Elsevier

Brain Research

Volume 1756, 1 April 2021, 147276
Brain Research

Impact of ovariectomy and CO2 inhalation on microglia morphology in select brainstem and hypothalamic areas regulating breathing in female rats

https://doi.org/10.1016/j.brainres.2021.147276Get rights and content

Highlights

  • Ovariectomy promoted a reactive microglial form in two CO2/H+ sensing regions in CNS (cNTS and LC).

  • In the cNTS of OVX rats, microglia increased morphological index due larger soma size.

  • In LC of OVX rats microglia increased morphological index by reducing the mean arborization area.

  • In the cNTS, CO2 exposure attenuated the increased microglia morphological index of OVX animals.

  • In the LC, hypercapnia promotes an increase in the NND of microglial cells.

  • In the c audal medullary raphe and PVN neither OVX nor CO2 altered the microglial morphology.

  • Our results shows a region-specific effect of microglia reactivity.

Abstract

The neural network that regulates breathing shows a significant sexual dimorphism. Ovarian hormones contribute to this distinction as, in rats, ovariectomy reduces the ventilatory response to CO2. Microglia are neuroimmune cells that are sensitive to neuroendocrine changes in their environment. When reacting to challenging conditions, these cells show changes in their morphology that reflect an augmented capacity for producing pro- and anti-inflammatory cytokines. Based on evidence suggesting that microglia contribute to sex-based differences in reflexive responses to hypercapnia, we hypothesized that ovariectomy and hypercapnia promote microglial reactivity in selected brain areas that regulate breathing. We used ionized calcium-binding-adapter molecule-1 (Iba1) immunolabeling to compare the density and morphology of microglia in the locus coeruleus (LC), the caudal medullary raphe, the caudal part of the nucleus of the tractus solitarius (cNTS), and the paraventricular nucleus of the hypothalamus (PVN). Tissue was obtained from SHAM (metaestrus) female rats or following ovariectomy. Rats were exposed to normocapnia or hypercapnia (5% CO2, 20 min). Ovariectomy and hypercapnia did not affect microglial density in any of the structures studied. Ovariectomy promoted a reactive phenotype in the cNTS and LC, as indicated by a larger morphological index. In these structures, hypercapnia had a relatively modest opposing effect; the medullary raphe or the PVN were not affected. We conclude that ovarian hormones attenuate microglial reactivity in CO2/H+ sensing structures. These data suggest that microglia may contribute to neurological diseases in which anomalies of respiratory control are associated with cyclic fluctuations of ovarian hormones or menopause.

Introduction

Microglia are neuroimmune-competent cells that are in contact with astrocytes and neurons (Nakagawa and Chiba, 2015). Within the central nervous system (CNS), the density of these “macrophage-like” cells changes over the course of development and varies significantly between structures (Hart et al., 2012, Lawson et al., 1990). In mature mammals under basal conditions, microglia survey their immediate environment by projecting their long processes to neighboring cells. Thus, the morphology of “resting” microglia is characterized by having a small soma with a well-spread arborization of its processes (Baldy et al., 2018, Tremblay et al., 2010, Verdonk et al., 2016, Wake et al., 2013). Microglia are highly sensitive to changes in their environment, and when facing stressors such as infections or traumas, they react rapidly, as indicated by changes in their morphology. Reactive microglia are characterized by having a larger soma area with a reduced arborization (small branches). Under that state, the microglia have an “ameboid” appearance, which indicates an increased capacity for secretion; this morphology may also facilitate proliferation and migration (Habib and Beyer, 2015, Lawson et al., 1990, Masgrau et al., 2017).

Microglial cells are sexually differentiated (Villa et al., 2018). Interestingly, the same stimulus can elicit different microglial responses, depending on the action of sex hormones or age (Crain et al., 2013, Crain et al., 2009, Luo and Chen, 2012, Nissen, 2017, Sohrabji and Williams, 2013). Microglia express estrogen receptors, and their activation inhibits the secretion of pro-inflammatory cytokines. This response is an important aspect of the anti-inflammatory actions of estrogens in the CNS (Habib et al., 2013, Habib and Beyer, 2015, Kipp and Beyer, 2009, Sierra et al., 2008, Villa et al., 2018) which, in turn, may be involved in sex-based differences in the prevalence of respiratory disorders with neural control dysfunction such as panic disorder (Yang et al., 2013).

By comparison with other neurological functions, our understanding of the role of microglia in cardiorespiratory homeostasis is in its infancy; yet, there is growing evidence to indicate that microglia influence the respiratory system. In this context, Lorea-Hernández et al. (2016) showed that, in young mice (7–8 postnatal days), intracisternal injection of LPS promoted microglial reactivity and reduced the respiratory frequency and amplitude suggesting that microglia may participate in the modulation of ventilation. Silva et al. (2018) reported that microglia are important for the maintenance of autonomic adjustments during hypoxia and during cardiorespiratory reflex activation elicited by peripheral chemoreceptors. The authors showed that acute hypoxia increased mRNA levels of pro-inflammatory cytokines in the rostral ventrolateral medulla and the paraventricular nucleus of the hypothalamus (PVN), which are important regulators of cardiorespiratory responses. Additionally, microglia of the subfornical organ are highly responsive to fluctuations of CO2/H+. Because excessive behavioral and cardiorespiratory response to CO2 is a hallmark of panic disorders (Griez et al., 1987, Sanderson et al., 1988, Sanderson and Wetzler, 1990, Schmidt et al., 2007, Woods et al., 1988), it was proposed that microglia might play a role in the pathophysiology of such diseases (Vollmer et al., 2016). In fact, this hypersensitivity to this stimulus is commonly used to diagnose PD and the levels used in CO2 inhalation tests range between 5% and 35% (Griez et al., 1987, Sanderson and Wetzler, 1990). Depending on the level used, the duration of the test varies between a few breaths to 20 min (Griez et al., 1987, Sanderson et al., 1988, Sanderson and Wetzler, 1990, Schmidt et al., 2007, Woods et al., 1988). The ventilatory response to CO2 results from a complex interplay between numerous structures that, in addition to having specialized “CO2-sensing properties”, are important for the integration of relevant sensory information to produce an adequate motor output. Central chemoreceptors are distributed in many locations in the brainstem, cerebellum and hypothalamus (Nattie and Li, 2012). The relative contribution of each region to the CO2-related respiratory drive depends on numerous factors, including sex, age, arousal state, and previous life experiences (Mitchell, 2004, Nattie and Li, 2012). The neural structures involved in the CO2 response cover broad areas within the CNS, and among those, the retrotrapezoid nucleus (RTN) has received the most attention, owing to its exquisite ability to detect changes in CO2/H+ (Guyenet et al., 2019). However, other structures, such as the locus coeruleus (LC), the caudal nucleus of the solitary tract (cNTS), and the medullary raphe, also have CO2-sensing properties (Biancardi et al., 2008, Corcoran and Milsom, 2009, de Carvalho et al., 2017, Dean and Putnam, 2010, Hodges and Richerson, 2010, Nattie and Li, 2012, Richerson, 2004), and unlike the RTN, they have been associated with the pathophysiology of panic disorder (Schenberg, 2016, Wemmie, 2011). Furthermore, the paraventricular nucleus (PVN) is activated during hypercapnia, owing to the numerous interactions between this structure, the LC and the NTS (Berquin et al., 2000, Cunningham et al., 1990, King et al., 2015, Sawchenko and Swanson, 1982).

Regardless, panic disorder show a significant sexual dimorphism; however, the effects of sex hormones on microglial function are still poorly explored (Asami et al., 2009, Ben Achour and Pascual, 2010, Habib et al., 2013).

Recently, we demonstrated in female rats that ovariectomy reduces the ventilatory response to inhalation of CO2-enriched (hypercapnic) gas by 43% relative to SHAM animals in estrus (Marques et al., 2015). In those experiments, hormone replacement with 17β-estradiol (E2) or E2 and progesterone (P) in physiological doses failed to restore the CO2 and hypoxic chemosensitivity. Keeping in mind that restoration of ovarian hormones and replication of their natural fluctuations is experimentally challenging, these data nonetheless raise the possibility that other mechanisms may be involved in these responses.

With that in mind, we aimed to further our understanding of the sexual dimorphism of the neurocircuitry regulating the hypercapnic ventilatory response. Based on the growing evidence linking microglia and sex hormones to this process, we hypothesized that ovariectomy and hypercapnia change the microglia morphology (“surveying” or “reactive”). This quantification was performed via optical methods after staining for ionized calcium binding adapter molecule 1 (Iba-1), a general marker of microglia (Korzhevskii and Kirik, 2016). Quantification of the selected structures was performed to evaluate areas where microglial function would be considered most relevant.

Section snippets

Results

The structures of interest (cNTS, and the caudal medullary raphe , PVN, LC) were identified using a rat brain atlas (Paxinos and Watson, 1998). Fig. 2, Fig. 3, Fig. 4, Fig. 5 show schematic representations of each structure (Panel a) and representative photomicrographs of Iba-1-immunopositive cells (Panels b-e). For each region of interest, cell density was expressed as a function of the size of the structure using standardized templates that were built according to the specific form of the

Discussion

Our understanding of the role of microglia in the respiratory network is limited and based mainly on research performed in immature rodents (Baldy et al., 2018, Lorea-Hernández et al., 2016, MacFarlane et al., 2016, Tenorio-Lopes et al., 2017a). Here, we show that neither OVX nor hypercapnia affected the density of microglia in the the cNTS, caudal medullary raphe, PVN or LC of adult female rats. However, we observed that OVX augmented the morphological index of the cNTS and LC. This

Animals

Experiments were performed on 40 adult female Sprague-Dawley rats (12 weeks old). Rats were supplied with food and water ad libitum, and were maintained under standard laboratory and animal care conditions (21 °C, 12:12 h light:dark cycle; lights on at 07:00 h and off at 19:00 h). Females were randomly assigned to one of the four experimental groups: 1) SHAM surgery exposed to normocapnia; 2) SHAM surgery exposed to hypercapnia; 3) ovariectomized (OVX) females exposed to normocapnia; and 4) OVX

Acknowledgements

The authors would like to express their gratitude to Elisa Maioqui Fonseca for her help with the experiments.

Funding.

This research was supported by operating grants from the Canadian Institutes of Health Research (RK & FB: MOP 133686; VJ: MOP 102715), Sao Paulo Research Foundation (FAPESP; 2019/09469-8) and Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq - 407490/2018-3). D.A.M. was the recipient of the scholarships, BEPE-FAPESP 2016/21730-5 in 2017/2018 and FRQ-S 268346

References (68)

  • P. Habib et al.

    Sex steroid hormone-mediated functional regulation of microglia-like BV-2 cells during hypoxia

    J. Steroid Biochem. Mol. Biol.

    (2013)
  • G.J. Harry

    Microglia during development and aging

    Pharmacol. Ther.

    (2013)
  • A.D. Hart et al.

    Age related changes in microglial phenotype vary between CNS regions: Grey versus white matter differences

    Brain Behav. Immun.

    (2012)
  • M. Kipp et al.

    Impact of sex steroids on neuroinflammatory processes and experimental multiple sclerosis

    Front. Neuroendocrinol.

    (2009)
  • L.J. Lawson et al.

    Heterogeneity in the distribution and morphology of microglia in the normal adult mouse brain

    Neuroscience

    (1990)
  • R. Masgrau et al.

    Should We Stop Saying ‘Glia’ and ‘Neuroinflammation’?

    Trends Mol. Med.

    (2017)
  • Y. Nakagawa et al.

    Diversity and plasticity of microglial cells in psychiatric and neurological disorders

    Pharmacol. Ther.

    (2015)
  • W.C. Sanderson et al.

    Panic induction via inhalation of 5.5% CO2 enriched air: a single subject analysis of psychological and physiological effects

    Behav. Res. Ther.

    (1988)
  • W.C. Sanderson et al.

    Five percent carbon dioxide challenge: Valid analogue and marker of panic disorder?

    Biol. Psychiatry

    (1990)
  • P.E. Sawchenko et al.

    The organization of noradrenergic pathways from the brainstem to the paraventricular and supraoptic nuclei in the rat

    Brain Res. Rev.

    (1982)
  • N.B. Schmidt et al.

    Reactivity to challenge with carbon dioxide as a prospective predictor of panic attacks

    Psychiatry Res.

    (2007)
  • H. Wake et al.

    Microglia: actively surveying and shaping neuronal circuit structure and function

    Trends Neurosci.

    (2013)
  • A. Ansorg et al.

    Immunohistochemistry and Multiple Labeling with Antibodies from the Same Host Species to Study Adult Hippocampal Neurogenesis

    J. Vis. Exp.

    (2015)
  • C. Baldy et al.

    The influence of sex and neonatal stress on medullary microglia in rat pups

    Exp .Physiol.

    (2018)
  • M. Battaglia

    Separation anxiety: at the neurobiological crossroads of adaptation and illness

    Dialogues Clin. Neurosci.

    (2015)
  • V. Biancardi et al.

    Locus coeruleus noradrenergic neurons and CO2 drive to breathing

    Pflugers Arch. – Eur. J. Physiol.

    (2008)
  • J.M. Crain et al.

    Microglia express distinct M1 and M2 phenotypic markers in the postnatal and adult central nervous system in male and female mice

    J. Neurosci. Res.

    (2013)
  • J.M. Crain et al.

    Expression of P2 nucleotide receptors varies with age and sex in murine brain microglia

    J. Neuroinflamm.

    (2009)
  • E.P. Cummins et al.

    NF-κB Links CO 2 Sensing to Innate Immunity and Inflammation in Mammalian Cells

    J. Immunol.

    (2010)
  • E.T. Cunningham et al.

    Organization of adrenergic inputs to the paraventricular and supraoptic nuclei of the hypothalamus in the rat

    J. Comp. Neurol.

    (1990)
  • D.S. Davies et al.

    Microglia show altered morphology and reduced arborization in human brain during aging and Alzheimer's disease: Microglial changes in ageing and Alzheimer's disease

    Brain Pathol.

    (2017)
  • Benjamin M Davis et al.

    Characterizing microglia activation: A spatial statistics approach to maximize information extraction

    Sci. Rep.

    (2017)
  • S. Fournier et al.

    Neonatal stress affects the aging trajectory of female rats on the endocrine, temperature, and ventilatory responses to hypoxia

    Am. J. Physiol.-Regul. Integr. Comp. Physiol.

    (2015)
  • P.G. Guyenet et al.

    The retrotrapezoid nucleus: central chemoreceptor and regulator of breathing automaticity

    Trends Neurosci.

    (2019)
  • Cited by (6)

    • Alterations in brainstem respiratory centers following peripheral inflammation: A systematic review

      2022, Journal of Neuroimmunology
      Citation Excerpt :

      The majority of articles specified the age where inflammation was induced (32 articles, 91%) and assessed changes in a specific brainstem nuclei (25 articles, 71%). Five articles reported randomisation (14%) (Chen et al., 2003; Chen et al., 2018; Del Rio et al., 2012; Marques et al., 2021; Na et al., 2017), two articles reported sample size calculations (6%) (Litvin et al., 2020; Litvin et al., 2018) and two articles reported blinding (6%) (Del Rio et al., 2012; Marques et al., 2021). Six articles used both sexes for their study (17%).

    • Subfornical organ interleukin 1 receptor: A novel regulator of spontaneous and conditioned fear associated behaviors in mice

      2022, Brain, Behavior, and Immunity
      Citation Excerpt :

      Mounting evidence within clinical and preclinical studies points to neuroimmune signaling in pathologies associated with CO2 sensitivity like PTSD and panic disorder (Vollmer et al., 2016; Michopoulos et al., 2017; Sumner et al., 2020; Won and Kim, 2020; Deslauriers et al., 2018; Jones et al., 2015; Furtado and Katzman, 2015). Limited studies have investigated the direct role of neuroimmune signaling in mediating CO2 responsivity and lasting CO2-dependent effects, nor the central nodes through which this signaling acts (Vollmer et al., 2016; van Duinen et al., 2008; Marques et al., 2021). Our recent work reported a role for the subfornical organ (SFO) in facilitating CO2-evoked fear (Vollmer et al., 2016).

    View full text