Neuroprotective Effect of VEGF-Mimetic Peptide QK in Experimental Brain Ischemia Induced in Rat by Middle Cerebral Artery Occlusion.

We investigated the effect of the VEGF-mimetic peptide, QK, on ischemic brain damage and on blood-brain barrier permeability in the rat. QK administered by the intracerebroventricular, intravenous, or intranasal route caused a 40% decrease in ischemic brain damage induced by permanent occlusion of the middle cerebral artery relative to that in controls. No increase in the volume of the ischemic hemisphere compared to that of the contralateral nonischemic hemisphere was observed in rats treated with QK, suggesting that this peptide did not cause brain edema. The effect of QK on vessel permeability was evaluated by intravital pial microvessel videoimaging, a technique that allows the pial vessels to be visualized through a surgically prepared open cranial window. The results showed that QK did not cause any leakage of intravenously injected fluorescein-dextran conjugates after intracarotid administration or topical application to the brain cortex. Collectively, these data suggest that QK may exert neuroprotective activity in the context of stroke without promoting any increase in vascular permeability. Because VEGF's neuroprotective activity may be overshadowed by the appearance of brain edema and microbleeds, QK could represent a significant step forward in stroke treatment.

Synaptic plasticity during recovery from permanent occlusion of the middle cerebral artery.

Synaptic rearrangements in the peri-infarct regions are believed to contribute to the partial recovery of function that takes place after stroke. Here, we performed neurophysiological recordings from single neurons of rats with permanent occlusion of the middle cerebral artery (pMCAO) during the resolution of their neurological deficits. Our results show that complex and dynamic changes of glutamate transmission in the peri-infarct area parallel the recovery from brain infarct. We have observed that frequency and duration of spontaneous glutamate-mediated synaptic events were markedly increased in striatal neurons during the early phase of the recovery (3 days after pMCAO), due to potentiation of both NMDA (N-methyl-d-aspartate) and non-NMDA receptor-mediated transmission. In the late phase of recovery (7 days after pMCAO), glutamate transmission was still enhanced because of a selective facilitation of non-NMDA receptor-mediated transmission. Spiny projection neurons but not aspiny interneurons underwent detectable changes of synaptic excitability in the striatum following pMCAO, indicating that the process of neuronal adaptation after focal brain ischemia is cell-type-specific. Our results provide a synaptic correlate of the long-lasting brain hyperexcitability mediating recovery described with noninvasive neurophysiological approaches.

Involvement of the potassium-dependent sodium/calcium exchanger gene product NCKX2 in the brain insult induced by permanent focal cerebral ischemia.

Sodium/calcium exchangers are neuronal plasma membrane transporters, which by coupling Ca2+ and Na+ fluxes, may play a relevant role in brain ischemia. The exchanger gene superfamily comprises two arms: the K+-independent (NCX) and K+-dependent (NCKX) exchangers. In the brain, three different NCX (NCX1, NCX2, NCX3) and three NCKX (NCKX2, NCKX3, NCKX4) family members have been described. Up to now, no sutides about the role played by NCKX proteins in cerebral ischemia have been published. The aim of the present study was to investigate the role of NCKX2 in an in vivo model of permanent middle cerebral artery occlusion (pMCAO). The role of this protein in the development of ischemic damage was assessed by knocking-down its expression with an antisense oligodeoxynucleotide (AS-ODN), intracerebroventricularly infused by an osmotic minipump for 48 h, starting from 24 h before pMCAO. The results showed that NCKX2 knocking-down by using antisense strategy increased the extent of the ischemic lesion. The results of this study suggest that NCKX2 could exert a neuroprotective effect during ischemic injury.

Na+/Ca2+ exchanger maintains ionic homeostasis in the peri-infarct area.

BACKGROUND AND PURPOSE: A prominent feature of cerebral ischemia is the excessive intracellular accumulation of both Na(+) and Ca(2+) ions, which results in subsequent cell death. The plasma membrane Na(+)/Ca(2+) exchanger (NCX), regulates the distribution of these ions acting either in the forward mode or in its reverse mode and it can play a critical role in brain ischemia. However, it is unclear whether the activity of NCX leads to detrimental or beneficial effects. METHODS: Extracellular field potentials and whole-cell patch clamp recordings were obtained from rat corticostriatal brain-slice preparations in the peri-infarct area 24 hours after the permanent middle cerebral artery occlusion. Ischemia was induced in rats by permanent middle cerebral artery occlusion. RESULTS: Bepridil, an inhibitor of NCX, reduced in a concentration-dependent manner (IC(50)=68 micromol/L) the field potential amplitude recorded from the peri-infarct area of corticostriatal slices. Conversely, no change was observed in sham-operated animals. The effect of bepridil was mimicked by 5-(N-4-chlorobenzyl)-2',4'-dimethylbenzamil (CB-DMB) (IC(50)=6 micromol/L), a more selective inhibitor of NCX. In whole-cell patch clamp experiments, bepridil and CB-DMB caused an inward current in spiny neurons recorded from the peri-infarct area but not in the same cells recorded from controls. Interestingly, cholinergic interneurons recorded from the striatal peri-infarct area did not develop an inward current after the application of NCX inhibitors, suggesting that the electrophysiological alterations induced by NCX inhibition are cell-type specific. Bepridil and CB-DMB also induced a suppression of excitatory synaptic currents in most of spiny neurons recorded from the peri-infarct area. This effect was not coupled to a significant change of paired-pulse facilitation suggesting a postsynaptic site of action. CONCLUSIONS: Our data indicate that NCX plays a critical role in the maintenance of ionic homeostasis in the peri-infarct area.

NR2B subunit exerts a critical role in postischemic synaptic plasticity.

BACKGROUND AND PURPOSE: We characterized the differential effect of the NR2B subunit antagonist ifenprodil in the induction of activity-dependent long-term potentiation (LTP) and of postischemic LTP as well as in the neuronal damage induced by focal ischemia. METHODS: Intracellular recordings were obtained from rat corticostriatal slice preparations. High-frequency stimulation of corticostriatal fibers was used as a LTP-inducing protocol. In vitro ischemia was induced by oxygen and glucose deprivation. In vivo ischemia was induced by permanent middle cerebral artery occlusion. Intracellular recordings were also performed in the ischemic penumbra. RESULTS: Antagonists selectively targeting N-methyl-d-aspartate receptors containing the NR2B subunit blocked postischemic LTP without affecting activity-dependent LTP. In a model of focal ischemia, blockade of NR2B subunit in vivo caused reduction of brain damage, amelioration of neurological outcome, and normalization of the synaptic levels of NR2B subunits. Moreover, the antagonism of NR2B subunit was able to rescue the activity-dependent LTP in the ischemic penumbra. CONCLUSIONS: We suggest that NR2B subunits contribute to the striatal damage caused by in vivo and in vitro ischemia and play a critical role in the induction of postischemic LTP as well as in the suppression of activity-dependent LTP in the ischemic penumbra.

Two sodium/calcium exchanger gene products, NCX1 and NCX3, play a major role in the development of permanent focal cerebral ischemia.

BACKGROUND AND PURPOSE: The Na+/Ca2+ exchanger, by mediating Ca2+ and Na+ fluxes in a bidirectional way across the synaptic plasma membrane, may play a pivotal role in the events leading to anoxic damage. In the brain, there are 3 different genes coding for 3 different proteins: NCX1, NCX2, and NCX3. The aim of this study was to determine whether NCX1, NCX2, and NCX3 might play a differential role in the development of cerebral injury induced by permanent middle cerebral artery occlusion (pMCAO). METHODS: By means of Western blotting, NCX1, NCX2, and NCX3 protein expression was evaluated in the ischemic core and in the remaining nonischemic area of the slice at different time intervals starting from ischemia induction. The role of each isoform was also assessed with antisense oligodeoxynucleotides (ODNs) targeted for each isoform. These ODNs were continuously intracerebroventricularly infused with an osmotic minipump (1 microL/h) for 48 hours, 24 hours before pMCAO. RESULTS: The results showed that after pMCAO all 3 NCX proteins were downregulated in ischemic core; NCX3 decreased in periinfarctual area whereas NCX1 and NCX2 were unchanged. The ODNs for NCX1 and NCX3 gene products were capable of inducing an increase in the ischemic lesion and to worsen neurological scores. CONCLUSIONS: The results of this study suggest that in the neuroprotective effect exerted by NCX during ischemic injury, the major role is prevalently exerted by NCX1 and NCX3 gene products.

Evidence for a protective role played by the Na+/Ca2+ exchanger in cerebral ischemia induced by middle cerebral artery occlusion in male rats.

In the present paper, the role played by Na+/Ca2+ exchanger (NCX) in focal cerebral ischemia was investigated. To this aim, permanent middle cerebral artery occlusion (pMCAO) was performed in male rats. The effects on the infarct volume of some inhibitors, such as tyrosine-6 glycosylated form of the exchanger inhibitory peptide (GLU-XIP), benzamil derivative (CB-DMB) and diarylaminopropylamine derivative (bepridil), and of the NCX activator, FeCl3, were examined. FeCl3, CB-DMB, bepridil and GLU-XIP, a modified peptide synthesized in our laboratory in order to facilitate its entrance into the cells through the glucose transporter, were intracerebroventricularly (i.c.v.) infused. FeCl3 (10 microg/kg) was able to reduce the extension of brain infarct volume. This effect was counteracted by the concomitant icv administration of CB-DMB (120 microg/kg). All NCX inhibitors, GLU-XIP, CB-DMB and bepridil, caused a worsening of the brain infarct lesion. These results suggest that a stimulation of NCX activity may help neurons and glial cells that are not irreversibly damaged in the penumbral zone to survive, whereas its pharmacological blockade can compromise their survival.