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Cited by 413Genomics, phytochemicals, and oxidative stressAcute Ischemic Stroke ManagementNeurological Disease Mechanisms and Treatments

A Small-Molecule-Inducible Nrf2-Mediated Antioxidant Response Provides Effective Prophylaxis against Cerebral Ischemia In Vivo

Open access
Shih, Andy Y., Li, Ping, Murphy, Timothy H.·2005·The Journal of Neuroscience
DOI 10.1523/jneurosci.4014-05.2005PMC PMC6725780
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Abstract

The transcription factor nuclear factor erythroid 2-related factor 2 (Nrf2) coordinates expression of genes required for free radical scavenging, detoxification of xenobiotics, and maintenance of redox potential. Previously, activation of this pleiotropic response was neuroprotective in cell culture models that simulate components of stroke damage. However, the role of Nrf2 in limiting stroke damage in vivo remained unclear. We report that Nrf2 activation protects the brain from cerebral ischemia in vivo. Acute (1-3 d) intracerebroventricular or intraperitoneal pretreatment with tert-butylhydroquinone (tBHQ), an Nrf2 activity inducer, reduced cortical damage and sensorimotor deficit at 24 h and even 1 month after ischemia-reperfusion in rats. Cortical glutathione levels robustly increased with tBHQ administration to rats and Nrf2-expressing mice, but not Nrf2-/-mice. Basal and inducible activities of antioxidant/detoxification enzymes in Nrf2-/-mice were reduced when compared with Nrf2+/+controls. Interestingly, larger infarcts were observed in Nrf2-/-mice at 7 d after stroke, but not at 24 h, suggesting that Nrf2 may play a role in shaping the penumbra well after the onset of ischemia. Neuronal death caused by a “penumbral” model of stroke, using intracortical endothelin-1 microinjection, was attenuated by tBHQ administration to Nrf2+/+, but not to Nrf2-/-mice, confirming the Nrf2-specific action of tBHQ in vivo. We conclude that Nrf2 plays a role in modulating ischemic injury in vivo. Accordingly, Nrf2 activation by small molecule inducers may be a practical preventative treatment for stroke-prone patients.

AI snapshot

Written from the paper’s full text.

TL;DR

Prophylactic tBHQ activates Nrf2-dependent antioxidant/detoxification enzymes and reduces cortical ischemic damage and sensorimotor deficits after ischemia-reperfusion in rats, with benefit lasting up to 1 month. Nrf2-knockout mice show larger delayed infarcts and fail to respond to tBHQ in a penumbral endothelin-1 stroke model.

Key findings

  • Intracerebroventricular tBHQ (1 mM, 1 µl/h for 72 h) before transient MCAO reduced cortical infarct volume as a percentage of total ischemic hemisphere at 24 h: vehicle 9.2 ± 2.9 vs tBHQ 1.1 ± 0.7 (p = 0.008); no significant subcortical protection was observed, and sensorimotor deficit was lower in tBHQ-treated rats at 24 h.
  • Intraperitoneal tBHQ (16.7 mg/kg, three injections 8 h apart) reduced cortical infarct at 24 h: vehicle 14.76 ± 3.83 vs tBHQ 7.50 ± 3.04 (*p < 0.05), and at 1 month: vehicle 24.9 ± 3.9 vs tBHQ 10.1 ± 4.5 (*p < 0.05); neurological scores were lower in tBHQ-treated animals throughout the survival period (p = 0.002).
  • tBHQ increased cortical GSH in rats and Nrf2-expressing mice but not Nrf2−/− mice; Nrf2−/− mice had reduced basal GST and NQO1 activity in multiple brain regions and liver, while basal brain GSH was not altered.
  • Nrf2−/− mice had larger infarcts at 7 d after permanent distal MCA occlusion (approximately twofold) but not at 24 h; in the intracortical endothelin-1 model, tBHQ reduced infarct size by 66.1 ± 15.5% in Nrf2+/+ mice versus control diet (*p < 0.05) but did not reduce infarct size in Nrf2−/− mice.

Methods

  • In vitro: astrocyte-enriched cultures from postnatal rats or Nrf2−/− mice were used for ARE reporter assays, GSH/GST/NQO1/LDH enzyme assays, adenoviral Nrf2 overexpression, and oxidative glutamate toxicity with MTT viability.
  • In vivo rats: Wistar or Sprague Dawley rats underwent 1.5 h transient middle cerebral artery occlusion; tBHQ was delivered by intracerebroventricular osmotic pump or intraperitoneal injection, with sensorimotor scoring and TTC or cresyl violet infarct measurement.
  • In vivo mice: Nrf2+/+, Nrf2+/−, and Nrf2−/− mice received permanent distal MCA cauterization or intracortical endothelin-1 microinjection; tBHQ was given as 1% dietary supplementation, with laser Doppler flow, TTC/cresyl violet, and FluoroJade assessment. Statistical analyses used means ± SEM, two-tailed t tests, one-way ANOVA with Bonferroni post hoc, or two-way ANOVA.

Limitations

  • Stated: Nrf2−/− mice were less tolerant to tBHQ feeding, consuming about half as much food and losing ~20% body weight, suggesting possible systemic toxicity; the authors note no evidence of tBHQ brain toxicity in Nrf2−/− mice based on FluoroJade, LDH, and antioxidant enzyme measures.
  • Stated: Nrf2−/− mice may have reduced sensitivity to the endothelin-1 model, possibly because Nrf2 overexpression upregulated the endothelin B receptor ~30-fold in astrocytes, which could alter ET-1 signaling.
  • Stated/evident: tBHQ did not protect against permanent focal ischemia in Nrf2-expressing mice, a negative result the authors say fails to directly link Nrf2 and tBHQ in protection against stroke damage in mice; intraperitoneal or dietary tBHQ also did not produce detectable cortical GST or NQO1 induction, possibly due to longer time requirements or dilution from restricted cell-type induction.

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

Integrity signals

Registry

Crossref · 10.1523/jneurosci.4014-05.2005

Cites

116 of 116 references verified0 citation errors · 0 not checkable

Statistics

Nothing to recompute.

Claims the paper makes

0 of 57 cited sources checked · 0 supported · 0 flagged

  • Findingown data

    Induction of GSH, GST, and NQO1 markers could be rescued by Nrf2 overexpression in Nrf2-/- astrocytes.

    “Importantly, induction of these markers could be rescued by Nrf2 overexpression (Fig. 1 E).”
    Quote in the paperSubstantial claim
  • Findingcitation

    In cultured astrocytes, tBHQ treatment induced GSH, GST, and NQO1 within 10-24 h.

    “Consistent with other reports, all three markers were induced in astrocytes within 10 -24 h in response to tBHQ treatment (Fig. 1 A) (Ahlgren-Beckendorf et al., 1999;Lee et al., 2003a;Kraft et al., 2004).”
    Quote in the paperSubstantial claim0 of 3 sources checked
    • cites Ahlgren-Beckendorf et al., 1999

      Not checked in the free run.

    • cites Lee et al., 2003a

      Not checked in the free run.

    • cites Kraft et al., 2004

      Not checked in the free run.

  • Findingown data

    tBHQ pretreatment reduced sensorimotor deficit at 24 h after ischemia-reperfusion in rats.

    “Acute (1-3 d) intracerebroventricular or intraperitoneal pretreatment with tert-butylhydroquinone (tBHQ), an Nrf2 activity inducer, reduced cortical damage and sensorimotor deficit at 24 h and even 1 month after ischemia-reperfusion in rats.”
    • Also stated in: § Intracerebroventricular infusion of tBHQ is protective in a rat model of ischemia-reperfusion · plain
      “However, 24 h after stroke, we found a significant reduction in the neurological scores in the tBHQ-treated group (indicating a more mild deficit) when compared with vehicletreated controls, consistent with infarct volume measurements (Fig. 2 D).”
    Quote in the paperSubstantial claim
  • Findingown data

    In vehicle-treated rats, a subcortical (striatum) and cortical infarct was observed 24 h after stroke using TTC.

    “In animals treated with vehicle, we observed a subcortical (i.e., striatum) and cortical infarct 24 h after stroke using vital staining with TTC (Fig. 2 A-C).”
    Quote in the paperSubstantial claim
  • Findingown data

    Cortical glutathione levels robustly increased with tBHQ administration to rats.

    “Cortical glutathione levels robustly increased with tBHQ administration to rats and Nrf2-expressing mice, but not Nrf2 Ϫ/Ϫ mice.”
    • Also stated in: § Induction of cortical glutathione levels in vivo after tBHQ treatment · hedged
      “Using a protocol consisting of three 16.7 mg/kg injections, each spaced 8 h apart, we could robustly and consistently increase GSH levels at 24 h after the first injection (Fig. 3).”
    Quote in the paperSubstantial claim
  • May combine two claims. 35
  • Classified as probably not a substantial claim. 19
  • The quote only partly states this claim. 1

  • May combine two claims.

    Findingown data

    Neuronal death caused by a penumbral model of stroke using intracortical endothelin-1 microinjection was attenuated by tBHQ administration to Nrf2+/+ mice, but not to Nrf2-/- mice.

    “Neuronal death caused by a “penumbral” model of stroke, using intracortical endothelin-1 microinjection, was attenuated by tBHQ administration to Nrf2 ϩ/ϩ , but not to Nrf2 Ϫ/Ϫ mice, confirming the Nrf2-specific action of tBHQ in vivo.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Larger infarcts were observed in Nrf2-/- mice at 7 d after stroke, but not at 24 h.

    “Interestingly, larger infarcts were observed in Nrf2 Ϫ/Ϫ mice at 7 d after stroke, but not at 24 h, suggesting that Nrf2 may play a role in shaping the penumbra well after the onset of ischemia.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    tBHQ treatment or Nrf2 overexpression did not increase cellular LDH activity.

    “Importantly, these effects were specific, because no increase in cellular LDH activity (not regulated by Nrf2) was observed with either tBHQ treatment or overexpression of Nrf2 (Fig. 1 A, B).”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingown data

    Astrocytes from Nrf2-/- mice have reduced basal ARE-mediated gene expression (23.8 ± 4.3% of Nrf2+/+ control) that fails to be induced by tBHQ.

    “Consistent with this report, we find that astrocytes prepared from Nrf2 Ϫ/Ϫ mice have reduced basal ARE-mediated gene expression (23.8 Ϯ 4.3% of Nrf2 ϩ/ϩ control; n ϭ 3), which fails to be induced by tBHQ, as assessed by reporter assay (Fig. 1C).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingcitation

    Adenoviral-mediated overexpression of Nrf2 increased GSH, GST, and NQO1 compared with GFP control, whereas a dominant-negative Nrf2 produced no change.

    “As a positive control, adenoviral-mediated overexpression of Nrf2 (Ad-Nrf2) was sufficient to increase all three markers, compared with GFP control (Ad-GFP), whereas Ad-Nrf2DN produced no change (Fig. 1 B) (Shih et al., 2003).”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites Shih et al., 2003

      Not checked in the free run.

  • May combine two claims.

    Findingown data

    tBHQ pretreatment reduced cortical damage and sensorimotor deficit at 1 month after ischemia-reperfusion in rats.

    “Acute (1-3 d) intracerebroventricular or intraperitoneal pretreatment with tert-butylhydroquinone (tBHQ), an Nrf2 activity inducer, reduced cortical damage and sensorimotor deficit at 24 h and even 1 month after ischemia-reperfusion in rats.”
    • Also stated in: § Induction of cortical glutathione levels in vivo after tBHQ treatment · plain
      “Prophylactic intraperitoneal injection of tBHQ confers protection both 24 h and 1 month after ischemia-reperfusion”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Mice lacking Nrf2 have increased sensitivity to permanent focal ischemia and fail to respond to Nrf2 inducers.

    “Conversely, mice lacking Nrf2 have increased sensitivity to permanent focal ischemia and fail to respond to Nrf2 inducers.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    The 16.7 mg/kg dose, but not the 3.3 mg/kg dose, of intraperitoneal tBHQ significantly increased cortical GSH levels in rats.

    “The 16.7 mg/kg dose, but not the 3.3 mg/kg dose, significantly increased cortical GSH levels.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    In Nrf2-/- mice in the ET-1 model, tBHQ appeared to exacerbate injury with infarcts on average 280.8 ± 132.2% larger than control diet, but the apparent increase was not significantly different from controls because of high infarct variability.

    “In fact, tBHQ appeared to exacerbate injury with infarcts, on average, 280.8 Ϯ 132.2% larger than in Nrf2 Ϫ/Ϫ mice receiving control diet (n ϭ 5 for control diet; n ϭ 6 for tBHQ diet); however, the apparent increase in sensitivity was not significantly different from controls because of high infarct variability between Nrf2 Ϫ/Ϫ mice on tBHQ diets.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    tBHQ-treated animals had, on average, lower neurological scores throughout the entire 1-month survival period and recovered sensorimotor function more quickly within the first 4 d of reperfusion compared with vehicle controls.

    “tBHQ-treated animals had, on average, lower neurological scores throughout the entire survival period (**p ϭ 0.002; two-way ANOVA) (Fig. 5B) but recovered sensorimotor function more quickly within the first 4 d of reperfusion, compared with vehicle controls.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    The 16.7 mg/kg dose of tBHQ produced a significant increase in cortical GSH, whereas 3.3 mg/kg did not.

    “The 16.7 mg/kg dose (total 50 mg/kg injected) was used for subsequent stroke experiments because this treatment produced a significant increase in cortical GSH, whereas a dose of 3.3 mg/kg was unable to produce this induction (Fig. 3).”
    Quote in the paperSubstantial claim

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

    Findingreasoning

    LDH activity did not differ between Nrf2 genotypes in brain.

    “LDH is not regulated by Nrf2 and, accordingly, no difference in activity was observed between genotypes (Fig. 6G,H ).”
    Quote in the paperNot a substantial claim

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

    Findingown data

    The 16.7 mg/kg dose of tBHQ sometimes produced a mild and transient ataxia within the first 5 min of injection, lasting less than 10 min.

    “The 16.7 mg/kg dose sometimes produced a mild and transient ataxia observed within the first 5 min of injection, which lasted for Ͻ10 min.”
    Quote in the paperNot a substantial claim

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

    Findingown data

    No changes were observed with the negative control enzyme LDH in any of the tissues examined.

    “No changes were observed with the negative control enzyme LDH in any of the tissues examined (Fig. 7F ).”
    Quote in the paperNot a substantial claim

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

    Findingown data

    Average animal weight in the two treatment groups was not different when examined both 24 h after intraperitoneal injection of tBHQ and 24 h after stroke.

    “Average animal weight in the two treatment groups was not different when examined both 24 h after intraperitoneal injection of tBHQ and 24 h after stroke.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingcitation

    Nrf2-/- mice were less tolerant to tBHQ feeding, consuming less food and losing more weight than Nrf2-expressing controls.

    “First, Nrf2 Ϫ/Ϫ mice were found to be less tolerant to tBHQ feeding, consuming less food and losing more weight than Nrf2expressing controls (Fig. 7 A, B) (Shih et al., 2005).”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites Shih et al., 2005

      Not checked in the free run.

  • May combine two claims.

    Findingown data

    In astrocyte-enriched cultures, tBHQ treatment induced GSH, GST, and NQO1 within 10-24 h, whereas LDH activity was unchanged.

    “Induction of GSH, GST, and NQO1 was observed within 10 -24 h, whereas LDH activity (negative control) was unchanged.”
    Quote in the paperSubstantial claim

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

    Findingown data

    Increased extracellular glutamate may be a novel mediator of Nrf2 activation.

    “Our preliminary in vitro studies suggest that increased extracellular glutamate may be a novel mediator of Nrf2 activation (A.Y. Shih and T. H. Murphy, unpublished observation).”
    Quote in the paperNot a substantial claim

  • The quote only partly states this claim.

    Findingown data

    Infarction of the anterior cortical regions was attenuated in tBHQ-treated animals 1 month after stroke.

    “Infarction of the anterior cortical regions was attenuated in the tBHQ-treated animals.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Intraperitoneal injection of 16.7 mg/kg tBHQ, but not 3.3 mg/kg, significantly increased cortical GSH levels in rats.

    “The 16.7 mg/kg dose, but not the 3.3 mg/kg dose, significantly increased cortical GSH levels.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    The neuroprotective effects of tBHQ were observed with two different models of ischemia-reperfusion (MCAO and ET-1 vasoconstriction) in rats and mice, using three different routes of administration (intracerebroventricular, intraperitoneal, and dietary).

    “We observed the neuroprotective effects of tBHQ, a well characterized Nrf2 inducer, with two different models of ischemia-reperfusion (MCAO and ET-1 vasoconstriction) in rats and mice, using three different routes of administration (intracerebroventricular, intraperitoneal, and dietary).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingcitation

    Adenoviral Nrf2 overexpression increased GSH content and NQO1 and GST activity compared with GFP control, whereas Ad-Nrf2DN produced no change.

    “As a positive control, Ad-Nrf2 was sufficient to increase GSH content, as well as NQO1 and GST activity, compared with GFP control (Ad-GFP), whereas Ad-Nrf2DN produced no change(Shih et al., 2003).”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites Shih et al., 2003

      Not checked in the free run.

  • May combine two claims.

    Findingown data

    Total GSH content was reduced in the liver but not in any brain regions examined of Nrf2-/- mice.

    “Total GSH content, although reduced in the Nrf2 Ϫ/Ϫ liver, was not reduced in any brain regions examined.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    On average, Nrf2-/- mice fed 1% tBHQ lost about 20% of their starting body weight and consumed approximately one-half as much food as Nrf2-/- mice fed control diet.

    “On average, Nrf2 Ϫ/Ϫ mice lost ϳ20% of their starting body weight and consumed approximately one-half as much food as Nrf2 Ϫ/Ϫ mice fed control diet.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingown data

    Seven days after stroke, Nrf2-/- mice sustained considerably more damage, which usually extended to medial and dorsal cortical regions, whereas Nrf2+/+ and Nrf2+/- controls exhibited no increase in infarct size beyond the 24 h time point.

    “However, 7 d after stroke, Nrf2 Ϫ/Ϫ mice sustained considerably more damage, which usually extended to medial and dorsal cortical regions, whereas Nrf2 ϩ/ϩ and Nrf2 ϩ/Ϫ controls exhibited no increase in infarct size beyond the 24 h time point.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Study design and conductown data

    The tBHQ solution was active for the entire duration of in vivo administration because it protected cultured neurons from oxidative glutamate toxicity even when extracted from the pump after 4 d at 37°C.

    “The tBHQ solution was active for the entire duration of in vivo administration because it was capable of protecting cultured neurons from oxidative glutamate toxicity even when extracted from the pump after 4 d at 37°C (supplemental Fig. 1, available at www. jneurosci.orgas supplemental material).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Premisecitation

    tBHQ is a well-characterized Nrf2 inducer and a metabolite of butylated hydroxyanisole that is already approved for human use.

    “One well characterized inducer, tertbutylhydroquinone (tBHQ), a metabolite of the widely used food antioxidant butylated hydroxyanisole, is already approved for human use (National Toxicology Program, 1997; Food and”
    Quote in the paperSubstantial claim0 of 2 sources checked
    • cites National Toxicology Program, 1997

      Not checked in the free run.

    • cites Food and Agriculture Organization of the United Nations/World Health Organization, 1999

      Not checked in the free run.

  • Classified as probably not a substantial claim.

    Premisecitation

    Increasing Nrf2 activity in mixed neuronal/glial cultures is highly neuroprotective during in vitro models that simulate components of stroke damage.

    “Previous studies have shown that increasing Nrf2 activity in mixed neuronal/glial cultures is highly neuroprotective during in vitro models that simulate components of stroke damage, such as oxidative glutamate toxicity (a model involving GSH depletion), H 2 O 2 exposure, metabolic inhibition by rotenone, and Ca 2ϩ overload (Murphy et al., 1991;Duffy et al., 1998;Lee et al., 2003b;Shih et al., 200”
    Quote in the paperSubstantial claim0 of 3 sources checked
    • cites Murphy et al., 1991

      Not checked in the free run.

    • cites Duffy et al., 1998

      Not checked in the free run.

    • cites Lee et al., 2003b

      Not checked in the free run.

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

    Premisecitation

    Nrf2 is normally localized to the cytoplasm, tethered to the regulatory protein Keap1.

    “Nrf2 is normally localized to the cytoplasm, tethered to the regulatory protein, kelch-like erythroid cellderived protein with CNC homology-associated protein 1 (Keap1) (Itoh et al., 1999).”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites Itoh et al., 1999

      Not checked in the free run.

  • May combine two claims.

    Study design and conductown data

    The three markers tested (GSH, GST, and NQO1) do not represent the full scope of Nrf2 gene targets but indicate that the phase 2 response is activated.

    “The three markers tested (GSH, GST, and NQO1) do not represent the full scope of Nrf2 gene targets but nonetheless indicate that the phase 2 response is activated.”
    Quote in the paperNot a substantial claim

  • Classified as probably not a substantial claim.

    Premisecitation

    A wide range of natural and synthetic small molecules are potent inducers of Nrf2 activity.

    “A wide range of natural and synthetic small molecules are potent inducers of Nrf2 activity (Fahey et al., 1997(Fahey et al., , 2001;;Rushmore and Kong, 2002).”
    Quote in the paperSubstantial claim0 of 3 sources checked
    • cites Fahey et al., 1997

      Not checked in the free run.

    • cites Fahey et al., 2001

      Not checked in the free run.

    • cites Rushmore and Kong, 2002

      Not checked in the free run.

  • Classified as probably not a substantial claim.

    Premisecitation

    When activated, Nrf2 specifically targets genes bearing an antioxidant response element (ARE) within their promoters.

    “When activated, Nrf2 specifically targets genes bearing an antioxidant response element (ARE) within their promoters (Venugopal and Jaiswal, 1996;Itoh et al., 1997).”
    Quote in the paperSubstantial claim0 of 2 sources checked
    • cites Venugopal and Jaiswal, 1996

      Not checked in the free run.

    • cites Itoh et al., 1997

      Not checked in the free run.

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

    Premisecitation

    Nrf2 gene targets, collectively known as phase 2 genes, are involved in glutathione production and use, detoxification of ROS/RNS and xenobiotics, and NADPH production.

    “Nrf2 gene targets, collectively known as phase 2 genes, are involved in glutathione (GSH) production and use, detoxification of ROS/RNS and xenobiotics, and NADPH production (Chan and Kwong, 2000;Ishii et al., 2000;Talalay, 2000;Thimmulappa et al., 2002).”
    Quote in the paperSubstantial claim0 of 3 sources checked
    • cites Chan and Kwong, 2000

      Not checked in the free run.

    • cites Ishii et al., 2000

      Not checked in the free run.

    • cites Talalay, 2000

      Not checked in the free run.

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

    Study design and conductnone given

    Proper cannula placement was verified by eye during brain sectioning at the time the animals were killed.

    “Proper cannula placement was verified by eye during brain sectioning at the time the animals were killed.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Interpretationown data

    At short time points (24 h), Nrf2-mediated gene induction is not complete or impaired in permanent ischemia models, whereas over time (7 d), Nrf2 activity in perhaps surviving glial cells restricts infarct expansion in Nrf2-expressing mice but not in Nrf2-/- mice.

    “These results suggest a model in which, at short time points (24 h), Nrf2-mediated gene induction is not complete or impaired in permanent ischemia models, whereas over time (7 d), Nrf2 activity in perhaps surviving glial cells restricts infarct expansion in Nrf2-expressing mice but not in Nrf2 Ϫ/Ϫ mice.”
    Quote in the paperSubstantial claim

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

    Interpretationreasoning

    The transient ataxia from 16.7 mg/kg tBHQ is unlikely to be related to Nrf2 because of its rapid and transient nature and may be attributable to discomfort from the injected solution.

    “However, this effect is unlikely to be related to Nrf2 because of its rapid and transient nature and may be attributable to discomfort from the injected solution.”
    Quote in the paperNot a substantial claim

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

    Study design and conductnone given

    An experimenter blinded to the animal treatment groups performed behavioral assessments and surgeries.

    “An experimenter blinded to the animal treatment groups performed behavioral assessments and surgeries.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Interpretationown data

    Nrf2-/- mice exhibit some tBHQ-induced systemic toxicity but not necessarily brain toxicity.

    “Together, these results indicate that phase 2 induction in response to tBHQ is abrogated in Nrf2 Ϫ/Ϫ mice, and these mice exhibit some tBHQ-induced systemic toxicity but not necessarily brain toxicity.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Study design and conductown data

    No significant difference in body core temperature or reduction of regional cerebral blood flow was observed between Nrf2 genotypes during the induction of ischemia.

    “No significant difference in body core temperature (Table 1) or reduction of regional cerebral blood flow was observed between genotypes during the induction of ischemia (Fig. 8 B).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Study design and conductown data

    Heat cauterization itself produced negligible tissue damage in sham surgeries, in which no change in regional cerebral blood flow was observed.

    “Heat cauterization itself produced negligible tissue damage in sham surgeries, in which no change in regional cerebral blood flow was observed.”
    Quote in the paperNot a substantial claim

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

    Study design and conductnone given

    Infarct quantification was performed by a researcher blinded to the experimental conditions.

    “Infarct quantification was performed by a researcher blinded to the experimental conditions.”
    Quote in the paperNot a substantial claim

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

    Study design and conductreasoning

    Nrf2-/- mice were derived from breeding Nrf2+/- heterozygotes to avoid selection for reduced sensitivity to oxidative stress.

    “All animals in this study were derived from breeding Nrf2 ϩ/Ϫ heterozygotes, because it was conceivable that some selection for mice with reduced sensitivity to oxidative stress may occur if the knock-out animals were allowed to inbreed over several generations.”
    Quote in the paperNot a substantial claim

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

    Interpretationreasoning

    Nrf2-/- mice may have reduced sensitivity to the ET-1-induced ischemia model.

    “A second caveat is that Nrf2 Ϫ/Ϫ mice may have reduced sensitivity to the ET-1-induced ischemia model.”
    Quote in the paperNot a substantial claim

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

    Premisecitation

    Glutamate released during anoxic depolarization is thought to cause neuronal excitotoxicity.

    “This finding is highly relevant to ischemia because glutamate released during anoxic depolarization is thought to cause neuronal excitotoxicity (Dirnagl et al., 1999;Lee et al., 1999).”
    Quote in the paperNot a substantial claim0 of 2 sources checked
    • cites Dirnagl et al., 1999

      Not checked in the free run.

    • cites Lee et al., 1999

      Not checked in the free run.

  • May combine two claims.

    Interpretationown data

    The protective effect of tBHQ can be clearly observed in ET-1-induced stroke, in which a partial reduction of blood flow produces a larger penumbra.

    “This idea is supported by our findings using ET-1-induced stroke, in which a partial reduction of blood flow produces a larger penumbra and the protective effect of tBHQ can be clearly observed.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Interpretationown data

    Nrf2-/- mice were more sensitive than controls during permanent focal ischemia, suggesting that Nrf2-mediated phase 2 gene induction may be a physiological response to mitigate additional damage.

    “In this study, we found that Nrf2 Ϫ/Ϫ mice were more sensitive than controls during permanent focal ischemia, suggesting that Nrf2-mediated phase 2 gene induction may be a physiological response to mitigate additional damage.”
    Quote in the paperSubstantial claim

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

    Interpretationcitation

    Peri-infarct depolarizations could further propagate anoxic glutamate release into penumbral zones and activate Nrf2 within surviving astrocytes.

    “Peri-infarct depolarizations could further propagate anoxic glutamate release into penumbral zones (Hossmann, 1996) and activate Nrf2 within surviving astrocytes.”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites Hossmann, 1996

      Not checked in the free run.

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

    Interpretationreasoning

    The smaller infarct size in Nrf2-/- mice on control diet in the ET-1 model could be explained by differential sensitivity of the Nrf2-/- genotype to ET-1-induced vasoconstriction.

    “This result could be explained by differential sensitivity of the Nrf2 Ϫ/Ϫ genotype to ET-1-induced vasoconstriction.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Interpretationreasoning

    Within transient MCAO, tBHQ administration was able to salvage the cortical penumbra but not the striatal stroke core.

    “Within this injury paradigm, tBHQ administration was able to salvage the cortical penumbra but not the striatal stroke core.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Interpretationown data

    tBHQ offers no protection to Nrf2-/- mice from ET-1 injection, confirming the Nrf2-specific action of tBHQ in vivo.

    “Importantly, tBHQ offers no protection to Nrf2 Ϫ/Ϫ mice from ET-1 injection, confirming the Nrf2-specific action of tBHQ in vivo.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Interpretationown data

    Loss of Nrf2 function in vivo exacerbates ischemic damage and abrogates the protective effects of tBHQ (in the ET-1 model), consistent with a specific role for Nrf2.

    “Conversely, loss of Nrf2 function in vivo exacerbates ischemic damage and abrogates the protective effects of tBHQ (in the ET-1 model), consistent with a specific role for Nrf2.”
    Quote in the paperSubstantial claim
51 of 51

  • The paper relies on this without support.

    The role of Nrf2 in limiting stroke damage in vivo was unclear or had not been examined before this study.

    “However, the role of Nrf2 in limiting stroke damage in vivo remained unclear.”
    “However, the protective role of Nrf2 activity during in vivo stroke has not been examined.”

  • The paper relies on this without support.

    Rodent models of focal cerebral ischemia and tBHQ prophylaxis are predictive of human stroke prophylaxis.

    “Accordingly, Nrf2 activation by small molecule inducers may be a practical preventative treatment for stroke-prone patients.”
    “Our data suggest that Nrf2 function is important for controlling stroke damage, and application of Nrf2 inducers may be an effective prophylactic treatment for those at risk for stroke.”

  • The paper relies on this without support.

    Rodent models of focal cerebral ischemia are predictive of human stroke for prophylactic neuroprotection.

    “Together, these results highlight the potential of Nrf2 activation by small molecule inducers such as tBHQ as a new prophylactic strategy for stroke.”

  • The paper relies on this without support.

    Nrf2-/- mice do not have compensatory developmental or genetic differences that alter ischemic injury independently of Nrf2.

    “In the current study, Nrf2 Ϫ/Ϫ mice exhibited lower phase 2 enzyme levels in brain and liver, and the neuroprotective effects of tBHQ were lost in an ET-1-induced ischemia model (Figs.6, 7, 9), providing additional evidence that tBHQ function is dependent on Nrf2 in vivo.”
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?

  • Nrf-2 over expression is protective in a rat model of cerebral ischemia.

    “Nrf-2 over expression is protective in a rat model of cerebral ischemia183.”

    From Mitochondrial Approaches for Neuroprotection (2008) · cites this paper as [183]

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.

  • Pump-study group sizes are reversed

    Minor

    The figure assigns nine animals to tBHQ and seven to vehicle, whereas Table 1 assigns nine to vehicle and seven to tBHQ.

1 of 1

Cites

Flagged first, then by citations

Research works printed but not linked: 8

  1. 1975, FAO Nutr Meet Rep Ser, 55A, 1
  2. 1999, World Health Organ Tech Rep Ser, 884, i
  3. 1996, Cerebrovasc Brain Metab Rev, 8, 195
  4. 2000, Cancer Res, 60, 957
  5. 2001, Cancer Res, 61, 3299
  6. 1997, Natl Toxicol Program Tech Rep Ser, 459, 1
  7. 1998, Folia Neuropathol, 36, 185
  8. 2002, Cancer Res, 62, 5196

Cited by

Sorted by citations

Related

Authors
Shih, A. Y., Li, P., & Murphy, T. H.
Affiliations
University of British Columbia (CA)
Corresponding author
Murphy, Timothy H.
ORCID
Venue
The Journal of Neuroscience
Publisher
Society for Neuroscience
Published
2005-11-02
Volume
25
Issue
44
Pages
10321-10335
ISSN
  • 0270-6474 (ISSN-L)
  • 1529-2401
DOI
10.1523/jneurosci.4014-05.2005
PMC
PMC6725780
OpenAlex
W2047499088
Semantic Scholar
30389f3f6cf8bd099bb4e0c52e48a260d1e6c0a7
Cited by
413
References
116
Citations by year
2026: 8 · 2025: 9 · 2024: 9 · 2023: 16 · 2022: 11 · 2021: 16 · 2020: 20 · 2019: 25 · 2018: 17 · 2017: 27 · 2016: 34 · 2015: 13 · 2014: 25 · 2013: 32 · 2012: 24
FWCI
4.77
Type
journal article
Language
en
Open access
Licence
Locations
  • Journal of Neuroscience (publishedVersion, cc-by-nc-sa)
  • Europe PMC (PubMed Central) (publishedVersion)
  • PubMed Central (publishedVersion)
  • PubMed (publishedVersion)
Funders
  • Heart and Stroke Foundation of Canada
  • Heart and Stroke Foundation of British Columbia and Yukon
  • Canadian Stroke Network
  • Michael Smith Health Research BC
  • Canadian Institutes of Health Research
Topics
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Genomics, phytochemicals, and oxidative stress
  • Health Sciences › Medicine › Epidemiology › Acute Ischemic Stroke Management
  • Life Sciences › Neuroscience › Neurology › Neurological Disease Mechanisms and Treatments
Keywords
Nrf2, cerebral ischemia, stroke, tert-butylhydroquinone, antioxidant response, glutathione, ischemia-reperfusion injury, penumbra, endothelin-1, neuroprotection, stroke prevention, antioxidant enzymes, Nrf2 knockout mice
MeSH terms
Animals, Antioxidants, Cells, Cultured, Brain Ischemia, Humans, Rats, Sprague-Dawley, Rats, Wistar, Mice, Knockout, Oxidative Stress, NF-E2-Related Factor 2, Mice, Rats

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 2026-10-10.

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

Statistics and arithmetic are recomputed, not judged.

Confidence bands are shown only for fields calibrated on our annotated test set. None is calibrated yet, so no band is shown.

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