Juraj Culman

Angiotensin AT2 receptor protects against cerebral ischemia-induced neuronal injury

Several lines of clinical and experimental evidence suggest an important role of the reninangiotensin system in ischemic brain injury although the cellular regulation of the angiotensin AT1 and AT2 receptors and their potential relevance in this condition have not yet been clearly defined. We first assessed the regulation of brain AT1 and AT2 receptors in response to transient unilateral medial cerebral artery occlusion in rats by real‐time RT‐PCR, Western blot, and immunofluorescence labeling. AT2 receptors in the peri‐infarct zone were significantly upregulated 2 days after transient focal cerebral ischemia. Increased AT2 receptors, which were abundantly distributed in a large number of brain regions adjacent to the infarct area including cerebral frontal cortex, piriform cortex, striatum, and hippocampus, were exclusively expressed in neurons. By contrast, AT1 receptors, which remained unaltered, were mainly expressed in astrocytes. In neurons of ischemic striatum, increased AT2 receptors were associated with intense neurite outgrowth. Blockade of central AT2 receptors with PD123177 abolished the neuroprotective effects of central AT1 receptor blockade with irbesartan on infarct size and neurological outcome. In primary cortical neurons, stimulation of AT2 receptors supported neuronal survival and neurite outgrowth. Our data indicate that cerebral AT2 receptors exert neuroprotective actions in response to ischemia‐induced neuronal injury, possibly by supporting neuronal survival and neurite outgrowth in peri‐ischemic brain areas.

Blockade of Central Angiotensin AT1 Receptors Improves Neurological Outcome and Reduces Expression of AP-1 Transcription Factors After Focal Brain Ischemia in Rats

BACKGROUND AND PURPOSE Angiotensin-converting enzyme inhibitors have been shown to protect against stroke in hypertensive rats and to improve neurological outcome after cerebral ischemia in normotensive rats. The present study was designated to test the hypothesis that blockade of brain AT(1) receptors improves the recovery from focal cerebral ischemia and reduces expression of AP-1 transcription factors c-Fos and c-Jun, which have been associated with programmed cell death and neurodegeneration. METHODS Experiments were carried out in normotensive male Wistar rats. Focal cerebral ischemia was induced by middle cerebral artery occlusion lasting for 90 minutes and followed by reperfusion. The selective AT(1) receptor antagonist irbesartan was infused intracerebroventricularly over a 5-day period before the induction of ischemia at a dose that inhibited brain but not vascular AT(1) receptors. Twenty-four hours after ischemia, neurological outcome was evaluated and expression of c-Fos and c-Jun proteins in the brain was studied immunocytochemically. RESULTS Focal brain ischemia resulted in a strong induction of c-Fos and c-Jun proteins in the cortex, which positively correlated with the degree of neurological deficits. Treatment of rats with irbesartan significantly improved neurological outcome of focal cerebral ischemia when compared with the vehicle-treated group and markedly reduced the expression of c-Fos and c-Jun proteins in the cortex on the ligated side of the brain. Irbesartan pretreatment completely abolished the ischemia-induced c-Fos expression in the hippocampus. CONCLUSIONS The present study shows a relationship between c-Fos and c-Jun expression and neurological outcome after focal brain ischemia. Our data indicate that long-term blockade of central AT(1) receptors improves the recovery from brain ischemia and reduces the expression of c-Fos and c-Jun proteins in the brain. Pretreatment with an AT(1) receptor antagonist has beneficial effects after cerebral ischemia.

Activation of cerebral peroxisome proliferator-activated receptors gamma promotes neuroprotection by attenuation of neuronal cyclooxygenase-2 overexpression after focal cerebral ischemia in rats

Up‐regulation of cyclooxygenase (COX)‐2 exacerbates neuronal injury after cerebral ischemia and contributes to neuronal cell death. The present study clarifies the function of cerebral peroxisome‐proliferator‐activated receptor(s) gamma (PPARγ) in the expression of COX‐2 in neurons of the rat brain after middle cerebral artery occlusion (MCAO) with reperfusion by immunohistochemistry, Western blot, and immunofluorescence staining. In peri‐infarct cortical areas the PPARγ was located in both microglia and neurons, whereas COX‐2 was almost exclusively expressed in neurons. PPARγ immunolabeling reached the peak 12 h after MCAO, whereas the number of COX‐2 immunostained cells gradually rose and reached its peak at 48 h. Intracerebroventricular infusion of pioglitazone, an agonist of the PPARγ, over a 5‐day period before and 2 days after MCAO, reduced the infarct size, the expression of tumor necrosis factor α (TNF‐α), COX‐2, and the number of cells positively stained for COX‐1 and COX‐2 in the peri‐infarct cortical regions. COX‐2 induction was also attenuated in the ipsilateral but not in the contralateral hippocampus. In primary cortical neurons expressing the PPARγ, pioglitazone suppressed COX‐2 expression in response to oxidative stress. This protective effect was reversed after cotreatment with GW 9662, a selective antagonist of the PPARγ, clearly demonstrating a PPARγ‐dependent mechanism. Our data provide evidence that activation of neuronal PPARγ considerably contributes to neuroprotection by prevention of COX‐2 up‐regulation in vitro and in peri‐infarct brain areas.—Zhao, Y., Patzer, A., Herdegen, T., Gohlke, P., Culma, J. Activation of cerebral peroxisome proliferator‐activated receptors gamma (PPARγ) promotes neuroprotection by attenuation of neuronal cyclooxygenase‐2 overexpression after focal cerebral ischemia in rats. FASEB J. 20, 1162–1175 (2006)

Chronic Treatment With a Low Dose of Lithium Protects the Brain Against Ischemic Injury by Reducing Apoptotic Death

Background and Purpose— In vitro and in vivo studies have demonstrated neuroprotective actions of lithium. The present study investigated the effect of a low dose of lithium on infarct volume and neurological outcome as well as on apoptotic and inflammatory processes in rats exposed to focal ischemia. Methods— Cerebral ischemia was induced by middle cerebral artery occlusion (MCAO) for 90 minutes followed by reperfusion. Lithium (1 mmol/kg) was given subcutaneously daily for 14 days before the onset of MCAO and 2 days thereafter. Blood parameters and cerebral blood flow were assessed before, during, and after MCAO. Rats were examined for neurological deficits 24 and 48 hours after MCAO. Two days after MCAO, the brains were removed for immunohistochemical evaluation of caspase-3, terminal deoxynucleotidyl transferase–mediated dUTP nick end labeling (TUNEL), activated microglia, and the expression of AP-1 proteins (c-Fos and c-Jun). Infarct volume was assessed by cresyl violet staining. Results— Pretreatment with lithium did not alter cerebral blood flow or blood parameters. Neurological deficits were significantly decreased in rats treated with lithium at 24 and 48 hours after ischemia. Infarct volume was reduced in rats treated with lithium at 48 hours after ischemia. Lithium significantly decreased the ischemia-induced caspase-3 immunoreactivity and TUNEL staining as well as the AP-1 protein expression in the penumbra of the ischemic cortex. No changes in activated microglia were observed. Conclusions— The present study demonstrates that chronic treatment with lithium at a low dose exhibits neuroprotection in transient focal cerebral ischemia. Antiapoptotic mechanisms are involved in the lithium-induced neuroprotective effects.

The intracerebral application of the PPARγ‐ligand pioglitazone confers neuroprotection against focal ischaemia in the rat brain

The present study addresses the neuroprotective function of intracerebroventricular (i.c.v.) application of pioglitazone, a selective ligand of the peroxisome proliferator‐activated receptor gamma (PPARγ) in the rat brain after ischaemia. Pioglitazone or vehicle were i.c.v. infused via osmotic minipumps over a 5‐day period before, and 2 days after transient middle cerebral artery occlusion (MCAO) for 90 min. This i.c.v. application of pioglitazone in the brain significantly reduced the infarct size and brain oedema, and attenuated in the peri‐infarct cortical regions the invasion of activated microglia and macrophages. Moreover, pioglitazone improved the recovery of sensory deficits 48 h after MCAO. Our data demonstrate for the first time that it is the activation of intracerebral PPARγ that can confer neuroprotection, anti‐inflammatory and neurological improvement following ischaemic injury.

The renin-angiotensin system in the brain: possible therapeutic implications for AT(1)-receptor blockers.

Biochemical, physiological and functional studies suggest that the brain renin-angiotensin system (RAS) is regulated independently of the peripheral RAS. The classical actions of angiotensin II in the brain include blood pressure control, drinking behaviour, natriuresis and the release of vasopressin into the circulation. At least two subtypes of G-protein coupled receptors, the AT1 and the AT2 receptor, have been identified. Most of the classic actions of angiotensin II in the brain are mediated by AT1 receptors. The AT2 receptor is involved in brain development and neuronal regeneration and protection. Additionally, AT2 receptors can modulate some of the classic angiotensin II actions in the brain. Selective non-peptide AT1 receptor blockers, applied systemically, have been shown to inhibit both peripheral and brain AT1receptors. In genetically hypertensive rats, inhibition of brain AT1 receptors may contribute to the blood pressure lowering effects of AT1 receptor blockers. Animal studies have shown that AT1 receptor antagonists enable endogenous angiotensin II to stimulate neuronal regeneration via activation of AT2 receptors. In animal models, inhibition of the brain RAS proved to be beneficial with respect to stroke incidence and outcome. Blockade of brain and cerebrovascular AT1 receptors by AT1 receptor blockers prevents the reduction in blood flow during brain ischaemia, reduces the volume of ischaemic injury and improves neurological outcome after brain ischaemia. This paper reviews the actions of angiotensin II and its receptors in the brain, and discusses the possible consequences of AT1 receptor blockade in neuroprotection, neuroregeneration, cerebral haemodynamics and ischaemia.

Sustained Blockade of Brain AT1 Receptors before and after Focal Cerebral Ischemia Alleviates Neurologic Deficits and Reduces Neuronal Injury, Apoptosis, and Inflammatory Responses in the Rat

In the present study, we investigate whether a long-term blockade of brain AT1 receptors in male Wistar rats before and after ischemic injury exerts neuroprotective effects and modulates apoptosis and inflammatory responses, which are associated with the post-ischemic progression of brain damage. The AT1 receptor antagonist irbesartan was continuously infused intracerebroventricularly using osmotic minipumps over a 5-day period before and for 3 or 7 days after middle cerebral artery occlusion (MCAO) for 90 minutes. Neurologic status was evaluated daily, starting 24 hours after MCAO. After MCAO (3 and 7 days), brains were removed for the measurement of infarct size and immunohistochemical evaluation of apoptosis and accumulation of reactive microglia and macrophages. Treatment with irbesartan before ischemia improved motor functions, whereas post-ischemic treatment improved sensory functions. Blockade of brain AT1 receptors reduced the infarct size on days 3 and 7 after MCAO. In the peri-infarct cortex, irbesartan treatment decreased the number of apoptotic cells on day 3 and attenuated the invasion of activated microglia and macrophages on days 3 and 7 after ischemia. Long-term blockade of brain AT1 receptors improves the recovery from cerebral ischemia. Antiapoptotic mechanisms and inhibition of post-ischemic inflammation are involved in the AT1 receptor blockade-induced neuroprotective effects in ischemic brain tissue.

Effects of orally applied candesartan cilexetil on central responses to angiotensin II in conscious rats.

Objective In the present study, we investigated the ability of the peripherally administered angiotensin II type 1 (AT1) receptor antagonist, candesartan cilexetil, to block central effects of angiotensin II (Ang II) in conscious rats. Design and methods Candesartan cilexetil was administered orally by gavage at doses of 0.1, 1, 10 and 30 mg/kg. Drinking response, pressor response and release of vasopressin into the circulation following intracerebroventricular (i.c.v.) Ang II (10 or 100 ng) were measured at 0.5, 2, 4 and 24 h following the drug application. The same parameters were measured after chronic treatment with candesartan cilexetil for 1 week. In a separate experiment, the release of vasopressin induced by microinjection of Ang II (100 ng) into the paraventricular nucleus (PVN) was determined 4 h after oral administration of candesartan cilexetil (1 mg/kg) or vehicle. Results Oral treatment with candesartan cilexetil inhibited all central responses to i.c.v. Ang II in a dose- and time-dependent manner. The Ang II-induced responses were inhibited 4 h after acute or chronic treatment with 0.1 mg/kg candesartan cilexetil, but had returned to control levels 24 h after drug application. In contrast, the highest dose of candesartan cilexetil (30 mg/kg) nearly abolished the central responses to Ang II for 24 h. Candesartan cilexetil completely blocked vasopressin release into the circulation induced by Ang II microinjection into the PVN. Conclusions Our results demonstrate that the AT1 receptor antagonist, candesartan cilexetil, very effectively inhibits the centrally mediated effects of Ang II upon peripheral application.

Tachykinin receptor inhibition and c-Fos expression in the rat brain following formalin-induced pain

Recent pharmacological evidence has implicated substance P and neurokinin A, natural ligands for neurokinin-1 and neurokinin-2 receptors, respectively, as neurotransmitters in brain neuronal circuits activated upon noxious stimulation. The expression of the inducible transcription factor, c-Fos, was used to identify areas in the brain activated by a noxious stimulus (the subcutaneous injection of formalin), and to investigate the effects of intracerebroventricular administration of selective, nonpeptide antagonists for neurokinin-1 and neurokinin-2 tachykinin receptors on the neural activity in these areas and on the behavioural response to formalin-induced pain. Formalin (5%, 50 microl), injected subcutaneously through a chronically implanted catheter in the region of the lower hindlimb, increased c-Fos expression in a number of brain areas related to nociceptive transmission or the integration of stress responses. Grooming behaviour, licking and biting directed to the injected site, was the most frequent behavioural response. Intracerebroventricular pretreatment of rats with either RP 67580 (500 pmol), the active enantiomer of a neurokinin-1 receptor antagonist, or with SR 48968 (500 pmol), the active enantiomer of a neurokinin-2 receptor antagonist, reduced the formalin-induced c-Fos staining in the prefrontal cortex, dorsomedial and ventromedial nuclei of the hypothalamus, the locus coeruleus and the periaqueductal gray. The neurokinin-1, but not the neurokinin-2, receptor antagonist attenuated the formalin-induced activation of c-Fos in the paraventricular nucleus of the hypothalamus. Simultaneous intracerebroventricular pretreatment with both neurokinin-1 and neurokinin-2 receptor antagonists did not produce any additional inhibitory effect on the post-formalin c-Fos expression. None of the tachykinin receptor antagonists had an effect on the formalin-induced c-Fos expression in the septohypothalamic nucleus, medial thalamus, parabrachial nucleus and central amygdaloid nucleus, indicating that neurotransmitters other than neurokinins are most probably responsible for the activation of these areas in response to noxious stimulation. While both tachykinin receptor antagonists reduced the grooming behaviour to formalin, the neurokinin-1 receptor antagonist was clearly more effective than the neurokinin-2 receptor antagonist. Intracerebroventricular pretreatment of rats with the inactive enantiomers of the tachykinin receptor antagonists, RP 68651 and SR 48965, was without effect. Our results show that (i) the modified formalin test elicited an intense grooming behaviour and expression of c-Fos in numerous forebrain and brainstem areas, (ii) both tachykinin receptor antagonists were able to attenuate the behavioural response to pain and to reduce the formalin-induced c-Fos expression in some, but not all, brain areas, and (iii) the neurokinin-1 antagonist, RP 67580, was more effective in inhibiting the behavioural response to formalin and the pain-induced activation of c-Fos than the antagonist for neurokinin-2 receptors, SR 48968, indicating that neurokinin-1 receptors are preferentially activated in neurokinin-containing pathways responding to noxious stimuli. Our results demonstrate that blockade of brain tachykinin receptors, especially of the neurokinin-1 receptor, reduces the behavioural response to pain and the pain-induced c-Fos activation in distinct brain areas which are intimately linked with nociceptive neurotransmission and the initiation and integration of central stress responses. Together with the previous findings of the inhibition of hypertensive and tachycardic responses to pain, the present data indicate that tachykinin receptor antagonists can effectively inhibit the generation of an integrated cardiovascular and behavioural response pattern to noxious stimuli.

Comparison between early and delayed systemic treatment with candesartan of rats after ischaemic stroke.

Objective The effects of candesartan treatment starting early (3 h) and delayed (24 h) after middle cerebral artery occlusion (MCAO) with reperfusion was investigated in normotensive rats. Methods Subcutaneous treatment with candesartan (0.3 and 3 mg/kg) or vehicle was initiated 3 or 24 h after the onset of MCAO and continued for seven consecutive days (n = 20 per group and timepoint). Neurological outcome was evaluated daily using two different scoring systems. Infarct and oedema volumes were determined in rats 2 or 7 days after MCAO. Mean arterial, systolic and diastolic blood pressures were recorded before and after the application of candesartan. Results Mean arterial, systolic and diastolic blood pressures were markedly decreased with the high dose, but only moderately decreased with the low dose of candesartan. Vehicle-treated rats showed marked neurological deficits 24 h after MCAO, which gradually improved with time. Candesartan improved neurological outcomes at all timepoints only when treatment was started 3, but not 24 h after MCAO. The infarct volume was reduced on days 2 and 7 after MCAO in rats treated with the low but not the high dose of candesartan. Conclusion The present study demonstrates that only an early but not a delayed onset of treatment with candesartan exerts neuroprotection after focal ischaemia. The degree of neurological impairments did not correlate with the infarct volume, which was reduced only after the low dose of candesartan. The high dose of candesartan failed to reduce the infarct volume, probably because of an excessive blood pressure decrease.