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Cited by 53Barrier Structure and Function StudiesProteoglycans and glycosaminoglycans researchNeuroinflammation and Neurodegeneration Mechanisms

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

Open access
Härtig, Wolfgang, Mages, Bianca, Aleithe, Susanne, Nitzsche, Björn, Altmann, Stephan, Barthel, Henryk, Krueger, Martin, Michalski, Dominik·2017·Frontiers in Integrative Neuroscience
DOI 10.3389/fnint.2017.00015PMC PMC5559442
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Abstract

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 understoodAnd 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 one day after ischemia onset from a) mice after filament-based permanent middle cerebral artery occlusion, b) rats subjected to thromboembolic middle cerebral artery occlusion 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. Subsequent semi-quantitative analyses in mice confirmed significantly decreased WFA-staining at the ischemic border zone and a relative decrease in the directly ischemia-affected neocortex. Triple fluorescence labeling throughout the three animal models revealed up-regulatedcollagen 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 as well as markers in net-bearing neurons such as the potassium channel subunit Kv3.1b and NeuN. In summary, the consistent 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.

AI snapshot

Written from the paper’s full text.

TL;DR

In three experimental focal cerebral ischemia models (mice, rats, sheep), ischemic neocortex showed up-regulated collagen IV and decomposed or erased perineuronal nets (PNs), alongside glial and neuronal changes. The authors conclude PNs are highly sensitive neurovascular unit constituents and potential future stroke-treatment targets.

Key findings

  • Across mouse, rat, and sheep models, ischemic neocortical areas displayed increased collagen IV immunoreactivity and decomposed/erased WFA-stained PNs; triple labeling associated these changes with activated astroglia, microglia/macrophages, and altered parvalbumin immunoreactivity.
  • In mice, semi-quantification showed significantly increased Coll IV integrated density from the second (7.1 × 10^11 ± 4.3 × 10^11) to third (13.1 × 10^11 ± 6.7 × 10^11) and fourth (24.2 × 10^11 ± 9.3 × 10^11) ROIs (each p = 0.043; Wilcoxon test, n = 5).
  • WFA staining in ischemic mouse cortex decreased significantly from the first (17.7 × 10^11 ± 11.6 × 10^11) to third (9.28 × 10^11 ± 10.7 × 10^11) ROI and then increased toward the fifth (17.5 × 10^11 ± 11.4 × 10^11) ROI (each p = 0.043); direct interhemispheric WFA comparison in regions 4–8 barely missed significance (each p = 0.080).
  • Ischemic rodent neocortex also showed less intense staining for aggrecan, neurocan, versican, cartilage link protein (CRTL), Kv3.1b, and NeuN, with only few or no PNs around parvalbumin-containing neurons.

Methods

  • Three focal cerebral ischemia models were used: filament-based permanent middle cerebral artery occlusion (pMCAO) in n = 5 C57BL/6 mice, thromboembolic MCAO in n = 3 Wistar rats, and electrosurgically induced pMCAO in three adult sheep; rodent tissue was analyzed 1 day after ischemia and sheep tissue 14 days after ischemia.
  • Multiple fluorescence labeling combined biotinylated WFA with antibodies against Coll IV, GFAP, Iba, parvalbumin, aggrecan, neurocan, versican, CRTL, Kv3.1b, and NeuN, followed by fluorescence and confocal microscopy.
  • Semi-quantification was performed in mice using eight equally spaced ROIs per hemisphere across three sections, measuring integrated density in ImageJ for Coll IV and WFA, with Wilcoxon tests and p < 0.05 considered significant.

Limitations

  • The paper states that sheep brains were not perfusion-fixed because of experimental constraints, and that limited rat and sheep tissue allowed only qualitative investigations in those species; semi-quantification was feasible only in mice based on n = 5.
  • The paper states that experiments were limited to a single time point (24 h in rodents, 14 days in sheep), so the longitudinal course and whether changes are primary or secondary remain unclear.
  • Evident from the text: rat and sheep sample sizes were very small (n = 3 each), and cross-species comparisons are complicated by different ischemia models, tissue processing, and post-ischemia intervals.

Model-written summary. Check it against the paper before relying on it.

Integrity signals

Registry

Crossref · 10.3389/fnint.2017.00015

Cites

94 of 94 references verified0 citation errors · 0 not checkable

Statistics

Nothing to recompute.

Claims the paper makes

0 of 74 cited sources checked · 0 supported · 0 flagged

  • Findingown data

    Triple fluorescence labeling across three animal models revealed decomposed PNs.

    “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.”
    Quote in the paperSubstantial claim
    • Rests on an assumption: Loss of WFA-staining and other marker changes reflect decomposition of PNs rather than reversible downregulation or masking of epitopes.
  • Findingown data

    Coll IV immunoreactivity on the non-affected hemisphere provided a consistent pattern with nearly equal integrated density values from medial to lateral.

    “Thereby, the Coll IV-immunoreactivity on the non-affected hemisphere was found to provide a consistent pattern with nearly equal values for the integrated density from medial to lateral (gray bars in Figure 3B).”
    Quote in the paperNot a substantial claim
    • Rests on an assumption: The contralateral hemisphere is non-affected and valid as an internal control for the ischemic hemisphere.
  • Findingown data

    WFA-binding sites showed a trend toward diminished relative labeling starting at the fourth ROI.

    “The pattern of WFA-binding sites provided—at least starting at the fourth ROI—a trend towards a diminished relative labeling.”
    Quote in the paperNot a substantial claim
    • The authors state this as an assumption: The medial-to-lateral increase in WFA-staining in the non-ischemic hemisphere represents the natural course in this neocortical region.
  • Findingown data

    Triple fluorescence labeling across three animal models revealed up-regulated collagen IV.

    “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.”
    Quote in the paperSubstantial claim
  • Findingown data

    Direct inter-hemispheric comparison per ROI barely missed statistical significance for regions 4-8 (each p = 0.080; Wilcoxon test, n = 5).

    “The direct inter-hemispheric comparison on the level of each ROI, however, barely missed statistical significance (regions 4–8, each p = 0.080; Wilcoxon test, n = 5).”
    Quote in the paperNot a substantial claim
  • May combine two claims. 29
  • Classified as probably not a substantial claim. 37

  • May combine two claims.

    Findingown data

    In all three species investigated, nearly complementary distribution patterns for WFA and massively enhanced Coll IV immunoreactivity were found.

    “Thereby, nearly complementary distribution patterns for WFA and massively enhanced Coll IV-immunoreactivity was found in all three species investigated (Figures 4A, 5A, 6A,A′).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    A considerable loss of WFA-stainable ECM and decomposed PNs were noted in whole forebrain sections including affected somatosensory cortex and dorsal striatum, as well as visual and auditory cortical areas, hippocampal formation and pontine gray matter.

    “A considerable loss of WFA-stainable ECM and decomposed PNs were noted as exemplified by images from whole forebrain sections including affected somatosensory cortex and dorsal striatum (Figure 1A), as well as visual and auditory cortical areas, hippocampal formation and pontine gray matter (Figure 1B).”
    Quote in the paperSubstantial claim
    • Rests on an assumption: Loss of WFA-staining and other marker changes reflect decomposition of PNs rather than reversible downregulation or masking of epitopes.

  • May combine two claims.

    Findingown data

    Coll IV immunofluorescence is significantly up-regulated in the ischemia-affected hemisphere compared with the contralateral hemisphere, corresponding with relatively reduced WFA signal from medial to lateral neocortex.

    “shows a significant up-regulation of the Coll IV-fluorescence signal that corresponds with a relatively reduced lectin-histochemical signal for WFA alongside from the medial to the lateral part of the neocortex.”
    Quote in the paperSubstantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Findingown data

    Morphological changes of PN structures became visible at high magnification by confocal laser-scanning microscopy.

    “Morphological changes of PN structures became visible at high magnification by confocal laser-scanning microscopy.”
    Quote in the paperNot a substantial claim
    • Rests on an assumption: Loss of WFA-staining and other marker changes reflect decomposition of PNs rather than reversible downregulation or masking of epitopes.

  • May combine two claims.

    Findingown data

    In the contralateral (non-ischemic) hemisphere, WFA-binding significantly increases from the second to the third ROI and from the fourth to the fifth ROI (each p = 0.043; Wilcoxon test, n = 5).

    “Thereby, a significant increase occurred from the second (19.2 × 1011 ± 11.0 × 1011) to the third (27.3 × 1011 ± 15.8 × 1011) as well as between the fourth (34.8 × 1011 ± 18.2 × 1011) to the fifth (45.7 × 1011 ± 20.6 × 1011) ROI (each p = 0.043; Wilcoxon test, n = 5).”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingown data

    On the ischemia-affected hemisphere, WFA-binding significantly decreases from the first to the third ROI and then increases toward the fifth ROI (each p = 0.043; Wilcoxon test, n = 5).

    “On the ischemia-affected hemisphere (white bars in Figure 3C), WFA-binding exhibited a shaped course in the bordering zone of ischemia by decreasing significantly from the first (17.7 × 1011 ± 11.6 × 1011) to the third (9.28 × 1011 ± 10.7 × 1011), followed by an increase towards the fifth (17.5 × 1011 ± 11.4 × 1011) ROIs (each p = 0.043; Wilcoxon test, n = 5).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    In sheep 14 days after infarction, strongly Iba-labeled microglia/macrophages are observed in the Coll IV-stained region, contrasted by ameboid and ramified microglial cells near PNs.

    “However, in the representative image from the ischemic border zone of the sheep 14 days after infarction (Figure 6A), strongly Iba-labeled microglia/macrophages can be observed in the Coll IV-stained region, contrasted by ameboid and ramified microglial cells in vicinity to PNs.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingown data

    The ischemia-affected region contained structures with diminished NeuN immunoreactivity without an immunosignal for Kv3.1b, and only parvalbumin-stained coarse neuropil as well as a few remnants of shrunken neurons.

    “On the contrary, the ischemia-affected region contained structures with diminished NeuN-immunoreactivity (Figures 9B″,C′) without an immunosignal for Kv3.1b (Figure 9B‴), and only parvalbumin-stained coarse neuropil as well as a few remnants of shrunken neurons (Figure 9C″).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    In the same area, WFA-stained nets appeared decomposed or were even abolished.

    “whereas in the same area WFA-stained nets appeared decomposed (Figures 4C′, 6C′) or were even abolished (Figure 5C′).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Fluorescence labeling verified the occurrence of PNs double-positive for WFA and aggrecan or only for one of both markers.

    “The fluorescence labeling also verified the occurrence of PNs double-positive for WFA and aggrecan or only for one of both markers.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingown data

    In rat, parvalbumin immunolabeling was severely affected, only a few apparently intact PNs surrounded cells with detectable calcium-binding protein remained, and the neuropil appeared coarse.

    “Concomitantly, parvalbumin-immunolabeling (Figure 7B″) was severely affected, while only a few apparently intact PNs surrounded cells with detectable calcium-binding protein remained and the neuropil appeared again coarse.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    In mouse neocortex, healthy tissue had evenly distributed PNs, while ischemia-affected areas were either devoid of WFA-labeled structures or exhibited only some remnants of nets.

    “In contrast to the rather evenly distributed PNs in the healthy tissue, the ischemia-affected areas were either devoid of WFA-labeled structures (Figure 9A) or exhibited only some remnants of nets (Figures 9B,C).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    In mice, parvalbumin in affected tissue was diminished in cells and in the neuropil with a coarser appearance than in healthy tissue.

    “After merging of staining patterns for PNs and parvalbumin, the calcium-binding protein was also observed in affected tissue, i.e., diminished in cells and in the neuropil with a coarser appearance than in healthy tissue (Figure 7A″).”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Findingown data

    Merge of staining patterns visualized PNs double-labeled by WFA and anti-aggrecan.

    “As expected, the merge of staining patterns also visualized PNs double-labeled by WFA and anti-aggrecan (Figure 7B″).”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingown data

    Overlay elucidated triple-stained nets, numerous WFA/aggrecan co-labeled nets, and nets mono-labeled by WFA.

    “The overlay (Figures 8A‴,B‴,C‴) elucidated triple-stained nets (arrows), numerous WFA/aggrecan co-labeled nets (arrowheads) and nets mono-labeled by WFA (two-headed arrow).”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingown data

    Parvalbumin immunolabeling detected shrunken cells and, especially in the rat, enhanced labeling of neuropil in the same area.

    “Parvalbumin-immunolabeling (Figures 4C″, 5C″, 6C″) detected shrunken cells and especially in the rat (Figure 5C″) combined with enhanced labeling of neuropil in the same area.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingown data

    In mice, the ischemia-affected neocortical area had lowered lectin-staining of the neuropil and was devoid of PNs detected by WFA and aggrecan.

    “Thereby, mice displayed a neocortical ischemia-affected area with lowered lectin-staining of the neuropil and devoid of PNs detected by WFA (Figure 7A) and aggrecan (Figure 7A′).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    In the ischemia-affected zone, merged staining patterns revealed only a few PNs around parvalbumin-containing neurons or even the absence of such net-enwrapped neurons.

    “In the ischemia-affected zone, merged staining patterns revealed only a few PNs around parvalbumin-containing neurons (Figure 4C‴) or even the absence of such net-enwrapped neurons (Figures 5C‴, 6C‴)”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Overlay visualized co-localized WFA-binding sites and aggrecan immunoreactivity as well as parvalbumin-containing cells devoid of PNs.

    “The overlay further visualized co-localized WFA-binding sites and aggrecan-immunoreactivity as well as parvalbumin-containing cells devoid of PNs.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Premiseown data

    Previous histochemical analyses after experimental stroke in mice revealed significant alterations of vascular components, microtubule-associated protein tau, and vesicular neurotransmitter transporters.

    “Based on the NVU concept, our own histochemical analyses after experimental stroke in mice revealed significant alterations of vascular components (Hawkes et al., 2013; Krueger et al., 2015), microtubule-associated protein tau (Michalski et al., 2016) and vesicular neurotransmitter transporters (Michalski et al., 2013).”
    Quote in the paperNot a substantial claim0 of 3 sources checked
    • cites Hawkes et al., 2013

      Not checked in the free run.

    • cites Krueger et al., 2015

      Not checked in the free run.

    • cites Michalski et al., 2016

      Not checked in the free run.

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    Karetko-Sysa et al. (2011) reported disturbance of PNs after focal photothrombotic stroke.

    “Additionally, Karetko-Sysa et al. (2011) reported the disturbance of PNs after focal photothrombotic stroke.”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites Karetko-Sysa et al., 2011

      Not checked in the free run.

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Study design and conductown data

    Mice and rats were randomly included in the study.

    “The study was carried out with n = 5 C57BL/6 mice of about 25 g body weight and n = 3 Wistar rats of about 300 g body weight (both breed by Charles River, Sulzfeld, Germany), which were randomly included, and with three male adult sheep from the nucleus breed of the educational manor (Faculty for Veterinary Medicine, University of Leipzig).”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    The NVU concept also involves the adjacent extracellular matrix (ECM), which is known to critically impact a variety of cellular functions.

    “Of note, this concept also involves the adjacent extracellular matrix (ECM) which is known to critically impact on a variety of cellular functions (del Zoppo, 2009).”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites del Zoppo, 2009

      Not checked in the free run.

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    Ischemia-induced damage of PNs in rat neocortices was analyzed by Bidmon et al. (1998) and by Wieloch's research group using a similar animal model.

    “Using a similar animal model, ischemia-induced damage of PNs in rat neocortices was analyzed by Bidmon et al. (1998) and by Wieloch’s research group (Madinier et al., 2014; Quattromani et al., 2017).”
    Quote in the paperNot a substantial claim0 of 3 sources checked
    • cites Bidmon et al., 1998

      Not checked in the free run.

    • cites Madinier et al., 2014 source

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    • cites Quattromani et al., 2017 source

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  • Classified as probably not a substantial claim.

    Study design and conductnone given

    Thresholds were adjusted individually for each staining until background intensity could be nearly excluded without affecting visualization of targeted structures.

    “Thresholds had been adjusted individually for each staining until the background intensity could be nearly excluded without affecting the visualization of targeted structures to prevent adulterations of the applied measurements.”
    Quote in the paperSubstantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    Perineuronal nets (PNs) are polyanionic, chondroitin sulfate proteoglycan (CSPG)-rich coatings of certain neurons.

    “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.”
    • Also stated in: § Introduction · plain
      “PNs represent polyanionic lattice-like coatings of neurons (Brückner et al., 1993; Celio and Blümcke, 1994; Oohashi et al., 2015; Sorg et al., 2016).”
    Quote in the paperNot a substantial claim0 of 3 sources checked
    • cites Brückner et al., 1993 source

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    • cites Celio and Blümcke, 1994

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    • cites Oohashi et al., 2015 source

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  • Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

    Premisecitation

    PNs frequently surround neurons containing parvalbumin, which are fast-firing GABAergic neurons co-expressing the voltage-gated potassium channel Kv3.1b.

    “PNs frequently surround neurons containing parvalbumin (Kosaka and Heizmann, 1989; Härtig et al., 1992), known as a marker for fast-firing GABAergic neurons co-expressing the voltage-gated potassium channel Kv3.1b (Härtig et al., 1999).”
    Quote in the paperNot a substantial claim0 of 3 sources checked
    • cites Kosaka and Heizmann, 1989

      Not checked in the free run.

    • cites Härtig et al., 1992 source

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    • cites Härtig et al., 1999 source

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  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    PNs are composed of chondroitin sulfate proteoglycans (CSPGs) and their glycosaminoglycans, hyaluronic acid and tenascins, produced by various neural cell types.

    “They are composed of chondroitin sulfate proteoglycans (CSPGs) and their glycosaminoglycans, hyaluronic acid and tenascins, produced by various neural cell types (Brückner et al., 1993).”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites Brückner et al., 1993 source

      Not checked in the free run.

  • Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

    Study design and conductnone given

    Animal experiments were performed according to the European Union Directive 2010/63/EU and the protocol was approved by the local authority.

    “Animal experiments were performed according to the European Union Directive 2010/63/EU and the protocol was approved by the local authority (Regierungspräsidium Leipzig; reference numbers: TVV 51/14 for mice, TVV 34/11 for rats and TVV 56/15 for sheep).”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Interpretationown data

    Consistent observations based on three different stroke models confirmed that PNs are highly sensitive constituents of the NVU along with impaired associated GABAergic neurons.

    “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.”
    Quote in the paperSubstantial claim
    • Rests on an assumption: The three stroke models and their different survival times are comparable enough to support a common conclusion about PN sensitivity.
    • Rests on an assumption: Loss of WFA-staining and other marker changes reflect decomposition of PNs rather than reversible downregulation or masking of epitopes.

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Study design and conductnone given

    Control experiments omitting primary antibodies or biotinylated WFA resulted in the expected absence of cellular labeling.

    “In control experiments, the omission of primary antibodies or biotinylated WFA resulted in the expected absence of cellular labeling.”
    Quote in the paperNot a substantial claim

  • Classified as probably not a substantial claim. May combine two claims.

    Premisecitation

    The type of ischemia induction and the selection of a single stroke model were discussed as reasons for the translational roadblock.

    “Notably, as another reason for the translational roadblock the type of ischemia induction, i.e., the widely used artificial stroke models (Young et al., 2007; Sommer, 2017) and the selection of a single stroke model (Fisher et al., 2009) were discussed.”
    Quote in the paperSubstantial claim0 of 3 sources checked
    • cites Young et al., 2007

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    • cites Sommer, 2017

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    • cites Fisher et al., 2009

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  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Study design and conductnone given

    Brightness and contrast of micrographs were slightly adjusted without deletion or creation of signals.

    “If necessary, brightness and contrast of micrographs were slightly adjusted without deletion or creation of signals.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    The neurovascular unit (NVU) concept comprises ischemia-affected neurons as well as vessels, micro-, astro- and oligodendroglia.

    “In this view, the concept of the “neurovascular unit” (NVU) as outlined by del Zoppo (2009, 2010) comprised ischemia-affected neurons as well as vessels, micro-, astro- and oligodendroglia.”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites del Zoppo (2009, 2010)

      Not checked in the free run.

  • Classified as probably not a substantial claim.

    Study design and conductreasoning

    For all fluorescence measurements, the threshold was adjusted to the contralateral hemisphere and kept constant at gray values of 1000 for WFA and 1250 for Coll IV to correct for background intensities.

    “For all measurements, the threshold was adjusted to the contralateral hemisphere and kept constant at gray values of 1000 for WFA and 1250 for Coll IV to correct for any background intensities relating to non-labeled areas, such as the lumen of cross-sectioned vessels.”
    Quote in the paperSubstantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    Chondroitinase ABC (ChABC) is an established tool for remodeling the extracellular matrix and especially perineuronal nets in vivo and under pathological conditions.

    “An established tool for the remodeling the ECM and especially PNs in vivo and even under pathological conditions are bacterial glycosaminoglycan-degrading enzymes such as chondroitinase ABC (ChABC) as reviewed by Burnside and Bradbury (2014).”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites Burnside and Bradbury (2014)

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  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    Such enzymes were observed in net-ensheathed parvalbumin-immunoreactive neurons.

    “Interestingly, such enzymes were observed in net-ensheathed parvalbumin-immunoreactive neurons (Rossier et al., 2015).”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites Rossier et al., 2015 source

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  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    The same enzymes later contribute to beneficial mechanisms of neurovascular remodeling.

    “However, the same enzymes later contribute to beneficial mechanisms of neurovascular remodeling (Lo, 2008; Rosell and Lo, 2008; del Zoppo, 2010)”
    Quote in the paperNot a substantial claim0 of 3 sources checked
    • cites Lo, 2008

      Not checked in the free run.

    • cites Rosell and Lo, 2008 source

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    • cites del Zoppo, 2010

      Not checked in the free run.

  • May combine two claims.

    Premisecitation

    The filament model in mice allows non-invasive induction of focal cerebral ischemia and leads to reproducible infarction in the middle cerebral artery territory, enabling robust quantifications on cellular reactions.

    “Thereby, the filament model in mice (Longa et al., 1989) allowed non-invasive induction of focal cerebral ischemia and led to reproducible infarction in the territory of the middle cerebral artery (Longa et al., 1989; Durukan and Tatlisumak, 2007; Engel et al., 2011), enabling robust quantifications on cellular reactions.”
    Quote in the paperSubstantial claim0 of 3 sources checked
    • cites Longa et al., 1989 source

      Not checked in the free run.

    • cites Durukan and Tatlisumak, 2007

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    • cites Engel et al., 2011

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  • May combine two claims.

    Premisecitation

    The applied thromboembolic rat model is currently considered the most patient-near animal model because the ischemic event is strictly caused by an embolus, matching the human situation where cardio-embolism and arterial embolism are the most frequent causes of stroke.

    “The applied thromboembolic rat model is currently considered as the most patient-near animal model, since the ischemic event is strictly caused by an embolus in excellent accordance to the human situation where cardio-embolism due to atrial fibrillation and arterial embolism—based on arteriosclerosis or carotid stenosis—accounts for the most frequent causes of stroke (Durukan and Tatlisumak, 2007;”
    Quote in the paperSubstantial claim0 of 3 sources checked
    • cites Durukan and Tatlisumak, 2007

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    • cites Young et al., 2007

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    • cites Sommer, 2017

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  • Classified as probably not a substantial claim.

    Premisecitation

    Current recommendations state that implementation of gyrencephalic, large animal models in preclinical stroke research is desirable.

    “According to current recommendations for improving experimental stroke research (Stroke Therapy Academic Industry Roundtable (STAIR), 1999; Fisher et al., 2009), an implementation of gyrencephalic, large animal models in preclinical stroke research is desirable.”
    Quote in the paperSubstantial claim0 of 2 sources checked
    • cites Stroke Therapy Academic Industry Roundtable (STAIR), 1999

      Not checked in the free run.

    • cites Fisher et al., 2009

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  • Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

    Premisecitation

    Immediately after stroke, matrix metalloproteinases become dysregulated and are associated with acute neurovascular disruption.

    “Immediately after stroke, matrix metalloproteinases are known to become dysregulated (Heo et al., 1999) and associated with acute neurovascular disruption.”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites Heo et al., 1999

      Not checked in the free run.

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    Injections of ChABC into the rat frontal or occipital cortex resulted in the erasure of perineuronal nets within 1 day.

    “Injections of ChABC into the rat frontal or occipital cortex resulted in the erasure of PNs within 1 day followed by the significant re-establishment of matrix components only after 4 weeks and the rebuilding of PNs only after 5 months (Brückner et al., 1998).”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites Brückner et al., 1998

      Not checked in the free run.

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Interpretationcitation

    The robustly observed up-regulation of Coll IV across the three animal models might be seen in the context of matrix metalloproteinases.

    “the robustly observed up-regulation of Coll IV across the three animal models might be seen in the context of these mediators.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Interpretationown data

    This finding is in line with the recently reported heterogeneity of PNs in the mouse hippocampus.

    “This finding is in line with the recently reported heterogeneity of PNs in the mouse hippocampus (Yamada and Jinno, 2017).”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites Yamada and Jinno, 2017

      Not checked in the free run.

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    Aggrecanases (ADAMTS-1, 4, 8, 15) and neprilysin are further candidate enzymes for stroke-induced degradation of net components.

    “Further candidates for the stroke-induced degradation of net components are aggrecanases, e.g., disintegrin and metalloproteinase with thrompospondin motifs (ADAMTS-1, 4, 8 and 15) and neprilysin (Cross et al., 2006; Lemarchant et al., 2013; Levy et al., 2015; Rossier et al., 2015).”
    Quote in the paperNot a substantial claim0 of 3 sources checked
    • cites Cross et al., 2006

      Not checked in the free run.

    • cites Lemarchant et al., 2013 source

      Not checked in the free run.

    • cites Levy et al., 2015

      Not checked in the free run.

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Interpretationcitation

    Removal of PN coatings might allow formerly net-enwrapped parvalbumin-containing neurons to re-open a more plastic state.

    “The removal of their coatings might allow formerly net-enwrapped parvalbumin-containing neurons to re-open a more plastic state (Hensch, 2014).”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites Hensch, 2014

      Not checked in the free run.

  • Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

    Premisecitation

    Matrix metalloproteinases influence vascular integrity and the ischemic stimulus promotes production of matrix metalloproteinases.

    “as matrix metalloproteinases have been considered to influence the vascular integrity and the ischemic stimulus is known to promote the production of matrix metalloproteinases (del Zoppo, 2009, 2010)”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites del Zoppo, 2009, 2010

      Not checked in the free run.

  • Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

    Premisecitation

    Aggrecan is known as the predominant CSPG in PNs, occurring in several isoforms with differential distribution as shown for the human cerebral cortex.

    “aggrecan known as predominant CSPG in PNs occurring in several isoforms (Matthews et al., 2002; Morawski et al., 2012a) with differential distribution as shown for the human cerebral cortex (Virgintino et al., 2009).”
    Quote in the paperNot a substantial claim0 of 3 sources checked
    • cites Matthews et al., 2002 source

      Not checked in the free run.

    • cites Morawski et al., 2012a

      Not checked in the free run.

    • cites Virgintino et al., 2009

      Not checked in the free run.

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    Enhanced levels of synaptophysin and growth-associated protein 43 indicate augmented axonal growth and synaptic plasticity.

    “After thalamic stroke in hypertensive rats, the continuous intra-infarct infusion of ChABC rescued neuronal loss and enhanced levels of synaptophysin and growth-associated protein 43 indicating augmented axonal growth as well as synaptic plasticity to finally ensure overall neuronal function (Chen et al., 2014).”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites Chen et al., 2014

      Not checked in the free run.

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Study design and conductreasoning

    Sheep brains were not perfusion-fixed due to experimental constraints of the underlying sheep studies.

    “In detail, sheep brains were not perfusion-fixed, which was due to the experimental constraints of the underlying sheep studies.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    ChABC injections into perirhinal cortices from transgenic mice with tau pathology restored object recognition memory.

    “Remarkably, ChABC injections into perirhinal cortices from transgenic mice with tau pathology restored even the object recognition memory of these animals (Yang et al., 2015).”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites Yang et al., 2015 source

      Not checked in the free run.

  • Classified as probably not a substantial claim.

    Premisecitation

    Post-stroke reactions have been described in a multi-phasic fashion.

    “Given the fact that post-stroke reactions were described in a multi-phasic fashion (Dirnagl et al., 1999), additional work is necessary to explore the longitudinal course of addressed markers.”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites Dirnagl et al., 1999

      Not checked in the free run.

  • May combine two claims.

    Premisecitation

    Following experimentally induced stroke in aged rats, timely fine-tuned delayed treatment with ChABC promoted functional recovery and neuroplasticity.

    “Following experimentally induced stroke in aged rats, the timely fine-tuned delayed treatment with ChABC promoted functional recovery and neuroplasticity, but did not protect the peri-infarct region (Soleman et al., 2012).”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites Soleman et al., 2012

      Not checked in the free run.

  • Classified as probably not a substantial claim.

    Study design and conductreasoning

    Frozen tissue sections from these blocs allowed for the detection of perineuronal nets and multiple vascular, glial and neuronal markers.

    “Consequently, frozen tissue sections from these blocs allowed for the detection of PNs and multiple vascular, glial and neuronal markers.”
    Quote in the paperSubstantial claim

  • Classified as probably not a substantial claim. May combine two claims.

    Study design and conductnone given

    Experiments were limited to a single time point: 24 h after ischemia induction in rodents, and 14 days after ischemia induction in sheep.

    “It is also noteworthy that experiments were limited to a single time point (i.e., 24 h after ischemia induction in rodents, and 14 days after ischemia induction in sheep).”
    Quote in the paperSubstantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    ChABC-induced removal of perineuronal nets in the medial prefrontal cortex impaired acquisition and reconsolidation of conditioned place preference memory after its impairment by cocaine.

    “Moreover, the ChABC-induced removal of PNs in the medial prefrontal cortex impaired acquisition and reconsolidation of conditioned place preference memory after its impairment by cocaine (Slaker et al., 2015).”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites Slaker et al., 2015 source

      Not checked in the free run.

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    ChABC-induced removal of negatively charged extracellular matrix components such as glycosaminoglycans resulted in enhanced calcium diffusion within brain tissue.

    “Notably, the ChABC-induced removal of negatively charged ECM components such as glycosaminoglycans resulted in enhanced calcium diffusion within brain tissue which influences synaptic transmission, neuronal excitability and other physiological processes (Hrabětová et al., 2009).”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites Hrabětová et al., 2009 source

      Not checked in the free run.

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    Enhanced calcium diffusion within brain tissue influences synaptic transmission, neuronal excitability and other physiological processes.

    “Notably, the ChABC-induced removal of negatively charged ECM components such as glycosaminoglycans resulted in enhanced calcium diffusion within brain tissue which influences synaptic transmission, neuronal excitability and other physiological processes (Hrabětová et al., 2009).”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites Hrabětová et al., 2009 source

      Not checked in the free run.

59 of 59

  • The paper relies on this without support.

    The contralateral hemisphere is non-affected and valid as an internal control for the ischemic hemisphere.

    “For controls, the pattern of the eight ROIs in the ischemic hemisphere was mirrored to the contralateral, non-affected hemisphere, resulting in 16 ROIs in each of the three brain sections per animal.”
    “To consider the natural patterns of PN components, the mirrored ROIs on the contralateral hemisphere served as controls (Figure 3A).”

  • The paper relies on this without support.

    The three stroke models and their different survival times are comparable enough to support a common conclusion about PN sensitivity.

    “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.”
    “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.”

  • The paper relies on this without support.

    Loss of WFA-staining and other marker changes reflect decomposition of PNs rather than reversible downregulation or masking of epitopes.

    “A considerable loss of WFA-stainable ECM and decomposed PNs were noted as exemplified by images from whole forebrain sections including affected somatosensory cortex and dorsal striatum (Figure 1A), as well as visual and auditory cortical areas, hippocampal formation and pontine gray matter (Figure 1B).”
    “Morphological changes of PN structures became visible at high magnification by confocal laser-scanning microscopy.”

  • The authors state this as an assumption.

    The medial-to-lateral increase in WFA-staining in the non-ischemic hemisphere represents the natural course in this neocortical region.

    “which is assumed to represent the natural course in this neocortical region.”
4 of 4

Claims other papers make about this one

Each is checked against this paper’s own text: does it say what the citing paper says it does?

  • Similar degradation of WFA-expressing PNNs in the neocortex has also been observed following thromboembolic middle cerebral artery occlusion in rats, permanent middle artery occlusion in mice, and focal cerebral ischemia in sheep.

    “Similar degradation of WFA-expressing PNNs in the neocortex has also been observed following thromboembolic middle cerebral artery occlusion in rats, permanent middle artery occlusion in mice, and focal cerebral ischemia in sheep (Härtig et al., 2017).”

    From The Perineuronal ‘Safety’ Net? Perineuronal Net Abnormalities in Neurological Disorders (2018) · cites this paper as Härtig et al., 2017

Self-check

Percentages, sums and reported differences re-computed from the paper’s own numbers.

Each contradiction between two passages listed here was confirmed by an independent check.

  • Figure 8 is assigned to both mouse and rat tissue

    Minor

    The results describe the sections shown in Figure 8 as mouse sections, but the Figure 8 caption identifies the tissue as rat neocortex.

  • Figure 5C′′′ is described as showing both absent and remaining net-enwrapped neurons

    Minor

    The results cite Figure 5C′′′ as showing an absence of net-enwrapped parvalbumin-containing neurons in the affected zone, while its caption says a few PNs remain around those neurons.

2 of 2

Cites

Flagged first, then by citations

Research works printed but not linked: 2

  1. Menzies, 1992, Middle cerebral artery occlusion in rats: a neurological and pathological evaluation of a reproducible model, Neurosurgery, 31, 100, 10.1097/00006123-199207000-00014 · DOI · Paper page
  2. Morawski, 2010, Perineuronal nets are largely unaffected in Alzheimer model Tg2576 mice, Neurobiol. Aging, 31, 1254, 10.1016/j.neurobiolaging.2008.07.023 · DOI

Cited by

Sorted by citations

Authors
Härtig, W., Mages, B., Aleithe, S., Nitzsche, B., Altmann, S., Barthel, H., Krueger, M., & Michalski, D.
Affiliations
Leipzig University (DE)
Corresponding author
Härtig, Wolfgang
ORCID
Venue
Frontiers in Integrative Neuroscience
Publisher
Frontiers Media SA
Published
2017-08-15
Volume
11
Pages
15-15
ISSN
1662-5145 (ISSN-L)
DOI
10.3389/fnint.2017.00015
PMC
PMC5559442
OpenAlex
W2748143122
Cited by
53
References
94
Citations by year
2026: 2 · 2025: 2 · 2024: 7 · 2023: 5 · 2022: 9 · 2021: 7 · 2020: 10 · 2019: 3 · 2018: 8
FWCI
2.86
Type
journal article
Language
en
Open access
Licence
cc-by
Locations
  • Frontiers in Integrative Neuroscience (publishedVersion, cc-by)
  • PubMed Central (publishedVersion, cc-by)
  • PubMed (publishedVersion)
  • DOAJ (DOAJ: Directory of Open Access Journals) (submittedVersion, cc-by-sa)
  • Qucosa (Saxon State and University Library Dresden) (submittedVersion, cc-by)
Funders
  • European Social Fund (100270131)
  • Deutsche Forschungsgemeinschaft (BA 3425/2-3)
Topics
  • Life Sciences › Neuroscience › Neurology › Barrier Structure and Function Studies
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Cell Biology › Proteoglycans and glycosaminoglycans research
  • Life Sciences › Neuroscience › Neurology › Neuroinflammation and Neurodegeneration Mechanisms
Keywords
perineuronal nets, focal cerebral ischemia, ischemic stroke, neurovascular unit, extracellular matrix, chondroitin sulfate proteoglycans, type IV collagen, astroglial activation, parvalbumin, GABAergic neurons, aggrecan, neurocan, versican, middle cerebral artery occlusion, stroke model

About these checks

Registry, retraction, Cites and statistics results are computed, not judged, with one exception: a printed reference that no index lists is read once by a model before it counts as a citation error. Claims are extracted by a model and kept only when their quote appears verbatim in the text; each cited source is then checked, with a model’s help, for whether it says what the claim relies on. Those verdicts can be wrong — read the claim’s quote and the cited source before relying on one.

Claims read by deepseek-v4.1-flash under definition version 3 (extract-v3) on 2026-10-06.

Classified by jev-1.13-20260917 (gold-guideline-v0.2) on 2026-10-06.

Statistics and arithmetic are recomputed, not judged.

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