Methylene blue post-treatment improves hypoxia-ischemic recovery in a neonatal rat model

https://doi.org/10.1016/j.neuint.2020.104782Get rights and content

Highlights

  • Methylene blue (MB) attenuates hypoxia-ischemia (HI)-induced brain loss and neuronal damage in neonatal rats.

  • MB attenuates blood-brain barrier disruption after HI injury.

  • MB represses HI-triggered inflammation and oxidative damage to the brain.

  • MB ameliorates mitochondrial fragmentation after HI brain injury.

  • MB preserves cognitive and motor functions of neonatal HI rats.

Abstract

Recent work suggested that methylene blue (MB) has beneficial effects in a variety of neurological disorders, while its role in neonatal hypoxic-ischemic (HI) encephalopathy is still unclear. The current study was designed to investigate the effects of MB on HI-induced brain damage and its underlying mechanisms. The results showed that MB treatment can strongly attenuate HI-induced brain loss and neuronal damage in the cortex and hippocampus of neonatal rats. Further mechanistic analysis suggested that MB treatment was able to significantly reduce blood-brain barrier disruption after HI insult. In addition, MB profoundly inhibited microglia and astrocyte activation and the pro-inflammatory cytokines production in neonatal cortex and hippocampus after HI. Further, MB treatment resulted in dramatic suppression of oxidative damage, as evidenced by robustly decreased DHE and protein carbonyls levels in HI brain. Moreover, MB strongly preserved mitochondrial function by repressing HI-induced mitochondrial fragmentation, and the following neuronal death in cortex and hippocampus. Finally, behavioral tests revealed that MB significantly improved the spatial reference memory and motor coordination of neonatal HI rats. Taken together, these findings demonstrate that the mechanisms behind neuroprotective actions of methylene blue are multifactorial, including suppression of oxidative stress and neuroinflammation, restoration of mitochondrial function, as well as attenuation of blood-brain barrier disruption. Our study might provide further directions for MB as a promising option in neonatal HI encephalopathy therapy.

Introduction

Neonatal hypoxic-ischemic brain injury is a newborn encephalopathy caused by impaired cerebral blood flow and oxygen deprivation (Kurinczuk et al., 2010; Vannucci, 1990). HI encephalopathy affects approximately 3 per 1000 births, and is a leading cause of long-term neurological sequelae (Kurinczuk et al., 2010). According to previous reports, 0.2%–0.4% of all full-term infants suffer from asphyxial injury during labor, and the asphyxiated neonates are related with increased risk of growth retardation, hypothermia, hyaline membrane disease, and seizures (MacDonald et al., 1980). Persistent treatment and health care for the neurological deficits in survivors impose heavy financial burden on their families and society. Seeking for effective therapeutic strategies is thus an urgent need for this neonatal brain disorder. Although there are abundant studies on HI, hypothermia is currently the only clinically effective intervention for HI. However, due to the narrow therapeutic window, complex management and clinical complication, application of hypothermia in clinic is still limited. Therefore, looking for alternative strategies is critical.

HI insult triggers a line of multifactorial and complex pathological events, including excitatory glutamate accumulation, activation of NMDA-type glutamate receptor-operated channels and subsequent massive Ca2+ influx, chronic glial activation, neuroinflammation, excessive reactive free radical (ROS) production and oxidative stress (Northington et al., 2011), blood-brain barrier (BBB) breakdown (Ek et al., 2015; Lee et al., 2018), mitochondrial dysfunction, as well as energy failure and consequent neuronal death (Lu et al., 2015). It seems mitochondrial dysfunction plays a central role in neurodegeneration following neonatal HI injury (Lu et al., 2015). Mitochondria are not only devoted to ATP synthesis, but also a primary source of oxidative stress (Cao et al., 1988) and neuroinflammatory processes (Bader and Winklhofer, 2019). Mitochondrial deficit leads to robust oxidative and inflammatory neural damage to the brain (Shin et al., 2018). BBB disruption is another factor for neonatal HI pathogenesis, and plays a critical role in neurological dysfunction after ischemic brain injury (Ek et al., 2015; Lee et al., 2018). Loss of BBB tight junction integrity results in enhanced BBB permeability, vasogenic edema and hemorrhagic transformation (Khatri et al., 2012). Interestingly, it has been demonstrated that disruption of BBB is triggered by inflammatory and oxidative damage to the brain (Blamire et al., 2000; Kim et al., 2001; Yang et al., 2019). Therefore, preserving mitochondrial integrity and functions after neonates HI might be a promising strategy to attenuate these neurotoxic cascades.

Methylene blue (MB) is a well-established drug that has been demonstrated to be neuroprotective with a wide range of use and minimal side effects (Stawicki et al., 2008; Tucker et al., 2018a). Importantly, its roles in mitochondria have received much attention in recent years owing to its properties in increasing mitochondrial function and inhibition of mitochondrial dysfunction-induced oxidative stress and inflammation (Tucker et al., 2018a; Wiklund et al., 2007). Although its neuroprotective effects have been widely reported in a variety of neurodegenerative diseases, its effects in neonatal HI encephalopathy remain elusive. The current was designed to investigate the roles of MB in neonatal HI brain injury in rat model. We examined both neuronal damage and functional changes after MB treatment, and analyzed the underlying mechanisms. Our study might provide important directions for MB application in future clinic trial.

Section snippets

Animals and neonatal brain hypoxia ischemia model

Ten-day-old unsexed neonatal Sprague-Dawley rats (Charles River Laboratories) were used in current studies. Brain hypoxia ischemia was performed as previously described (Zhang et al., 2018). Briefly, neonatal rats were anesthetized under isoflurane. Right common carotid artery was exposed through midline neck incision, and permanently ligated with nylon thread. Rats were allowed to recover for 1.5 h after wound closure. Next, rats were placed in a hypoxic environment (6% oxygen/94% nitrogen at

MB treatment attenuates HI-induced brain tissue loss and neuronal damage

To examine if MB treatment could attenuate the brain damage in neonatal rats, brain sections were subjected to CV staining at 14 days after HI injury (Fig. 1A, a), and tissue loss of the ipsilateral hemisphere was quantitatively analyzed (Fig. 1A and b). The results suggested that hypoxia-ischemia caused robustly increased brain loss in neonatal rats (n = 5) as compared to sham group (n = 5; p < 0.001, 95% CI [-24.167 to −14.233]). Interestingly, MB treatment (n = 6) strongly reduced HI-induced

Discussion

While the beneficial effects of MB have been widely reported, its precise roles in neonatal HI brain injury are not well understood. The current study provides leading evidences that MB yields profound neuroprotection and functional preservation following HI injury to neonatal rat brain. The neuroprotective effects of MB treatment demonstrated in our study are likely due to multifactoral beneficial effects, including suppression of mitochondrial fragmentation, reduction of neuroninflammatory

Conclusions

The current study demonstrates that MB has profound neuroprotective and neurological function-preserving effects in neonatal hypoxic ischemic rats. Although the neuroprotective effects could be attributed to complex mechanisms, the results from our study suggest that the beneficial effects of MB could be due to its functions to maintain mitochondrial integrity, suppress oxidative stress, inhibit neuroinflammation, and attenuate blood-brain barrier disruption in both cortex and hippocampus.

Author contributions

QZ, JX conceived most of the experimental design, GZ, YL, YD performed the experiments and analyzed the data. LY, JW performed parts of the experiments and analyzed the data. GZ, YL, QZ, JX wrote the paper.

Declaration of competing interest

The authors declare no competing financial interests.

Acknowledgment

This study was supported in part by an American Heart Association Innovative Project Award 18IPA34170148 (to QZ).

References (81)

  • A. Manaenko et al.

    Comparison Evans Blue injection routes: intravenous versus intraperitoneal, for measurement of blood-brain barrier in a mice hemorrhage model

    J. Neurosci. Methods

    (2011)
  • J.C. Martinou et al.

    Mitochondria in apoptosis: bcl-2 family members and mitochondrial dynamics

    Dev. Cell

    (2011)
  • P. Mergenthaler et al.

    Sugar for the brain: the role of glucose in physiological and pathological brain function

    Trends Neurosci.

    (2013)
  • A.S. Reichert et al.

    Contact sites between the outer and inner membrane of mitochondria-role in protein transport

    Biochim. Biophys. Acta

    (2002)
  • B.J. Song et al.

    Mitochondrial dysfunction and tissue injury by alcohol, high fat, nonalcoholic substances and pathological conditions through post-translational protein modifications

    Redox Biol.

    (2014)
  • S.M. Sullivan et al.

    Morphological changes in white matter astrocytes in response to hypoxia/ischemia in the neonatal pig

    Brain Res.

    (2010)
  • I. Torres-Cuevas et al.

    Brain oxidative damage in murine models of neonatal hypoxia/ischemia and reoxygenation

    Free Radic. Biol. Med.

    (2019)
  • T.M. Visarius et al.

    Stimulation of respiration by methylene blue in rat liver mitochondria

    FEBS Lett.

    (1997)
  • Y.C. Wang et al.

    Mitochondrial dysfunction and oxidative stress contribute to the pathogenesis of spinocerebellar ataxia type 12 (SCA12)

    J. Biol. Chem.

    (2011)
  • Y. Wen et al.

    Alternative mitochondrial electron transfer as a novel strategy for neuroprotection

    J. Biol. Chem.

    (2011)
  • G.Y. Yang et al.

    Tumor necrosis factor alpha expression produces increased blood-brain barrier permeability following temporary focal cerebral ischemia in mice

    Brain Res. Mol. Brain Res.

    (1999)
  • J. Zhang et al.

    Tert-butylhydroquinone post-treatment attenuates neonatal hypoxic-ischemic brain damage in rats

    Neurochem. Int.

    (2018)
  • K. Abe et al.

    Ischemic delayed neuronal death. A mitochondrial hypothesis

    Stroke

    (1995)
  • S.O. Algra et al.

    Cerebral ischemia initiates an immediate innate immune response in neonates during cardiac surgery

    J. Neuroinflammation

    (2013)
  • K.L. Arvin et al.

    Minocycline markedly protects the neonatal brain against hypoxic-ischemic injury

    Ann. Neurol.

    (2002)
  • A.A. Baburamani et al.

    Vulnerability of the developing brain to hypoxic-ischemic damage: contribution of the cerebral vasculature to injury and repair?

    Front. Physiol.

    (2012)
  • V. Bader et al.

    Mitochondria at the interface between neurodegeneration and neuroinflammation

    Semin. Cell Dev. Biol.

    (2019)
  • A.L. Betz et al.

    Attenuation of stroke size in rats using an adenoviral vector to induce overexpression of interleukin-1 receptor antagonist in brain

    J. Cerebr. Blood Flow Metabol.

    (1995)
  • A.M. Blamire et al.

    Interleukin-1beta -induced changes in blood-brain barrier permeability, apparent diffusion coefficient, and cerebral blood volume in the rat brain: a magnetic resonance study

    J. Neurosci.

    (2000)
  • H. Boutin et al.

    Role of IL-1alpha and IL-1beta in ischemic brain damage

    J. Neurosci.

    (2001)
  • E. Bratek et al.

    The activation of group II metabotropic glutamate receptors protects neonatal rat brains from oxidative stress injury after hypoxia-ischemia

    PLoS One

    (2018)
  • L.H. Chang et al.

    Effect of dichloroacetate on recovery of brain lactate, phosphorus energy metabolites, and glutamate during reperfusion after complete cerebral ischemia in rats

    J. Cerebr. Blood Flow Metabol.

    (1992)
  • Z.W. Chen et al.

    MEF2D mediates the neuroprotective effect of methylene blue against glutamate-induced oxidative damage in HT22 hippocampal cells

    Mol. Neurobiol.

    (2017)
  • Y.S. Cho et al.

    Ischemic preconditioning maintains the immunoreactivities of glucokinase and glucokinase regulatory protein in neurons of the gerbil hippocampal CA1 region following transient cerebral ischemia

    Mol. Med. Rep.

    (2015)
  • Y. Di et al.

    Methylene blue reduces acute cerebral ischemic injury via the induction of mitophagy

    Mol. Med.

    (2015)
  • C.J. Ek et al.

    Brain barrier properties and cerebral blood flow in neonatal mice exposed to cerebral hypoxia-ischemia

    J. Cerebr. Blood Flow Metabol.

    (2015)
  • A.M. Fenn et al.

    Methylene blue attenuates traumatic brain injury-associated neuroinflammation and acute depressive-like behavior in mice

    J. Neurotrauma

    (2015)
  • S. Gabel et al.

    Inflammation promotes a conversion of astrocytes into neural progenitor cells via NF-kappaB activation

    Mol. Neurobiol.

    (2016)
  • S.D. Gan et al.

    Enzyme immunoassay and enzyme-linked immunosorbent assay

    J. Invest. Dermatol.

    (2013)
  • T.W. Gauthier

    Methylene blue-induced hyperbilirubinemia in neonatal glucose-6-phosphate dehydrogenase (G6PD) deficiency

    J. Matern. Fetal Med.

    (2000)
  • Cited by (0)

    1

    These authors contribute equally to this work, and should be considered as co-first authors.

    View full text