Cerebral ischemia is a multifactorial pathology characterized by different events evolving in time. The acute injury, characterized by a massive increase of extracellular glutamate levels, is followed by activation of resident immune cells and production or activation of inflammation mediators. Although after ischemia precocious activation of immune cells may be neuroprotective and supportive for regeneration, protracted neuroinflammation is now recognized as the predominant mechanism of secondary brain injury progression. In this thesis I investigated on the putative protective effects of ligands at histamine and adenosine receptors. Hstamine is recognized as an important neurotransmitter or neuromodulator in the Central Nervous System (CNS). Numerous studies have indeed suggested that histamine acting on the H1 and H2 receptors may play a protective effect during ischemia while no informations there were on the role of the H4 receptors that is considered the main immune system histamine receptor with a pro-inflammatory role in the periphery while its role in brain immune responses was scarcely studied. In the experimental model of focal cerebral ischemia induced by occlusion of the middle cerebral artery (MCAo) in rats, the levels of histamine evaluated by microdialysis increase in the ischemic areas. The histamine H4 receptor is predominantly expressed in cell types of immune system where it is involved in the regulation of immunological and inflammatory responses, and in numerous areas of the CNS including cortex and striatum. Therefore a first aim of my thesis was to investigate on the putative neuroprotective effects of the selective H4 receptor antagonist, JNJ7777120. Chronic treatment with JNJ7777120, significantly protects from the neurological deficit 1, 5 and 7 days after tMCAo (p<0.001 at each time point) and significantly reduced the body weight loss at 5 and 7 days after tMCAo with respect to vehicle-treated rats (respectively p<0.05; p<0.01). Seven days after the ischemic insult, JNJ7777120 reduces the volume of the ischemic cortical damage (p<0.0005) and the volume of the ischemic striatal damage (p<0.0001). Moreover, H4 antagonist reconstitutes the cytoarchitecture of ischemic cortex and striatum and decreases the number of heterochromatic small nuclei 7 days after transient ischemia. JNJ7777120, significantly reduces the number of IBA1+ (specific for microglia) and GFAP+ (specific for astrocytes) cells in the ischemic core and boundary zones of striatum and cortex with respect to vehicle-treated rats, and also decreases the plasma levels of the proinflammatory cytokines, IL-1β and TNF-α, while increases the levels of IL-10 regulatory cytokine with anti-inflammatory action , 7 days after tMCAo. Two days after ischemia, JNJ7777120 reduces granulocytes (evaluated as HIS-48+ cells) infiltration . Our results have demonstrated that the selective antagonist of histamine H4 receptor, JNJ7777120, systemically and chronically administered after ischemia, reduces the ischemic brain damage and improves the neurological deficit, by controlling a secondary inflammatory damage after brain ischemia Results demonstrate that the histamine H4 receptor represents a new interesting target after brain ischemia. Extracellular adenosine concentration dramatically increases during cerebral ischemia and a protective role is recognized to adenosine by acting on A1 receptors. However the use of adenosine A1 agonists is hampered by peripheral and central side effects. Most recent evidence indicated that the adenosine A2A receptor subtype is of critical importance in stroke. An overexpression of A2A receptors was found in neurons and microglia of the striatum and cortex after focal ischemia induced by permanent MCAo. Previous results from the laboratory where I carried out my thesis work have demonstrated that adenosine A2A receptor antagonists repeatedly administered after brain ischemia have protected from the ischemic damage evaluated 24 hours thereafter likely reducing early acute excitotoxic phenomena. Most recently, the question has been raised if A2A receptor continuous blockade maintain protection at a more distant time after ischemia. A second aim of my thesis was therefore to investigate if the selective A2A receptor antagonist, SCH58261 maintains the protective effects 7 days after ischemia in a model of transient cerebral ischemia in the rat. The adenosine A2A receptor antagonist, SCH58261, administered twice/day for 7 days protects from neurological deficit 1 day after tMCAo (p<0.01), but no more after 5 and 7 days. Moreover, seven days after tMCAo, SCH58261 does not protect ischemic areas from damage and does not ameliorate myelin organization into the ischemic striatum. Two days after tMCAo, SCH58261 does not reduce blood cell infiltration into ischemic striatal and cortical tissue. The lack of protection by the A2A receptor antagonist 7 days after ischemia could be explained by with the fact that the early protective effect from acute excitotoxicity is overwhelmed by the subsequent damage brought about by massive cell infiltration and neuroinflammation. Indeed neuroinflammation is now recognized as a predominant mechanism of secondary progression of brain injury after ischemia. Clarification of the time-limit in which A2A receptor antagonism is protective after brain ischemia might let to devise a correct therapeutic strategy with A2A receptor antagonists. Few studies are present in literature on the role of A2B receptors in brain ischemia. An overexpression of A2B receptors occurs on endothelial cells, neurons and astrocytes 24 hours after tMCAo in the rat. To date there are no evidences in literature on the protective effects of A2B receptor agonists at more distant times from ischemia when a defined neuroinflammation develops. A further aim of my thesis was to investigate, in the model of transient cerebral ischemia in the rat, the putative protective effects of the A2B receptor agonist, BAY 60-6583 7 days after ischemia, when a clear inflammatory response has developed. Treatment with adenosine A2B receptor, BAY 60-6583, chronically administered, improves the neurological deficit evaluated 1 and 5 and up to 7 days after tMCAo (p<0.001-0.02), but does not protect from the body weight loss. Seven days after ischemia, BAY 60-6583 significantly reduces the infarct area within the cortex (p<0.04) and causes a trend towards a reduction of damage also in striatum. Seven days after transient ischemia, BAY 60-6583 has reconstituted the cortical and striatal cytoarchitecture. Both peripheral antiadhesion and central antinflammatory effects could account for protective effect of A2B agonists but further experiments will investigate on the intimate brain cell transduction mechanisms involved in the A2B protective effects after transient ischemia.

Protection by adenosinergic and histaminergic drugs from ischemic damage induced by middle cerebral artery occlusion in the rat / Ilaria Dettori. - (2019).

Protection by adenosinergic and histaminergic drugs from ischemic damage induced by middle cerebral artery occlusion in the rat.

Ilaria Dettori
2019

Abstract

Cerebral ischemia is a multifactorial pathology characterized by different events evolving in time. The acute injury, characterized by a massive increase of extracellular glutamate levels, is followed by activation of resident immune cells and production or activation of inflammation mediators. Although after ischemia precocious activation of immune cells may be neuroprotective and supportive for regeneration, protracted neuroinflammation is now recognized as the predominant mechanism of secondary brain injury progression. In this thesis I investigated on the putative protective effects of ligands at histamine and adenosine receptors. Hstamine is recognized as an important neurotransmitter or neuromodulator in the Central Nervous System (CNS). Numerous studies have indeed suggested that histamine acting on the H1 and H2 receptors may play a protective effect during ischemia while no informations there were on the role of the H4 receptors that is considered the main immune system histamine receptor with a pro-inflammatory role in the periphery while its role in brain immune responses was scarcely studied. In the experimental model of focal cerebral ischemia induced by occlusion of the middle cerebral artery (MCAo) in rats, the levels of histamine evaluated by microdialysis increase in the ischemic areas. The histamine H4 receptor is predominantly expressed in cell types of immune system where it is involved in the regulation of immunological and inflammatory responses, and in numerous areas of the CNS including cortex and striatum. Therefore a first aim of my thesis was to investigate on the putative neuroprotective effects of the selective H4 receptor antagonist, JNJ7777120. Chronic treatment with JNJ7777120, significantly protects from the neurological deficit 1, 5 and 7 days after tMCAo (p<0.001 at each time point) and significantly reduced the body weight loss at 5 and 7 days after tMCAo with respect to vehicle-treated rats (respectively p<0.05; p<0.01). Seven days after the ischemic insult, JNJ7777120 reduces the volume of the ischemic cortical damage (p<0.0005) and the volume of the ischemic striatal damage (p<0.0001). Moreover, H4 antagonist reconstitutes the cytoarchitecture of ischemic cortex and striatum and decreases the number of heterochromatic small nuclei 7 days after transient ischemia. JNJ7777120, significantly reduces the number of IBA1+ (specific for microglia) and GFAP+ (specific for astrocytes) cells in the ischemic core and boundary zones of striatum and cortex with respect to vehicle-treated rats, and also decreases the plasma levels of the proinflammatory cytokines, IL-1β and TNF-α, while increases the levels of IL-10 regulatory cytokine with anti-inflammatory action , 7 days after tMCAo. Two days after ischemia, JNJ7777120 reduces granulocytes (evaluated as HIS-48+ cells) infiltration . Our results have demonstrated that the selective antagonist of histamine H4 receptor, JNJ7777120, systemically and chronically administered after ischemia, reduces the ischemic brain damage and improves the neurological deficit, by controlling a secondary inflammatory damage after brain ischemia Results demonstrate that the histamine H4 receptor represents a new interesting target after brain ischemia. Extracellular adenosine concentration dramatically increases during cerebral ischemia and a protective role is recognized to adenosine by acting on A1 receptors. However the use of adenosine A1 agonists is hampered by peripheral and central side effects. Most recent evidence indicated that the adenosine A2A receptor subtype is of critical importance in stroke. An overexpression of A2A receptors was found in neurons and microglia of the striatum and cortex after focal ischemia induced by permanent MCAo. Previous results from the laboratory where I carried out my thesis work have demonstrated that adenosine A2A receptor antagonists repeatedly administered after brain ischemia have protected from the ischemic damage evaluated 24 hours thereafter likely reducing early acute excitotoxic phenomena. Most recently, the question has been raised if A2A receptor continuous blockade maintain protection at a more distant time after ischemia. A second aim of my thesis was therefore to investigate if the selective A2A receptor antagonist, SCH58261 maintains the protective effects 7 days after ischemia in a model of transient cerebral ischemia in the rat. The adenosine A2A receptor antagonist, SCH58261, administered twice/day for 7 days protects from neurological deficit 1 day after tMCAo (p<0.01), but no more after 5 and 7 days. Moreover, seven days after tMCAo, SCH58261 does not protect ischemic areas from damage and does not ameliorate myelin organization into the ischemic striatum. Two days after tMCAo, SCH58261 does not reduce blood cell infiltration into ischemic striatal and cortical tissue. The lack of protection by the A2A receptor antagonist 7 days after ischemia could be explained by with the fact that the early protective effect from acute excitotoxicity is overwhelmed by the subsequent damage brought about by massive cell infiltration and neuroinflammation. Indeed neuroinflammation is now recognized as a predominant mechanism of secondary progression of brain injury after ischemia. Clarification of the time-limit in which A2A receptor antagonism is protective after brain ischemia might let to devise a correct therapeutic strategy with A2A receptor antagonists. Few studies are present in literature on the role of A2B receptors in brain ischemia. An overexpression of A2B receptors occurs on endothelial cells, neurons and astrocytes 24 hours after tMCAo in the rat. To date there are no evidences in literature on the protective effects of A2B receptor agonists at more distant times from ischemia when a defined neuroinflammation develops. A further aim of my thesis was to investigate, in the model of transient cerebral ischemia in the rat, the putative protective effects of the A2B receptor agonist, BAY 60-6583 7 days after ischemia, when a clear inflammatory response has developed. Treatment with adenosine A2B receptor, BAY 60-6583, chronically administered, improves the neurological deficit evaluated 1 and 5 and up to 7 days after tMCAo (p<0.001-0.02), but does not protect from the body weight loss. Seven days after ischemia, BAY 60-6583 significantly reduces the infarct area within the cortex (p<0.04) and causes a trend towards a reduction of damage also in striatum. Seven days after transient ischemia, BAY 60-6583 has reconstituted the cortical and striatal cytoarchitecture. Both peripheral antiadhesion and central antinflammatory effects could account for protective effect of A2B agonists but further experiments will investigate on the intimate brain cell transduction mechanisms involved in the A2B protective effects after transient ischemia.
2019
Felicita Pedata
ITALIA
Ilaria Dettori
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Utilizza questo identificatore per citare o creare un link a questa risorsa: https://hdl.handle.net/2158/1151849
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