here, we refined SOAT1-expression in GBM and IDH-mutant astrocytoma, CNS WHO grade 4 (HGA), and assessed the distribution of LD in these tumors. Twenty-seven GBM and three HGA specimens were evaluated by multiple GFAP, Iba1, IDH1 R132H, and SOAT1 immunofluorescence labeling as well as Oil Red O staining. To a small extent SOAT1 was expressed by tumor cells in both tumor entities. In contrast, strong expression was observed in glioma-associated macrophages. Triple immunofluorescence labeling revealed, for the first time, evidence for SOAT1 colocalization with Iba1 and IDH1 R132H, respectively. Furthermore, a notable difference in the amount of LD between GBM and HGA was observed. Therefore, SOAT1 suppression might be a therapeutic option to target GBM and HGA growth and invasiveness. In addition, the high expression in cells related to neuroinflammation could be beneficial for a concomitant suppression of protumoral microglia/macrophages.
Subject(s)
Adrenal Cortex Neoplasms , Adrenocortical Carcinoma , Brain Neoplasms , Glioblastoma , Glioma , Sterol O-Acyltransferase/metabolism , Brain Neoplasms/metabolism , Glioblastoma/metabolism , Glioma/metabolism , Humans , Isocitrate Dehydrogenase/genetics , MutationABSTRACT
BACKGROUND: Scattered extracellular deposits of amyloid within the brain parenchyma can be found in a heterogeneous group of diseases. Its condensed accumulation in the white matter without evidence for systemic amyloidosis is known as primary brain amyloidoma (PBA). Although originally considered as a tumor-like lesion by its space-occupying effect, this condition displays also common hallmarks of a neurodegenerative disorder. CASE PRESENTATION: A 50-year-old woman presented with a mild cognitive decline and seizures with a right temporal, irregular and contrast-enhancing mass on magnetic resonance imaging. Suspecting a high-grade glioma, the firm tumor was subtotally resected. Neuropathological examination showed no glioma, but distinct features of a neurodegenerative disorder. The lesion was composed of amyloid AL λ aggregating within the brain parenchyma as well as the adjacent vessels, partially obstructing the vascular lumina. Immunostaining confirmed a distinct perivascular inflammatory reaction. After removal of the PBA, mnestic impairments improved considerably, the clinical course and MRI-results are stable in the 8-year follow-up. CONCLUSION: Based on our histopathological findings, we propose to regard the clinicopathological entity of PBA as an overlap between a neoplastic and neurodegenerative disorder. Since the lesions are locally restricted, they might be amenable to surgery with the prospect of a definite cure.
Subject(s)
Amyloidosis/pathology , Brain Diseases/pathology , Neurodegenerative Diseases/etiology , Neurodegenerative Diseases/pathology , Female , Humans , Magnetic Resonance Imaging , Middle AgedABSTRACT
Elephants are precocial mammals that are relatively mature as newborns and mobile shortly after birth. To determine whether the retina of newborn elephants is capable of supporting the mobility of elephant calves, we compared the retinal structures of 2 newborn elephants (1 African and 1 Asian) and 2 adult animals of both species by immunohistochemical and morphometric methods. For the first time, we present here a comprehensive qualitative and quantitative characterization of the cellular composition of the newborn and the adult retinas of 2 elephant species. We found that the retina of elephants is relatively mature at birth. All retinal layers were well discernible, and various retinal cell types were detected in the newborns, including Müller glial cells (expressing glutamine synthetase and cellular retinal binding protein; CRALBP), cone photoreceptors (expressing S-opsin or M/L-opsin), protein kinase Cα-expressing bipolar cells, tyrosine hydroxylase-, choline acetyltransferase (ChAT)-, calbindin-, and calretinin-expressing amacrine cells, and calbindin-expressing horizontal cells. The retina of newborn elephants contains discrete horizontal cells which coexpress ChAT, calbindin, and calretinin. While the overall structure of the retina is very similar between newborn and adult elephants, various parameters change after birth. The postnatal thickening of the retinal ganglion cell axons and the increase in ganglion cell soma size are explained by the increase in body size after birth, and the decreases in the densities of neuronal and glial cells are explained by the postnatal expansion of the retinal surface area. The expression of glutamine synthetase and CRALBP in the Müller cells of newborn elephants suggests that the cells are already capable of supporting the activities of photoreceptors and neurons. As a peculiarity, the elephant retina contains both normally located and displaced giant ganglion cells, with single cells reaching a diameter of more than 50 µm in adults and therefore being almost in the range of giant retinal ganglion cells found in aquatic mammals. Some of these ganglion cells are displaced into the inner nuclear layer, a unique feature of terrestrial mammals. For the first time, we describe here the occurrence of many bistratified rod bipolar cells in the elephant retina. These bistratified bipolar cells may improve nocturnal contrast perception in elephants given their arrhythmic lifestyle.
Subject(s)
Elephants/anatomy & histology , Neuroglia , Neurons , Retina/cytology , Retina/growth & development , Visual Pathways/anatomy & histology , Age Factors , Animals , Animals, Newborn , Calbindin 2/metabolism , Calbindins/metabolism , Cerebellum/growth & development , Choline O-Acetyltransferase/metabolism , Eye/anatomy & histology , Female , Male , Mice , Nerve Tissue Proteins/metabolism , Neuroglia/metabolism , Neurons/metabolism , Opsins/metabolism , Optic Nerve/anatomy & histology , Optic Nerve/growth & development , Species Specificity , Tyrosine 3-Monooxygenase/metabolismABSTRACT
Introduction: Spontaneous cervical artery dissection (sCAD) is a rare vasculopathy whose trigger is still unknown. We hypothesized that autoimmunity against components of the vascular wall might play a critical role in sCAD and examined anti-collagen type I antibodies in patients with sCAD, acute ischemic stroke, patients with thromboendarterectomy, and controls. Methods: Fifty-seven patients with sCAD (age 45.7 ± 10.2 years, female 18 (31.6%)) were prospectively enrolled in four German stroke centers. Blood samples were collected at baseline, at day 10 ± 3, and after 6 ± 1 months. Patients with ischemic stroke not related to CAD (n=54, age 56.7 ± 13.7 years, female 15 (27.8%)), healthy probands (n=80, age 57.4 ± 12.9 years, female 56 (70%)), and patients undergoing thromboendarterectomy of the carotid artery (n=9, age 70.7 ± 9.3 years, female 2 (22.2%)) served as controls. Anti-collagen type I antibodies were determined by enzyme-linked immunosorbent assays (ELISAs). Results: Patients with acute sCAD had higher serum levels of anti-collagen type I antibodies (33.9 ± 24.6 µg/ml) than probands (18.5 ± 11.0 µg/ml; p <0.001) but lower levels than patients with ischemic stroke not related to sCAD (47.8 ± 28.4 µg/ml; p=0.003). In patients with sCAD, serum levels of anti-collagen type I antibodies were similar in the acute, subacute, and chronic phase. Levels of anti-collagen type I antibodies significantly correlated with circulating collagen type I (rho=0.207, p=0.003). Conclusion: Anti-collagen type I antibodies seem not to represent a trigger for acute sCAD or ischemic stroke but may rather be linked to the metabolism and turnover of collagen type I.
Subject(s)
Autoantibodies , Collagen Type I , Ischemic Stroke , Humans , Female , Male , Middle Aged , Collagen Type I/immunology , Collagen Type I/blood , Prospective Studies , Ischemic Stroke/immunology , Ischemic Stroke/blood , Adult , Aged , Autoantibodies/blood , Autoantibodies/immunology , Vertebral Artery Dissection/immunology , Vertebral Artery Dissection/blood , Vertebral Artery Dissection/surgeryABSTRACT
Inhibitory (GABAergic) interneurons entrain assemblies of excitatory principal neurons to orchestrate information processing in the hippocampus. Disrupting the dynamic recruitment as well as the temporally precise activity of interneurons in hippocampal circuitries can manifest in epileptiform seizures, and impact specific behavioral traits. Despite the importance of GABAergic interneurons during information encoding in the brain, experimental tools to selectively manipulate GABAergic neurotransmission are limited. Here, we report the selective elimination of GABAergic interneurons by a ribosome inactivation approach through delivery of saporin-conjugated anti-vesicular GABA transporter antibodies (SAVAs) in vitro as well as in the mouse and rat hippocampus in vivo. We demonstrate the selective loss of GABAergic--but not glutamatergic--synapses, reduced GABA release, and a shift in excitation/inhibition balance in mixed cultures of hippocampal neurons exposed to SAVAs. We also show the focal and indiscriminate loss of calbindin(+), calretinin(+), parvalbumin/system A transporter 1(+), somatostatin(+), vesicular glutamate transporter 3 (VGLUT3)/cholecystokinin/CB(1) cannabinoid receptor(+) and neuropeptide Y(+) local-circuit interneurons upon SAVA microlesions to the CA1 subfield of the rodent hippocampus, with interneuron debris phagocytosed by infiltrating microglia. SAVA microlesions did not affect VGLUT1(+) excitatory afferents. Yet SAVA-induced rearrangement of the hippocampal circuitry triggered network hyperexcitability associated with the progressive loss of CA1 pyramidal cells and the dispersion of dentate granule cells. Overall, our data identify SAVAs as an effective tool to eliminate GABAergic neurons from neuronal circuits underpinning high-order behaviors and cognition, and whose manipulation can recapitulate pathogenic cascades of epilepsy and other neuropsychiatric illnesses.
Subject(s)
GABAergic Neurons/physiology , Hippocampus , Interneurons , Nerve Net/physiology , Neural Inhibition , Phagocytosis , Animals , Calbindin 2 , Calbindins , Cell Death/genetics , Cells, Cultured , Female , GABAergic Neurons/metabolism , GABAergic Neurons/pathology , Hippocampus/metabolism , Hippocampus/pathology , Hippocampus/physiology , Interneurons/metabolism , Interneurons/pathology , Interneurons/physiology , Male , Mice , Mice, Inbred C57BL , Nerve Net/metabolism , Nerve Net/pathology , Neural Inhibition/genetics , Phagocytosis/genetics , Rabbits , Rats , Rats, Sprague-Dawley , S100 Calcium Binding Protein G/geneticsABSTRACT
BACKGROUND/AIMS: The association between postoperative infection and prolonged survival in high-grade glioma is still a matter of debate. Previously we demonstrated that the intracerebral (i.c.) injection of heat-inactivated staphylococcal epitopes (HISE) resulted in a well-defined infux of immunocompetent cells across the blood-brain barrier. The present study investigated the potential antitumoral effect of HISE-immunostimulation in an experimental glioma model. METHODS: Wistar rats were intracerebrally implanted with 9L gliosarcoma cells (n=6), 9L cells mixed with HISE (n=12), or phosphate buffered saline (n=4). Tumor growth was measured by serial magnetic resonance imaging (MRI). After death due to the tumor burden, the brains were histopathologically assessed for inflammation and oncolysis. A toxicity assay was performed to quantify potential impairment of HISE on tumor cell growth in vitro. RESULTS: Animals treated by HISE showed a significant increase in average survival and even complete regression of an already established mass in one case. Naïve 9L gliosarcomas failed to recruit significant numbers of systemic immune cells. In contrast, concomitant intracerebral HISE inoculation lead to a oncolysis and a distinct peri- and intratumoral infiltration of macrophages, CD8 and CD4 co-expressing T-lymphocytes in two thirds of the tumor-bearing animals. The toxicity screening showed HISE-mediated oncolysis to be ineffective ex vivo. CONCLUSION: This study describes a novel approach for combatting malignant glioma using inactivated staphylococci as potent immunomodulators. Our results provide an outline for investigating the strategic potential of bacteria as emerging future therapeutics.
Subject(s)
Brain Neoplasms/therapy , Gliosarcoma/therapy , Immunologic Factors/therapeutic use , Staphylococcus epidermidis/metabolism , Animals , Brain Neoplasms/mortality , Brain Neoplasms/pathology , CD4-Positive T-Lymphocytes/immunology , CD4-Positive T-Lymphocytes/physiology , CD8-Positive T-Lymphocytes/immunology , CD8-Positive T-Lymphocytes/physiology , Cell Line, Tumor , Disease Models, Animal , Gliosarcoma/mortality , Gliosarcoma/pathology , Immunotherapy , Kaplan-Meier Estimate , Magnetic Resonance Imaging , Rats , Rats, Wistar , Staphylococcus epidermidis/immunology , Transplantation, HomologousABSTRACT
Acute measles may lead in rare instances to the chronic progressive central nervous system disease process subacute sclerosing panencephalitis (SSPE). SSPE results from a persistent measles virus (MV) infection with incomplete virus replication involving the entire human brain. The experimental encephalitis model in Lewis rats was used to define affected cell populations after infection with the neurotropic MV strain CAM/RB. Distribution patterns of MV were analysed by appropriate cell markers in the brain sections of infected animals employing multiple immunofluorescence labelling and confocal laser scanning microscopy. MV was detected in neurones but not in astrocytes, oligodendrocytes, microglia, and endothelial cells. GABAergic and glutamatergic neurons displayed MV antigen whereas cholinergic and catecholaminergic neurons appeared devoid of MV immunoreactivity. Mapping of the rat brain has revealed MV-infected neurones predominantly in motor, somatosensory, auditory, and visual cortices as well as in the basal ganglia and thalamic nuclei of infected rats. The results indicate that MV apparently disseminates via GABAergic and glutaminergic neurones and their processes. The tightly restricted viral distribution pattern is consistent with both inefficient immune clearance from infected neurones and with the observed disease symptoms.
Subject(s)
Brain/pathology , Measles virus/physiology , Measles/pathology , Neurons/pathology , Subacute Sclerosing Panencephalitis/pathology , Animals , Animals, Newborn , Astrocytes/pathology , Biomarkers/analysis , Brain/virology , Endothelial Cells/pathology , Fluorescent Antibody Technique , Humans , Measles/virology , Measles virus/pathogenicity , Microglia/pathology , Neurons/virology , Oligodendroglia/pathology , Rats , Rats, Inbred Lew , Subacute Sclerosing Panencephalitis/virology , Virus ReplicationABSTRACT
Knowledge of the brain's structure and function is essential for understanding processes in health and disease. Histochemical and fluorescence-based techniques have proven beneficial in characterizing brain regions and cellular compositions in pre-clinical research. Atomic force microscopy (AFM) has been introduced for mechanical tissue characterization, which may also help investigate pathophysiological aspects in disease-related models such as stroke. While combining AFM and fluorescence-based techniques, this study explored the mechanical properties of naive and ischemic brain regions in mice. Ischemia-affected regions were identified by the green signal of fluorescein isothiocyanate-conjugated albumin. A semi-automated protocol based on a brain atlas allowed regional allocations to the neocortex, striatum, thalamus, hypothalamus, hippocampus, and fiber tracts. Although AFM led to varying measurements, intra-individual analyses indicated a gradually increased tissue stiffness in the neocortex compared to subcortical areas, i.e., the striatum and fiber tracts. Regions affected by ischemia predominantly exhibited an increased tissue stiffness compared to those of the contra-lateral hemisphere, which might be related to cellular swelling. This study indicated intra-individual differences in mechanical properties among naive and ischemia-affected brain regions. The combination of AFM, semi-automated regional allocations, and fluorescence-based techniques thus qualifies for mechanical characterizations of the healthy and disease-affected brain in pre-clinical research.
Subject(s)
Neocortex , Stroke , Mice , Animals , Microscopy, Atomic Force/methods , Ischemia , HippocampusABSTRACT
In the setting of stroke, ischemia not only impairs neuronal function, but also detrimentally affects the different components of the neurovascular unit, which are shown to be involved in the transition from reversible to long-lasting tissue damage. In this context, the glial proteins myelin basic protein (MBP) and the 2',3'-cyclic-nucleotide 3'-phosphodiesterase (CNP) as well as the vasculature-associated basement membrane proteins laminin and collagen IV have been identified as ischemia-sensitive elements. However, available data from immunofluorescence and Western blot analyses are often found to be contradictory, which renders interpretation of the respective data rather difficult. Therefore, the present study investigates the impact of tissue pre-treatment and antibody clonality on immunofluorescence measurements of the mentioned proteins in a highly reproducible model of permanent middle cerebral artery occlusion. Here, immunofluorescence labeling using polyclonal antibodies revealed an increased immunofluorescence intensity of MBP, CNP, laminin and collagen IV in ischemic areas, although Western blot analyses did not reveal increased protein levels. Importantly, contrary to polyclonal antibodies, monoclonal ones did not provide increased fluorescence intensities in ischemic areas. Further, we were able to demonstrate that different ways of tissue pre-treatment including paraformaldehyde fixation and antigen retrieval may not only impact on fluorescence intensity measurements in general, but rather one-sidedly affect either ischemic or unaffected tissue. Therefore, immunofluorescence intensity measurements do not necessarily correlate with the actual protein levels, especially in ischemia-affected tissue and should always be complemented by different techniques to enhance reproducibility and to hopefully overcome the translational roadblock from bench to bedside.
ABSTRACT
CSF protein levels are altered in neurological disorders, such as hydrocephalus of different etiologies. In this retrospective observational study, we analyzed cerebrospinal fluid (CSF) samples in hydrocephalic diseases such as aqueductal stenosis (AQS, n = 27), normal pressure hydrocephalus (NPH, n = 24), hydrocephalus communicans (commHC, n = 25) and idiopathic intracranial hypertension (IIH)/pseudotumor cerebri (PC, n = 7) in comparison with neurological patients without hydrocephalic configuration (control, n = 95). CSF was obtained through CSF diversion procedures and lumbar punction and analyzed for protein concentrations according to the institution's laboratory standards. We found significantly decreased CSF protein levels in patients suffering from AQS (0.13 mg/dL [0.1-0.16 mg/dL] p = 2.28 × 10-8) and from PC (0.18 mg/dL [0.12-0.24 mg/dL] p = 0.01) compared with controls (0.34 mg/dL [0.33-0.35 mg/dL]). Protein levels were not altered in patients suffering from commHC and NPH compared with neurologically healthy individuals. We propose that a decrease in CSF protein levels is part of an active counterregulatory mechanism to lower CSF volume and, subsequently, intracranial pressure in specific diseases. Research regarding said mechanism and more specific proteomic research on a cellular level must still be performed to prove this hypothesis. Differences in protein levels between different diseases point to different etiologies and mechanisms in different hydrocephalic pathologies.
ABSTRACT
BACKGROUND AND PURPOSE: CT and MR imaging techniques are frequently used for the diagnosis and progress monitoring of ischemic stroke in clinical practice and research. After stroke, both methods are characterized by a transient pseudo-normalized imaging signal, the so-called fogging phenomenon. This study evaluates potential pathophysiological changes associated with fogging, as well as its influence on the correct determination of the ischemic lesion in a rat stroke model. METHODS: Male spontaneously hypertensive rats were subjected to permanent middle cerebral artery occlusion. Ischemic lesion volume, brain edema and gray scale value spread within the ischemic lesion were determined on T2-weighted MR sequences at days 1, 4, 8, 11 and 29 after stroke onset, and compared with immunohistochemistry for astrogliosis, microglia/macrophage infiltration and angiogenesis. RESULTS: All animals showed MR fogging at days 4, 8 and 11 after stroke. The transient normalization of T2 signals occurred independently from the development of infarct volumes, but coincided well with the spatio-temporal occurrence of necrosis, angiogenesis and microglia/macrophage infiltration. CONCLUSIONS: Our results suggest that the fogging effect reflects the clearance of necrotic tissue within the ischemic lesion and is thus not relevant for the determination of the lesion volume.
Subject(s)
Infarction, Middle Cerebral Artery/pathology , Magnetic Resonance Imaging , Stroke/pathology , Animals , Disease Models, Animal , Image Processing, Computer-Assisted , Immunohistochemistry , Infarction, Middle Cerebral Artery/complications , Male , Rats , Rats, Inbred SHR , Stroke/etiologyABSTRACT
As chemically specialized forms of the extracellular matrix in the central nervous system, polyanionic perineuronal nets (PNs) contain diverse constituents, including chondroitin sulfate proteoglycans (CSPGs), hyaluronic acid, and tenascins. They are detectable by various histological approaches such as colloidal iron binding and immunohistochemical staining to reveal, for instance, the CSPGs aggrecan, neurocan, phosphacan, and versican. Moreover, biotin, peroxidase, or fluorescein conjugates of the lectins Vicia villosa agglutinin and soybean agglutinin enable the visualization of PNs. At present, the N-acetylgalactosamine-binding Wisteria floribunda agglutinin (WFA) is the most widely applied marker for PNs. Therefore, this article is largely focused on methodological aspects of WFA staining. Notably, fluorescent WFA labeling allows, after its conversion into electron-dense adducts, electron microscopic analyses. Furthermore, the usefulness of WFA conjugates for the oftentimes neglected in vivo and in vitro labeling of PNs is emphasized. Subsequently, we discuss impaired WFA-staining sites after long-lasting experiments in vitro, especially in autoptic brain samples with long postmortem delay and partial enzymatic degradation, while immunolabeling of aggrecan and CSPG link proteins under such conditions has proven more robust. In some hippocampal regions from perfusion-fixed mice, more PNs are aggrecan immunoreactive than WFA positive, whereas the retrosplenial cortex displays many WFA-binding PNs devoid of visible aggrecan immunoreactivity. Additional multiple fluorescence labeling exemplarily revealed in ischemic tissue diminished staining of WFA-binding sites and aquaporin 4 and concomitantly upregulated immunolabeling of neurofilament, light chains, and collagen IV. Finally, we briefly discuss possible future staining approaches based on nanobodies to facilitate novel technologies revealing details of net morphology.
ABSTRACT
The regulation of surface tension (ST) by surfactants plays an important role in the human respiratory system but is largely unexplored in brain homeostasis. The aim of this study was to evaluate changes in ST in relation to surfactant proteins (SPs) in children with intraventricular hemorrhage (IVH). CSF samples from 93 patients were analyzed for ST with a force tensiometer and SP-A-D and -G with ELISA assays. Patients belonged to six groups: (i) IVH before primary intervention (PI), (ii) IVH 4−28 days after PI, (iii) IVH 44−357 days after PI, (iv) hydrocephalus, (v) sepsis and (vi) controls. We found indirect correlations and significant differences in ST and SPs (all p < 0.001; except for SP-C, p = 0.007). Post hoc analyses showed significantly decreased ST in IVH patients before PI compared with patients with hydrocephalus, sepsis or controls (p < 0.001), but it increased in IVH patients over time. All SPs were significantly elevated when comparing IVH patients before PI with controls (all p < 0.001; except for SP-C, p = 0.003). Children suffering from IVH displayed an increase in SPs and a decrease in ST as coping mechanisms to preserve CSF flow. The increase in ST over time could serve as prognostic marker for the healing process.
ABSTRACT
(1) Background: Astrocytic gliomas present overlapping appearances in conventional MRI. Supplementary techniques are necessary to improve preoperative diagnostics. Quantitative DWI via the computation of apparent diffusion coefficient (ADC) histograms has proven valuable for tumor characterization and prognosis in this regard. Thus, this study aimed to investigate (I) the potential of ADC histogram analysis (HA) for distinguishing low-grade gliomas (LGG) and high-grade gliomas (HGG) and (II) whether those parameters are associated with Ki-67 immunolabelling, the isocitrate-dehydrogenase-1 (IDH1) mutation profile and the methylguanine-DNA-methyl-transferase (MGMT) promoter methylation profile; (2) Methods: The ADC-histograms of 82 gliomas were computed. Statistical analysis was performed to elucidate associations between histogram features and WHO grade, Ki-67 immunolabelling, IDH1 and MGMT profile; (3) Results: Minimum, lower percentiles (10th and 25th), median, modus and entropy of the ADC histogram were significantly lower in HGG. Significant differences between IDH1-mutated and IDH1-wildtype gliomas were revealed for maximum, lower percentiles, modus, standard deviation (SD), entropy and skewness. No differences were found concerning the MGMT status. Significant correlations with Ki-67 immunolabelling were demonstrated for minimum, maximum, lower percentiles, median, modus, SD and skewness; (4) Conclusions: ADC HA facilitates non-invasive prediction of the WHO grade, tumor-proliferation rate and clinically significant mutations in case of astrocytic gliomas.
ABSTRACT
The classic surfactant proteins (SPs) A, B, C, and D were discovered in the lungs, where they contribute to host defense and regulate the alveolar surface tension during breathing. Their additional importance for brain physiology was discovered decades later. SP-G, a novel amphiphilic SP, was then identified in the lungs and is mostly linked to inflammation. In the brain, it is also present and significantly elevated after hemorrhage in premature infants and in distinct conditions affecting the cerebrospinal fluid circulation of adults. However, current knowledge on SP-G-expression is limited to ependymal cells and some neurons in the subventricular and superficial cortex. Therefore, we primarily focused on the distribution of SP-G-immunoreactivity (ir) and its spatial relationships with components of the neurovascular unit in murine forebrains. Triple fluorescence labeling elucidated SP-G-co-expressing neurons in the habenula, infundibulum, and hypothalamus. Exploring whether SP-G might play a role in Alzheimer's disease (AD), 3xTg-AD mice were investigated and displayed age-dependent hippocampal deposits of ß-amyloid and hyperphosphorylated tau separately from clustered, SP-G-containing dots with additional Reelin-ir-which was used as established marker for disease progression in this specific context. Semi-quantification of those dots, together with immunoassay-based quantification of intra- and extracellular SP-G, revealed a significant elevation in old 3xTg mice when compared to age-matched wildtype animals. This suggests a role of SP-G for the pathophysiology of AD, but a confirmation with human samples is required.
Subject(s)
Alzheimer Disease , Pulmonary Surfactant-Associated Protein A/metabolism , Alzheimer Disease/genetics , Alzheimer Disease/metabolism , Amyloid beta-Peptides/metabolism , Animals , Brain/metabolism , Disease Models, Animal , Hippocampus/metabolism , Humans , Mice , Mice, Transgenic , Surface-Active Agents/metabolism , tau Proteins/genetics , tau Proteins/metabolismABSTRACT
Prion diseases are fatal neurodegenerative disorders causing motor dysfunctions, dementia and neuropathological changes such as spongiosis, astroglyosis and neuronal loss. The chain of events leading to the clinical disease and the role of distinct brain areas are still poorly understood. The role of nervous system integrity and axonal properties in prion pathology are still elusive. There is no evidence of both the functional axonal impairments in vivo and their connection with prion disease. We studied the functional axonal impairments in motor neurons at the onset of clinical prion disease using the combination of tracing as a functional assay for axonal transport with immunohistochemistry experiments. Well-established and novel confocal and ultramicroscopy techniques were used to image and quantify labeled neurons. Despite profound differences in the incubation times, 30% to 45% of neurons in the red nucleus of different mouse lines showed axonal transport impairments at the disease onset bilaterally after intracerebral prion inoculation and unilaterally -- after inoculation into the right sciatic nerve. Up to 94% of motor cortex neurons also demonstrated transport defects upon analysis by alternative imaging methods. Our data connect axonal transport impairments with disease symptoms for different prion strains and inoculation routes and establish further insight on the development of prion pathology in vivo. The alterations in localization of the proteins involved in the retrograde axonal transport allow us to propose a mechanism of transport disruption, which involves Rab7-mediated cargo attachment to the dynein-dynactin pathway. These findings suggest novel targets for therapeutic and diagnostic approaches in the early stages of prion disease.
Subject(s)
Axonal Transport/physiology , Motor Neurons/metabolism , Prion Diseases/metabolism , Amidines/metabolism , Animals , Immunohistochemistry , Luminescent Proteins/metabolism , Mice , Mice, Inbred C57BL , Motor Cortex/metabolism , Motor Cortex/pathology , Motor Neurons/pathology , Motor Neurons/ultrastructure , Nerve Tissue Proteins/metabolism , PrPSc Proteins/metabolism , Prion Diseases/pathology , Red Nucleus/metabolism , Red Nucleus/physiopathology , Sciatic Nerve/metabolism , Sciatic Nerve/ultrastructure , rab GTP-Binding Proteins/metabolism , rab7 GTP-Binding ProteinsABSTRACT
The deposition of amyloid-ß (Aß) peptides in the walls of leptomeningeal and cortical blood vessels as cerebral amyloid angiopathy (CAA) is present in normal ageing and the majority of Alzheimer's disease (AD) brains. The failure of clearance mechanisms to eliminate Aß from the brain contributes to the development of sporadic CAA and AD. Here, we investigated the effects of CAA and ageing on the pattern of perivascular drainage of solutes in the brains of naïve mice and in the Tg2576 mouse model of AD. We report that drainage of small molecular weight dextran along cerebrovascular basement membranes is impaired in the hippocampal capillaries and arteries of 22-month-old wild-type mice compared to 3- and 7-month-old animals, which was associated with age-dependent changes in capillary density. Age-related alterations in the levels of laminin, fibronectin and perlecan in vascular basement membranes were also noted in wild-type mice. Furthermore, dextran was observed in the walls of veins of Tg2576 mice in the presence of CAA, suggesting that deposition of Aß in vessel walls disrupts the normal route of elimination of solutes from the brain parenchyma. These data support the hypothesis that perivascular solute drainage from the brain is altered both in the ageing brain and as a consequence of CAA. These findings have implications for the success of therapeutic strategies for the treatment of AD that rely upon the health of the ageing cerebral vasculature.