Activation of acetylcholine receptors and microglia in hypoxic-ischemic brain damage in newborn rats

doi:10.1016/j.braindev.2012.10.006

Brain and Development

Volume 35, Issue 7, August 2013, Pages 607-613

https://doi.org/10.1016/j.braindev.2012.10.006 Get rights and content

Abstract

Objective: We previously showed that acetylcholine receptor (AChR) agonist reduced hypoxic-ischemic brain damage in the newborn rats. To further investigated the interaction between hypoxia and chorinergic anti-inflammatory pathway, we examined the effect of AChR antagonist on brain damage and to see the relation between microglial activation and protective effect of AChR agonist. Study design: Seven-day-old Wistar rats were divided into 2 groups, one receiving AChR antagonists to see if they have deleterious effects on hypoxic-ischemic brain damage, and the other receiving AChR agonist, carbachol, to investigate the emergence of microglia in the hippocampus. Rats were subjected to left carotid artery ligation followed by 8% hypoxia. Brains were analyzed histologically and immunohistochemically. Results: Antagonists of AChRs significantly enhanced brain damage in 1-h hypoxia-ischemia. In particular, the nicotinic AChR antagonist showed a marked enhancement of brain damage compared to the saline controls (p < 0.01). The hippocampal CA1 was most vulnerable to any AChR antagonists, while the cortex was least vulnerable and only responsive to a higher dose of non-selective nAChR antagonist. Carbachol showed significantly less accumulation of microglia in the hippocampus than the saline controls (p < 0.01) in hypoxia–ischemia. Conclusion: An AchR-responsive pathway in the brain plays an important role in modifying perinatal brain damage, in which microglial accumulation may be involved.

Introduction

Perinatal hypoxic-ischemic brain damage still remains one of the most important medical problems. The mechanisms involved to develop brain damage have been extensively studied and inflammatory response is one contributor to hypoxic-ischemic brain damage [1]. Recently, we have also demonstrated that peripheral injection of carbachol, known as an acetylcholine receptor (AChR) agonist, reduces brain damage in a newborn rat model of hypoxia-ischemia [2]. However, the role of AChR in the developing brain against hypoxia-ischemia is currently inconclusive. For example, whether AChR antagonists have enhancing effects on hypoxic-ischemic brain damage has not been throughly studied. Besides, since carbachol does not cross the blood–brain-barrier under physiological conditions, it may act through systemic effects, it may stimulate afferent vagus activities to elicit central effects, or hypoxia-ischemia may damage the blood–barain-barrier to permeate it.

In the peripheral organs, macrophage is involved in the regulation of the hypoxia-induced inflammatory pathway via vagus nerve activity [3], [4]. Similarly, in the central nervous system, microglia is activated by hypoxia-ischemia [5], which is also related to inflammatory processes by alpha-7-nicotinic-AChR on microglia [6], [7]. Thus, we speculated that carbachol, an AChR agonist, stimulates cholinergic anti-inflammatory pathway via afferent vagus activities, resulting in protection against brain damage in our previous study. However, these interactions between microglial activation and AChR stimulation are currently unclear in the developing brain.

Therefore, we hypothesized that AChR antagonists have deleterious effects on hypoxic-ischemic brain damage in the newborn rat, and that an AChR agonist, carbachol, has protecting effect through modifying microglial activities in the brain after hypoxia-ischemia.

Section snippets

Material and methods

Animal model: This study was performed in accordance with the Guidelines of the Experimental Animal Center of the University of Miyazaki, Faculty of Medicine. Pregnant Wistar rats were housed in the same animal center with free access to water and food under a 12-h on/off lighting schedule. The pups were reared with their dams until the time of the experiment.

The 7-day-old rat, whose cerebral maturity corresponds to a 32∼34 week gestation human fetus or newborn infant [8], [9], is subjected to

AChR antagonist study

All pups survived and were included in the histological analysis. Fig. 3 shows the relative difference of hemisphere area. The maximum blocking dose of nonselective nicotinic-AChR antagonist, MLC (10 mg), and selective alpha-7-nicotinic-AChR antagonist, MCA (10 mg), exhibited significantly greater shrinkage of the ligated side than those of the controls (p < 0.01). Both antagonists showed a dose-dependent deteriorating effects to cause brain damage. On the other hand, muscarinic AChR antagonist,

Discussion

The mechanism involved in the development of hypoxic-ischemic brain damage is not fully understood. Possible contributing mechanisms are, for example, excitotoxicity of neurotransmitters, free-radical production, and inflammatory responses. The inflammatory pathways are activated by hypoxia-ischemia, and cholinergic anti-inflammatory responses are also involved to decrease brain damage. We previously showed a marked reduction of hypoxic-ischemic brain damage by carbachol, compared with saline

Acknowledgement

This work was supported in part by a Grant (#79-258) from the Ministry of Education, Culture, Sports, Science and Technology, Japan.

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Cited by (21)

Atomoxetine, a selective norepinephrine reuptake inhibitor, improves short-term histological outcomes after hypoxic-ischemic brain injury in the neonatal male rat
2018, International Journal of Developmental Neuroscience
Despite the recent progress of perinatal medicine, perinatal hypoxic-ischemic (HI) insult remains an important cause of brain injury in neonates, and is pathologically characterized by neuronal loss and the presence of microglia. Neurotransmitters, such as norepinephrine (NE) and glutamate, are involved in the pathogenesis of hypoxic-ischemic encephalopathy via the interaction between neurons and microglia. Although it is well known that the monoamine neurotransmitter NE acts as an anti-inflammatory agent in the brain under pathological conditions, its effects on perinatal HI insult remains elusive. Atomoxetine, a selective NE reuptake inhibitor, has been used clinically for the treatment of attention-deficit hyperactivity disorder in children. Here, we investigated whether the enhancement of endogenous NE by administration of atomoxetine could protect neonates against HI insult by using the neonatal male rat model. We also examined the involvement of microglia in this process.
Unilateral HI brain injury was induced by the combination of left carotid artery dissection followed by ligation and hypoxia (8% O₂, 2 h) in postnatal day 7 (P7) male rat pups. The pups were randomized into three groups: the atomoxetine treatment immediately after HI insult, the atomoxetine treatment at 3 h after HI insult, or the vehicle treatment group. The pups were euthanized on P8 and P14, and the brain regions including the cortex, striatum, hippocampus, and thalamus were evaluated by immunohistochemistry.
HI insult resulted in severe brain damage in the ipsilateral hemisphere at P14. Atomoxetine treatment immediately after HI insult significantly increased NE levels in the ipsilateral hemisphere at 1 h after HI insult and reduced the neuronal damage via the increased phosphorylation of cAMP response element-binding protein (pCREB) in all brain regions examined. In addition, the number of microglia was maintained under atomoxetine treatment compared with that of the vehicle treatment group. To determine the involvement of microglia in the process of neuronal loss by HI insult, we further examined the influence of hypoxia on rat primary cultured microglia by the quantitative real-time polymerase chain reaction. Hypoxia did not cause the upregulation of interleukin-1beta (IL-1β) mRNA expression, but decreased the microglial intrinsic nitric oxide synthase (iNOS)/arginase1 mRNA expression ratio. NE treatment further decreased the microglial iNOS/arginase1 mRNA expression ratio. In contrast, no significant neuroprotective effect was observed at P14 when atomoxetine was administered at 3 h after HI insult.
These findings suggested that the enhancement of intrinsic neurotransmitter NE signaling by a selective NE reuptake inhibitor, atomoxetine, reduced the perinatal HI insult brain injury. In addition, atomoxetine treatment was associated with changes of TUNEL, pCREB, and BDNF expression levels, and microglial numbers, morphology, and responses.
Galantamine administration reduces reactive astrogliosis and upregulates the anti-oxidant enzyme catalase in rats submitted to neonatal hypoxia ischemia
2017, International Journal of Developmental Neuroscience
Neonatal hypoxia ischemia (HI) plays a role in the etiology of several neurological pathologies and causes severe sequelae. Acetylcholine is a neurotransmitter in the central nervous system and cholinesterase inhibitors have demonstrated a positive action over HI induced deficits. In order to evaluate the effects of pre and post-hypoxia administrations of galantamine, a cholinesterase inhibitor, in a model of perinatal HI, Wistar rats in the post-natal day 7 (PND7) were subjected to a combination of unilateral occlusion of the right carotid artery with the exposure to a 1 h hypoxia. Intraperitoneal injections of galantamine were administered in two different protocols: one pre and other post-hypoxia. The analysis of brain structures volume at PND45 showed that pre-hypoxia galantamine treatment prevented tissue injury to the ipsilesional hippocampus. Also, immunofluorescence showed HI-induced increase in the number of astrocytes that was prevented by pre-hypoxia treatment. Biochemical analysis was performed in the ipsilesional hippocampus at PND8 and revealed that pre-hypoxia galantamine treatment: 1) prevented the neuronal loss induced by HI; 2) reduced the HI-induced hypertrophy of astrocytes; and 3) caused an increase in the activity of the anti-oxidant enzyme catalase. Overall, treatment with galantamine was able to prevent the brain damage, increase the survival of neurons, reduce astrocytic reaction and increase the activity of the anti-oxidant enzyme catalase in rats submitted to neonatal hypoxia ischemia.
Galantamine, an acetylcholinesterase inhibitor, reduces brain damage induced by hypoxia-ischemia in newborn rats
2014, International Journal of Developmental Neuroscience
Citation Excerpt :
However, it was incapable of suppressing late microglial activation of the cortex, in which tissue damage was in progress after hypoxia-ischemia (HI) (Furukawa et al., 2014). On the other hand, repetitive administration of carbachol rescued progression of hypoxic-ischemic brain damage with a reduction of early and late microglial activation (Furukawa et al., 2013a,b). Thus, an activating cholinergic nervous system during and after HI seems to be important in preventing brain damage caused by HI.
Our aim is to elucidate whether galantamine, known as an acetylcholinesterase inhibitor, reduces brain damage induced by hypoxia-ischemia (HI).
7-day-old Wistar rats were used. Rats were subjected to left carotid artery ligation followed by 2 h of hypoxia (8% oxygen). We injected galantamine intraperitoneally just before hypoxia (5.0 mg/kg, n = 14; 2.5 mg/kg, n = 9; 1.0 mg/kg, n = 11) and after hypoxia (5.0 mg/kg, n = 7) to determine its neuroprotective effect. An equivalent volume of saline was administered as a control before (n = 31) and after hypoxic load (n = 7). We also examined the production of IL-1β in the ligated hemisphere side after injection of galantamine (prior hypoxia; 5.0 mg/kg, n = 7) or saline (n = 8). Brains were analyzed 7 days after HI.
Two of the 5.0 mg/kg galantamine pre-treated rats and a post-treated rat died during experiments. The remaining survived and 5.0 mg/kg galantamine pre-treated rats showed a marked reduction of brain damage (p < 0.01) compared with the control. The other galantamine groups had severe brain damage similar to controls. Microglial accumulation was significantly reduced in rats pre-treated with 5.0 mg/kg of galantamine compared to control rats on both the hippocampus (p = 0.02) and cortex (p < 0.01). In contrast, the other galantamine groups showed a lower suppressive effect on microglial accumulation compared to the control. Galantamine significantly reduced IL-1β productions when compared to the control (p < 0.01).
Pre-treatment of galantamine reduced brain damage with a suppressive effect on microglial accumulation and IL-1β production in a newborn rat model of HI.
Regional differences of microglial accumulation within 72 hours of hypoxia-ischemia and the effect of acetylcholine receptor agonist on brain damage and microglial activation in newborn rats
2014, Brain Research
Citation Excerpt :
Activated microglia have been recorded as a marked feature of white matter in all cases involving periventricular leukomalacia in the human preterm fetus (Hirayama et al., 2001). We recently demonstrated that stimulation or inhibition of AChR in the developing rat modifies hypoxic-ischemic brain damage 7 days after HI, as well as regional differences of susceptibility to AChR antagonists or agonists (Furukawa et al., 2011; Furukawa et al., 2013). Additionally, AChR agonist reduced activation of microglia in the hippocampal region 24 hours after HI in the developing rat (Furukawa et al., 2013).
We examined regional specificity of microglial activation in the developing rat brain for 72 hours after hypoxia-ischemia (HI) and the effect of acetylcholine receptor (AChR) agonist on microglial activation.
Seven-day-old Wistar rats were divided into two groups: one receiving a single dose of AChR agonist just before hypoxia (carbachol; 0.1 mg/kg) to investigate the reducing effect on brain damage with decreasing activation of microglia and the other group receiving saline as a control. Rats were subjected to left carotid artery ligation followed by 8% hypoxia. Brains were analyzed immunohistochemically at 24, 48, and 72 hours after HI. TNFα production was measured at respective times after HI.
Activation of microglia on the hippocampus of the control group was strong for the first 48 hours and then weakened. In contrast, activation of microglia on white matter and the cortex was weak at 24 hours and then became stronger. A single dose of carbachol significantly reduced brain damage with a marked reduction of microglial activation on the hippocampus, whereas it was less effective regarding microglial activation on white matter and the cortex. TNFα production was low in both groups.
Regional specificity was observed for both microglial activation and susceptibility to carbachol for the first 72 hours after HI. Our data suggested that timely intervention along with region-specific microglial activation, apart from TNFα production, may be critical for the prevention of further brain damage after HI in the newborn.
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Original articleActivation of acetylcholine receptors and microglia in hypoxic-ischemic brain damage in newborn rats

Abstract

Introduction

Section snippets

Material and methods

AChR antagonist study

Discussion

Acknowledgement

Neuropharmacology

Clin Ther

Am J Obstet Gynecol

Mol Cell Neurosci

Prog Brain Res

Brain Res Dev Brain Res

Does inflammation after stroke affect the developing brain differently than adult brain?

Dev Neurosci

Acetylcholine receptor agonist reduces brain damage induced by hypoxia-ischemia in newborn rats

Reprod Sci

Involvement of nicotinic acetylcholine receptors in suppression of antimicrobial activity and cytokine responses of alveolar macrophages to Legionella pneumophila infection by nicotine

J Immunol

Nicotinic acetylcholine receptor alpha7 subunit is an essential regulator of inflammation

Nature

Immunocytochemical study of an early microglial activation in ischemia

J Cereb Blood Flow Metab

Cholinergic modulation of microglial activation by alpha7 nicotinic receptors

J Neurochem

Original article
Activation of acetylcholine receptors and microglia in hypoxic-ischemic brain damage in newborn rats