Regionally Altered Immunosignals of Surfactant Protein-G, Vascular and Non-Vascular Elements of the Neurovascular Unit after Experimental Focal Cerebral Ischemia in Mice, Rats, and Sheep.

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.

SOAT1: A Suitable Target for Therapy in High-Grade Astrocytic Glioma?

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.

Surfactant Protein-G in Wildtype and 3xTg-AD Mice: Localization in the Forebrain, Age-Dependent Hippocampal Dot-like Deposits and Brain Content.

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.

Classification of Microglial Morphological Phenotypes Using Machine Learning.

Microglia are the brain's immunocompetent macrophages with a unique feature that allows surveillance of the surrounding microenvironment and subsequent reactions to tissue damage, infection, or homeostatic perturbations. Thereby, microglia's striking morphological plasticity is one of their prominent characteristics and the categorization of microglial cell function based on morphology is well established. Frequently, automated classification of microglial morphological phenotypes is performed by using quantitative parameters. As this process is typically limited to a few and especially manually chosen criteria, a relevant selection bias may compromise the resulting classifications. In our study, we describe a novel microglial classification method by morphological evaluation using a convolutional neuronal network on the basis of manually selected cells in addition to classical morphological parameters. We focused on four microglial morphologies, ramified, rod-like, activated and amoeboid microglia within the murine hippocampus and cortex. The developed method for the classification was confirmed in a mouse model of ischemic stroke which is already known to result in microglial activation within affected brain regions. In conclusion, our classification of microglial morphological phenotypes using machine learning can serve as a time-saving and objective method for post-mortem characterization of microglial changes in healthy and disease mouse models, and might also represent a useful tool for human brain autopsy samples.

The Cytoskeletal Elements MAP2 and NF-L Show Substantial Alterations in Different Stroke Models While Elevated Serum Levels Highlight Especially MAP2 as a Sensitive Biomarker in Stroke Patients.

In the setting of ischemic stroke, the neurofilament subunit NF-L and the microtubule-associated protein MAP2 have proven to be exceptionally ischemia-sensitive elements of the neuronal cytoskeleton. Since alterations of the cytoskeleton have been linked to the transition from reversible to irreversible tissue damage, the present study investigates underlying time- and region-specific alterations of NF-L and MAP2 in different animal models of focal cerebral ischemia. Although NF-L is increasingly established as a clinical stroke biomarker, MAP2 serum measurements after stroke are still lacking. Therefore, the present study further compares serum levels of MAP2 with NF-L in stroke patients. In the applied animal models, MAP2-related immunofluorescence intensities were decreased in ischemic areas, whereas the abundance of NF-L degradation products accounted for an increase of NF-L-related immunofluorescence intensity. Accordingly, Western blot analyses of ischemic areas revealed decreased protein levels of both MAP2 and NF-L. The cytoskeletal alterations are further reflected at an ultrastructural level as indicated by a significant reduction of detectable neurofilaments in cortical axons of ischemia-affected areas. Moreover, atomic force microscopy measurements confirmed altered mechanical properties as indicated by a decreased elastic strength in ischemia-affected tissue. In addition to the results from the animal models, stroke patients exhibited significantly elevated serum levels of MAP2, which increased with infarct size, whereas serum levels of NF-L did not differ significantly. Thus, MAP2 appears to be a more sensitive stroke biomarker than NF-L, especially for early neuronal damage. This perspective is strengthened by the results from the animal models, showing MAP2-related alterations at earlier time points compared to NF-L. The profound ischemia-induced alterations further qualify both cytoskeletal elements as promising targets for neuroprotective therapies.

Surfactant protein C is associated with perineuronal nets and shows age-dependent changes of brain content and hippocampal deposits in wildtype and 3xTg mice.

Surfactant protein C (SP-C) modulates cerebrospinal fluid (CSF) rheology. During ageing, its declining levels are accompanied by an increased burden of white matter lesions. Pulmonary SP-C intermediates harbouring the BRICHOS-domain prevent protein misfolding in the lungs. Thus, cerebral SP-C intermediates may counteract cerebral ß-amyloidosis, a hallmark of Alzheimer's disease (AD). However, data on the molecular neuroanatomy of SP-C and its alterations in wildtype and triple transgenic (3xTg) mice, featuring essential elements of AD-neuropathology, are lacking. Therefore, this study investigated SP-C-containing structures in murine forebrains and their spatial relationships with vascular, glial and neuronal components of the neurovascular unit. Fluorescence labelling demonstrated neuronal SP-C in the medial habenula, the indusium griseum and the hippocampus. Glial counterstaining elucidated astrocytes in the corpus callosum co-expressing SP-C and S100ß. Notably, perineuronal nets were associated with SP-C in the nucleus reticularis thalami, the lateral hypothalamus and the retrosplenial cortex. In the hippocampus of aged 3xTg mice, an increased number of dot-like depositions containing SP-C and Reelin, but devoid of BRICHOS-immunoreactivity were observed apart from AD-like lesions. Wildtype and 3xTg mice revealed an age-dependent increase of such deposits markedly pronounced in about 24-month-old 3xTg mice. SP-C levels of the intracellular and extracellular compartments in each group revealed an inverse correlation of SP-C and Reelin, with reduced SP-C and increased Reelin in an age-dependent fashion especially in 3xTg mice. Taken together, extracellular SP-C, as modulator of glymphatic clearance and potential ligand of PNs, declines in 3xTg mice, which show an accumulation of extracellular Reelin depositions during ageing.

Increased Immunosignals of Collagen IV and Fibronectin Indicate Ischemic Consequences for the Neurovascular Matrix Adhesion Zone in Various Animal Models and Human Stroke Tissue.

Ischemic stroke causes cellular alterations in the "neurovascular unit" (NVU) comprising neurons, glia, and the vasculature, and affects the blood-brain barrier (BBB) with adjacent extracellular matrix (ECM). Limited data are available for the zone between the NVU and ECM that has not yet considered for neuroprotective approaches. This study describes ischemia-induced alterations for two main components of the neurovascular matrix adhesion zone (NMZ), i.e., collagen IV as basement membrane constituent and fibronectin as crucial part of the ECM, in conjunction with traditional NVU elements. For spatio-temporal characterization of these structures, multiple immunofluorescence labeling was applied to tissues affected by focal cerebral ischemia using a filament-based model in mice (4, 24, and 72 h of ischemia), a thromboembolic model in rats (24 h of ischemia), a coagulation-based model in sheep (2 weeks of ischemia), and human autoptic stroke tissue (3 weeks of ischemia). An increased fibronectin immunofluorescence signal demarcated ischemia-affected areas in mice, along with an increased collagen IV signal and BBB impairment indicated by serum albumin extravasation. Quantifications revealed a region-specific pattern with highest collagen IV and fibronectin intensities in most severely affected neocortical areas, followed by a gradual decline toward the border zone and non-affected regions. Comparing 4 and 24 h of ischemia, the subcortical fibronectin signal increased significantly over time, whereas neocortical areas displayed only a gradual increase. Qualitative analyses confirmed increased fibronectin and collagen IV signals in ischemic areas from all tissues and time points investigated. While the increased collagen IV signal was restricted to vessels, fibronectin appeared diffusely arranged in the parenchyma with focal accumulations associated to the vasculature. Integrin α5 appeared enriched in the vicinity of fibronectin and vascular elements, while most of the non-vascular NVU elements showed complementary staining patterns referring to fibronectin. This spatio-temporal characterization of ischemia-related alterations of collagen IV and fibronectin in various stroke models and human autoptic tissue shows that ischemic consequences are not limited to traditional NVU components and the ECM, but also involve the NMZ. Future research should explore more components and the pathophysiological properties of the NMZ as a possible target for novel neuroprotective approaches.

Spatiotemporal Changes of Cerebral Monocarboxylate Transporter 8 Expression.

Background: Mutations of monocarboxylate transporter 8 (MCT8), a thyroid hormone (TH)-specific transmembrane transporter, cause a severe neurodevelopmental disorder, the Allan-Herndon-Dudley syndrome. In MCT8 deficiency, TH is not able to reach those areas of the brain where TH uptake depends on MCT8. Currently, therapeutic options for MCT8-deficient patients are missing, as TH treatment is not successful in improving neurological deficits. Available data on MCT8 protein and transcript levels indicate complex expression patterns in neural tissue depending on species, brain region, sex, and age. However, information on human MCT8 expression is still scattered and additional efforts are needed to map sites of MCT8 expression in neurovascular units and neural tissue. This is of importance because new therapeutic strategies for this disease are urgently needed. Methods: To identify regions and time windows of MCT8 expression, we used highly specific antibodies against MCT8 to perform immunofluorescence labeling of postnatal murine brains, adult human brain tissue, and human cerebral organoids. Results: Qualitative and quantitative analyses of murine brain samples revealed stable levels of MCT8 protein expression in endothelial cells of the blood-brain barrier (BBB), choroid plexus epithelial cells, and tanycytes during postnatal development. Conversely, the neuronal MCT8 protein expression that was robustly detectable in specific brain regions of young mice strongly declined with age. Similarly, MCT8 immunoreactivity in adult human brain tissue was largely confined to endothelial cells of the BBB. Recently, cerebral organoids emerged as promising models of human neural development and our first analyses of forebrain-like organoids revealed MCT8 expression in early neuronal progenitor cell populations. Conclusions: With respect to MCT8-deficient conditions, our analyses not only strongly support the contention that the BBB presents a lifelong barrier to TH uptake but also highlight the need to decipher the TH transport role of MCT8 in early neuronal cell populations in more detail. Improving the understanding of the spatiotemporal expression in latter barriers will be critical for therapeutic strategies addressing MCT8 deficiency in the future.

Endothelial edema precedes blood-brain barrier breakdown in early time points after experimental focal cerebral ischemia.

In the setting of stroke, ischemia-related blood-brain barrier (BBB) dysfunction aggravates the cerebral edema, which critically impacts on the clinical outcome. Further, an impaired vascular integrity is associated with the risk of intracranial bleeding, especially after therapeutic recanalization. Therefore, the present study was aimed to investigate early vascular alterations from 30 min to 4 h after experimental middle cerebral artery occlusion (MCAO) in mice. Here, an extravasation of the permeability marker FITC-albumin was detectable in animals 2 and 4 h after MCAO. Thereby, BBB breakdown correlated with alterations of the endothelial surface, indicated by a discontinuous isolectin-B4 staining, while tight junction strands remained detectable using electron and immunofluorescence microscopy. Noteworthy, already 30 min after MCAO, up to 60% of the ischemia-affected vessels showed an endothelial edema, paralleled by edematous astrocytic endfeet, clearly preceding FITC-albumin extravasation. With increasing ischemic periods, scores of vascular damage significantly increased with up to 60% of the striatal vessels showing loss of endothelial integrity. Remarkably, comparison of permanent and transient ischemia did not provide significant differences 4 h after ischemia induction. As these degenerations also involved penumbral areas of potentially salvageable tissue, adjuvant approaches of endothelial protection may help to reduce the vasogenic edema after ischemic stroke.

Simultaneous alterations of oligodendrocyte-specific CNP, astrocyte-specific AQP4 and neuronal NF-L demarcate ischemic tissue after experimental stroke in mice.

Ischemic stroke not only affects neurons, but also glial and vascular elements. The development of novel neuroprotective strategies thus requires an improved pathophysiological understanding of ischemia-affected cell types that comprise the 'neurovascular unit' (NVU). To explore spatiotemporal alterations of oligodendrocytes, astrocytes and neurons after experimental ischemic stroke, we applied a permanent middle cerebral artery occlusion model in mice for 4 and 24 h. Using fluorescence microscopy, the oligodendrocyte marker 2',3'-cyclic nucleotide phosphodiesterase (CNP), the neuronal neurofilament light chain (NF-L) and the astroglial aquaporin-4 (AQP4) were analyzed in regional relation to one another. Immunofluorescence intensities of CNP and NF-L were simultaneously increased in the ischemic neocortex and striatum. AQP4 immunoreactivity was decreased in the ischemic striatum, which represents the initial and potentially strongest affected site of infarction. The more distant ischemic neocortex and infarct border zones exhibited areas with alternately increased or decreased AQP4 immunoreactivity, leading to an increase of fluorescence intensity in total. Further, deformed CNP-immunopositive processes were found around axonal spheroids, indicating a combined affection of oligodendrocytes and neurons due to ischemia. Importantly, altered AQP4 immunosignals were not limited to the ischemic core, but were also detectable in penumbral areas. This applies for CNP and NF-L also, since altered immunosignals of all three markers coincided regionally at both time points. In conclusion, the present study provides evidence for a simultaneous affection of oligodendrocytes, astrocytes and neurons after experimental focal cerebral ischemia. Consequently, CNP, AQP4 and NF-L immunofluorescence alterations can be utilized to identify ischemia-affected tissue. The simultaneity of the described alterations further strengthens the concept of interdependent NVU components and distinguishes NF-L, CNP and AQP4 as highly ischemia-sensitive elements. Consequently, future therapeutic approaches might influence stroke evolution via strategies simultaneously addressing both neuronal and glial functions.

Transcriptional Response and Morphological Features of the Neurovascular Unit and Associated Extracellular Matrix After Experimental Stroke in Mice.

Experimental stroke studies yielded insights into single reactions of the neurovascular unit (NVU) and associated extracellular matrix (ECM). However, the extent of simultaneous processes caused by ischemia and their underlying transcriptional changes are still poorly understood. Strictly following the NVU and ECM concept, this study explored transcriptional responses of cellular and non-cellular components as well as their morphological characteristics following ischemia. Mice were subjected to 4 or 24 h of unilateral middle cerebral artery occlusion. In the neocortex and the striatum, cytoskeletal and glial elements as well as blood-brain barrier and ECM components were analyzed using real-time PCR. Western blot analyses allowed characterization of protein levels and multiple immunofluorescence labeling enabled morphological assessment. Out of 37 genes analyzed, the majority exhibited decreased mRNA levels in ischemic areas, while changes occurred as early as 4 h after ischemia. Down-regulated mRNA levels were predominantly localized in the neocortex, such as the structural elements α-catenin 2, N-cadherin, ß-catenin 1, and ßIII-tubulin, consistently decreasing 4 and 24 h after ischemia. However, a few genes, e.g., claudin-5 and Pcam1, exhibited increased mRNA levels after ischemia. For several components such as ßIII-tubulin, N-cadherin, and ß-catenin 1, matching transcriptional and immunofluorescence signals were obtained, whereas a few markers including neurofilaments exhibited opposite directions. In conclusion, the variety in gene regulation emphasizes the complexity of interactions within the ischemia-affected NVU and ECM. These data might help to focus future research on a set of highly sensitive elements, which might prospectively facilitate neuroprotective strategies beyond the traditional single target perspective.

Microglia contribute to the glia limitans around arteries, capillaries and veins under physiological conditions, in a model of neuroinflammation and in human brain tissue.

Microglia represent resident immune cells of the central nervous system (CNS), which have been shown to be involved in the pathophysiology of practically every neuropathology. As microglia were described to participate in the formation of the astroglial glia limitans around CNS vessels, they are part of the neurovascular unit (NVU). Since the NVU is a highly specialized structure, being functionally and morphologically adapted to differing demands in the arterial, capillary, and venous segments, the present study was aimed to systematically investigate the microglial contribution to the glia limitans along the vascular tree. Thereby, the microglial participation in the glia limitans was demonstrated for arteries, capillaries, and veins by immunoelectron microscopy in wild-type mice. Furthermore, analysis by confocal laser scanning microscopy revealed the highest density of microglial endfeet contacting the glial basement membrane around capillaries, with significantly lower densities around arteries and veins. Importantly, this pattern appeared to be unaltered in the setting of experimental autoimmune encephalomyelitis (EAE) in CX3CR1CreERT2:R26-Tomato reporter mice, although perivascular infiltrates of blood-borne leukocytes predominantly occur at the level of post-capillary venules. However, EAE animals exhibited significantly increased contact sizes of individual microglial endfeet around arteries and veins. Noteworthy, under EAE conditions, the upregulation of MHC-II was not limited to microglia of the glia limitans of veins showing infiltrates of leukocytes, but also appeared at the capillary level. As a microglial contribution to the glia limitans was also observed in human brain tissue, these findings may help characterizing microglial alterations within the NVU in various neuropathologies.

Radial glial elements in the cerebral cortex of the lesser hedgehog tenrec.

We investigated astroglial cells in several areas of the telencephalic cortex of the lesser hedgehog tenrec (Echinops telfairi). Compared to other mammals, the cortex of the tenrec has a relatively large paleocortex and a low encephalization index. We stained sections from tenrec forebrains with structural and functional glia markers focusing on selected cortical areas, the paleocortex, rhinal cortex, neocortex and the dentate gyrus of the hippocampal formation. We found that in all parts of the tenrec forebrain cortex, radial processes exist which are positive for glial fibrillary acidic protein (GFAP) although with differential localization: in the rhinal cortex and neocortical region radial glial fibers are located in the subventricular regions, whereas in the dentate gyrus and paleocortex they appear to arise from the cells in the respective granular layers. The relatively high abundance of the radial fibers in layer III of the paleocortex was very conspicuous. Only few of these radial processes were also co-labeled with doublecortin (DCX), yet most of the DCX-positive cells were negative for GFAP. The GFAP-positive radial fibers were in turn neither positive for glutamine synthetase, nor did they show immunoreactivity for the astroglia-specific water channel aquaporin-4 (AQP4). Star-shaped astrocytes, however, displayed the typical perivascular and subpial expression patterns for AQP4. We conclude that the radial glia in the adult tenrec represents an immature form of astroglia that persists in these animals throughout life.

Impaired Neurofilament Integrity and Neuronal Morphology in Different Models of Focal Cerebral Ischemia and Human Stroke Tissue.

As part of the neuronal cytoskeleton, neurofilaments are involved in maintaining cellular integrity. In the setting of ischemic stroke, the affection of the neurofilament network is considered to mediate the transition towards long-lasting tissue damage. Although peripheral levels of distinct neurofilament subunits are shown to correlate with the clinically observed severity of cerebral ischemia, neurofilaments have so far not been considered for neuroprotective approaches. Therefore, the present study systematically addresses ischemia-induced alterations of the neurofilament light (NF-L), medium (NF-M), and heavy (NF-H) subunits as well as of α-internexin (INA). For this purpose, we applied a multi-parametric approach including immunofluorescence labeling, western blotting, qRT-PCR and electron microscopy. Analyses comprised ischemia-affected tissue from three stroke models of middle cerebral artery occlusion (MCAO), including approaches of filament-based MCAO in mice, thromboembolic MCAO in rats, and electrosurgical MCAO in sheep, as well as human autoptic stroke tissue. As indicated by altered immunosignals, impairment of neurofilament subunits was consistently observed throughout the applied stroke models and in human tissue. Thereby, altered NF-L immunoreactivity was also found to reach penumbral areas, while protein analysis revealed consistent reductions for NF-L and INA in the ischemia-affected neocortex in mice. At the mRNA level, the ischemic neocortex and striatum exhibited reduced expressions of NF-L- and NF-H-associated genes, whereas an upregulation for Ina appeared in the striatum. Further, multiple fluorescence labeling of neurofilament proteins revealed spheroid and bead-like structural alterations in human and rodent tissue, correlating with a cellular edema and lost cytoskeletal order at the ultrastructural level. Thus, the consistent ischemia-induced affection of neurofilament subunits in animals and human tissue, as well as the involvement of potentially salvageable tissue qualify neurofilaments as promising targets for neuroprotective strategies. During ischemia formation, such approaches may focus on the maintenance of neurofilament integrity, and appear applicable as co-treatment to modern recanalizing strategies.

Damaged Neocortical Perineuronal Nets Due to Experimental Focal Cerebral Ischemia in Mice, Rats and Sheep.

As part of the extracellular matrix (ECM), perineuronal nets (PNs) are polyanionic, chondroitin sulfate proteoglycan (CSPG)-rich coatings of certain neurons, known to be affected in various neural diseases. Although these structures are considered as important parts of the neurovascular unit (NVU), their role during evolution of acute ischemic stroke and subsequent tissue damage is poorly understood and only a few preclinical studies analyzed PNs after acute ischemic stroke. By employing three models of experimental focal cerebral ischemia, this study was focused on histopathological alterations of PNs and concomitant vascular, glial and neuronal changes according to the NVU concept. We analyzed brain tissues obtained 1 day after ischemia onset from: (a) mice after filament-based permanent middle cerebral artery occlusion (pMCAO); (b) rats subjected to thromboembolic MACO; and (c) sheep at 14 days after electrosurgically induced focal cerebral ischemia. Multiple fluorescence labeling was applied to explore simultaneous alterations of NVU and ECM. Serial mouse sections labeled with the net marker Wisteria floribunda agglutinin (WFA) displayed largely decomposed and nearly erased PNs in infarcted neocortical areas that were demarcated by up-regulated immunoreactivity for vascular collagen IV (Coll IV). Subsequent semi-quantitative analyses in mice confirmed significantly decreased WFA-staining along the ischemic border zone and a relative decrease in the directly ischemia-affected neocortex. Triple fluorescence labeling throughout the three animal models revealed up-regulated Coll IV and decomposed PNs accompanied by activated astroglia and altered immunoreactivity for parvalbumin, a calcium-binding protein in fast-firing GABAergic neurons which are predominantly surrounded by neocortical PNs. Furthermore, ischemic neocortical areas in rodents simultaneously displayed less intense staining of WFA, aggrecan, the net components neurocan, versican and the cartilage link protein (CRTL) as well as markers in net-bearing neurons such as the potassium channel subunit Kv3.1b and neuronal nuclei (NeuN). In summary, theconsistent observations based on three different stroke models confirmed that PNs are highly sensitive constituents of the NVU along with impaired associated GABAergic neurons. These results suggest that PNs could be promising targets of future stroke treatment, and further studies should address their reorganization and plasticity in both stabilizing the acute stroke as well as supportive effects during the chronic phase of stroke.

Delayed histochemical alterations within the neurovascular unit due to transient focal cerebral ischemia and experimental treatment with neurotrophic factors.

Current stroke therapy is focused on recanalizing strategies, but neuroprotective co-treatments are still lacking. Modern concepts of the ischemia-affected neurovascular unit (NVU) and surrounding penumbra emphasize the complexity during the transition from initial damaging to regenerative processes. While early treatment with neurotrophic factors was shown to result in lesion size reduction and blood-brain barrier (BBB) stabilization, cellular consequences from these treatments are poorly understood. This study explored delayed cellular responses not only to ischemic stroke, but also to an early treatment with neurotrophic factors. Rats underwent 60 minutes of focal cerebral ischemia. Fluorescence labeling was applied to sections from brains perfused 7 days after ischemia. Analyses focused on NVU constituents including the vasculature, astrocytes and microglia in the ischemic striatum, the border zone and the contralateral hemisphere. In addition to histochemical signs of BBB breakdown, a strong up-regulation of collagen IV and microglia activation occurred within the ischemic core with simultaneous degradation of astrocytes and their endfeet. Activated astroglia were mainly depicted at the border zone in terms of a glial scar formation. Early treatment with pigment epithelium-derived factor (PEDF) resulted in an attenuation of the usually up-regulated collagen IV-immunoreactivity. However, glial activation was not influenced by treatment with PEDF or the epidermal growth factor (EGF). In conclusion, these data on ischemia-induced cellular reactions within the NVU might help to develop treatments addressing the transition from injury towards regeneration. Thereby, the integrity of the vasculature in close relation to neighboring structures like astrocytes appears as a promising target.