Periprocedural monitoring with regional cerebral oxygen saturation in carotid artery stenting.

SUMMARY: Hemodynamic instability during and after carotid artery stenting (CAS) may reduce cerebral blood flow (CBF), leading to cerebral ischemia. To investigate changes in CBF in the periprocedural period, we continuously recorded the regional cerebral oxygen saturation (rSO2) using near-infrared spectroscopy. In 46 consecutive patients with carotid artery stenosis, rSO2 was continuously recorded during and after CAS. In addition, the patients underwent SPECT to evaluate a change in CBF on the next day after CAS. Introprocedural bradycardia (heart rate < 50 bpm) occurred in 21 patients (46%) including one transient cardiac arrest. Intraprocedural hypotension (systolic blood pressure < 80 mmHg) occurred in 18 patients (39%), and 16 of them showed prolonged hypotension. The rSO2 in patients with bradycardia/hypotension during CAS was significantly less than that in patients without them (p < 0.01). Moreover, the SPECT on the next day after CAS demonstrated that the ipsilateral CBF in patients with bradycardia/hypotension during CAS significantly more than that in patients without them (p < 0.05). Intraprocedural hemodynamic instability resulted in a significant decrease in rSO2, leading to a possible severe cerebral ischemia. In addition, intraprocedural bradycardia/hypotension might be related with postprodedural hyperperfusion, causing the morbidity and mortality after CAS.

Transgenic CuZn-superoxide dismutase inhibits NO synthase induction in experimental subarachnoid hemorrhage.

BACKGROUND AND PURPOSE: The expression of inducible NO synthase (iNOS) after experimental subarachnoid hemorrhage (SAH) has been postulated to play a critical role in the pathogenesis of SAH and subsequent cerebral vasospasm. The inhibitory effect of CuZn-superoxide dismutase (CuZn-SOD) on the induction of iNOS after SAH was examined by using transgenic mice overexpressing CuZn-SOD. METHODS: SOD-transgenic mice and nontransgenic littermates were subjected to SAH by endovascular perforation of the left anterior cerebral artery. The iNOS mRNA expression after SAH was determined by reverse transcription-polymerase chain reaction, and the distribution of iNOS-positive cells was immunohistochemically examined. The nuclear expression of activated nuclear factor-kappaB, a major transcription factor of iNOS gene, was also immunohistochemically examined. RESULTS: In nontransgenic mice, SAH-induced iNOS protein and mRNA expressions in the arteries of basal cistern as well as in the cerebral cortex were demonstrated by immunohistochemistry and reverse transcription-polymerase chain reaction. SAH-induced iNOS protein and mRNA expressions in those tissues were much reduced in SOD-transgenic mice compared with nontransgenic mice. Moreover, the nuclear expression of the activated form of nuclear factor-kappaB was immunohistochemically detected in the cerebral cortices of nontransgenic mice but not in those of SOD-transgenic mice. CONCLUSIONS: These results indicate that oxygen-derived free radicals, particularly superoxide, play an important role in the iNOS gene expression after SAH and provide a molecular basis for the protective role of SOD against vasospasm after SAH.

Activation of Arc gene, a dendritic immediate early gene, by middle cerebral artery occlusion in rat brain.

The effect of middle cerebral artery (MCA) occlusion on the activity-regulated cytoskeleton-associated protein (Arc) mRNA expression has been investigated using in situ hybridization. It was induced in the extensive regions of cerebral cortex, medial striatum, and distant areas such as the ipsilateral lateral septal nucleus, bilateral hippocampal formation and contralateral amygdala following MCA occlusion. In the hippocampal formation, it was induced in the granule cell layer and the stratum pyramidale at 1 h and in the molecular layer and in the stratum oriens and stratum radiatum bilaterally at 4 h. MK-801 pretreatment strongly attenuated the induction of Arc mRNA. The present results suggest that Arc may play an important role in the neuronal plasticity through NMDA activation following focal cerebral ischemia.

Induction of Rheb mRNA following middle cerebral artery occlusion in the rat.

Rheb is a recently identified member of the Ras super-family and is an immediate early gene that is rapidly and transiently induced in the hippocampal granule cells by NMDA-dependent synaptic activity in the long term potentiation paradigm. The close homologies with Ras and its rapid inducibility strongly suggest that Rheb shares many biochemical and signaling properties with Ras. The present study investigated the effect of middle cerebral artery (MCA) occlusion on the expression of Rheb mRNA in the rat brain. In situ hybridization autoradiography showed that Rheb mRNA was induced in the extensive regions of cerebral cortex and medial striatum surrounding the ischemic region and bilateral hippocampal formation following MCA occlusion. The induction of Rheb mRNA in the cingulate cortex persisted prominently at 24 h of MCA occlusion. Although the Rheb mRNA induction in the medial striatum and hippocampal formation decreased after 8h of occlusion, it still remained significant at 24h of occlusion. The data suggest the possibility that Ras signaling pathways can be implicated in the cerebral ischemia-elicited events through NMDA receptor activation.

Amelioration of vasospasm after subarachnoid hemorrhage in transgenic mice overexpressing CuZn-superoxide dismutase.

BACKGROUND AND PURPOSE: To clarify the effect of superoxide dismutase (SOD) on vasospasm after subarachnoid hemorrhage (SAH), we investigated sequential changes in arterial diameter after SAH in transgenic mice overexpressing CuZn-SOD (SOD-1). METHODS: SOD-transgenic mice and nontransgenic littermates (35 to 40 g) were subjected to SAH produced by endovascular perforation of left anterior cerebral artery. At 4 hours and 1, 3, 7, and 14 days after SAH, the mice were perfused with 10% formalin and consequently with a mixture of carbon black and 10% gelatin to cast all vessels. Vasospasm was evaluated by measuring the diameter of the left middle cerebral artery (MCA) with a microscope. RESULTS: In nontransgenic mice, the diameter of the MCA on day 3 after SAH (110.5+/-20.5 microm [mean+/-SD]; n=16) was significantly reduced compared with that without SAH (138.5+/-14.5 microm; n=12) (P<0.01). Moreover, on day 3 after SAH, the diameter of the MCA in SOD-transgenic mice (127. 9+/-20.2 microm; n=20) was significantly larger than that in nontransgenic mice (110.5+/-20.5 microm; n=16) (P<0.05). CONCLUSIONS: These results suggest that SOD is effective on the amelioration of vasospasm after SAH and that oxygen free radicals, particularly superoxide, play an important role in the pathogenesis of vasospasm after SAH.

Role of superoxide dismutase in ischemic brain injury: a study using SOD-1 transgenic mice.

1. Nitric oxide radicals (NO) play an important role in the pathophysiology of focal cerebral ischemia. 2. Vascular NO can reduce ischemic brain injury by increasing CBF, whereas neuronal NO may mediate neurotoxicity following brain ischemia, mainly by its reaction with superoxide to generate peroxynitrite. 3. These findings could contribute to a strategy for the treatment of cerebral ischemia.

Attenuation of acute and chronic damage following traumatic brain injury in copper, zinc-superoxide dismutase transgenic mice.

To elucidate the role of oxygen-derived free radicals and superoxide dismutase in traumatic brain injury (TBI), blood-brain barrier (BBB) permeability, brain edema, behavioral function, and necrotic cavity volume (CV) were evaluated after TBI using nontransgenic (nTg) mice and heterozygous and homozygous transgenic (Tg) mice with a 1.5- (Tg 1.5x), 3.1-(Tg3.1x) and five- (Tg5x) fold increase in human copper, zinc-superoxide dismutase (CuZn-SOD) activity. Traumatic brain injury was produced by the weight-drop method. Evans blue dye leakage 4 hours after injury was attenuated in a CuZn-SOD dose-dependent manner with decreases of 18.6%, 40.9%, and 48.8%, in the Tg1.5x, Tg3.1x, and Tg5x groups, respectively. The water content 6 hours after injury in the Tg3.1x (79.64%) and Tg5x (79.45%) groups was significantly lower than in nTg mice (81.37%). There was an initial decrease in body weight and in motor performance, as measured by beam walk and beam balance tasks undertaken 1 day after TBI. However, the average reduction in beam balance and beam walk performance deficits and changes in body weight postinjury were significantly ameliorated in Tg mice. The CV was significantly smaller in Tg mice than in nTg mice (p < 0.01). These results indicate that superoxide radicals play a deleterious role following TBI. Furthermore, Tg mice provide a useful model for demonstrating the beneficial role of an antioxidant enzyme in TBI without the confounding effect of pharmacokinetics, toxicity, and BBB permeability associated with exogenous agents.

Effects of nitric oxide synthase inhibition on brain infarction in SOD-1-transgenic mice following transient focal cerebral ischemia.

To investigate the role of superoxide in the toxicity of nitric oxide (NO), we examined the effect of nitric oxide synthase (NOS) inhibition on brain infarction in transgenic mice overexpressing CuZn-superoxide dismutase (SOD-1). Male SOD-transgenic mice and non-transgenic littermates (30-35 g) were subjected to 60 min of middle cerebral artery occlusion followed by 24 h of reperfusion. Either NG-nitro-L-arginine methyl ester (L-NAME; 3 mg/kg), a mixed neuronal and endothelial NOS inhibitor, or 7-nitroindazole (7-NI; 25 mg/kg), a selective neuronal NOS inhibitor, was administered intraperitoneally 5 min after the onset of ischemia. At 24 h of reperfusion, the mice were decapitated and the infarct volume was evaluated in each group. In the nontransgenic mice, L-NAME significantly increased the infarct volume as compared with the vehicle, while 7-NI significantly decreased it. In the SOD-transgenic mice, L-NAME-treated animals showed a significantly larger infarct volume than vehicle-treated ones, whereas there were no significant differences between 7-NI- and vehicle-treated mice. Our findings suggest that selective inhibition of neuronal NOS ameliorates ischemic brain injury and that both neuronal and endothelial NOS inhibition may result in the deterioration of ischemic injury due to vasoconstriction of the brain. Since L-NAME increased infarct volume even in SOD-transgenic mice, the protective effect of SOD could result from the vasodilation by increased endothelial NO as well as the reduction of neuronal injury due to less production of peroxynitrite compared to wild-type mice. Moreover, the neurotoxic role of NO might not be dependent on NO itself, but the reaction with superoxide to form peroxynitrite, because of no additive effects of SOD and a neuronal NOS inhibitor.

Induction of brain-derived neurotrophic factor (BDNF) and the receptor trk B mRNA following middle cerebral artery occlusion in rat.

Middle cerebral artery (MCA) occlusion in halothane-anesthetized rats induced brain-derived neurotrophic factor (BDNF) and the receptor, trk B mRNA, in brain. In situ hybridization studies showed that BDNF and trk B mRNAs were induced in a widespread region of the ipsilateral cortex outside the infarct at 4 h following MCA occlusion. They were also induced in the bilateral hippocampi which are remote from the ischemic MCA region. These data show that changes in neurotrophic factor and receptor gene expressions can occur in the areas outside the infarct which could survive.

Expression of c-fos and hsp70 mRNA after traumatic brain injury in transgenic mice overexpressing CuZn-superoxide dismutase.

The aim of this study was to determine the role of oxidative stress on c-fos and hsp70 gene expression in transgenic (Tg) mice overexpressing CuZn-superoxide dismutase (SOD-1) following traumatic brain injury (TBI). hsp70 mRNA, as investigated using in situ hybridization, was induced around the lesion at 4 and 24 h, but not at 1 and 48 h, in both Tg and non-transgenic (nTg) mice littermates. The degree of hsp70 induction was somewhat greater in nTg than Tg mice at 4 and 24 h after TBI. c-fos mRNA was induced throughout cortex, hippocampus, caudate putamen and the ventricular wall in Tg and nTg mice. TBI induced c-fos bilaterally in the cortex in both animals. There was a time-dependent difference in cortical c-fos expression between nTg and Tg mice. The induction of c-fos mRNA in the striatum was greater in nTg at 24 h and decreased in both animals by 48 h. Edema of the injured cortex was significantly attenuated in Tg mice at all time points (1-48 h). These data show that the degree of hsp70 induction and the degree, extent, and duration of c-fos induction produced by TBI are affected by levels of superoxide dismutase activity. It is proposed that superoxide radicals affect spreading depression and brain edema produced by TBI and that this effect may either directly or indirectly modulate the expression of the c-fos and hsp70 genes after TBI.

Alcohol dehydrogenase (ADH) isozymes in the AdhN/AdhN strain of Peromyscus maniculatus (ADH-deermouse) and a possible role of class III ADH in alcohol metabolism.

Although the AdhN/AdhN strain of Peromyscus maniculatus (so-called ADH- deermouse) has been previously considered to be deficient in ADH, we found ADH isozymes of Classes II and III but not Class I in the liver of this strain. On the other hand, the AdhF/AdhF strain (so-called ADH+ deermouse), which has liver ADH activity, had Class I and III but not Class II ADH in the liver. In the stomach, Class III and IV ADHs were detected in both deermouse strains, as well as in the ddY mouse, which has the normal mammalian ADH system with four classes of ADH. These ADH isozymes were identified as electrophoretic phenotypes on the basis of their substrate specificity, pyrazole sensitivity, and immunoreactivity. Liver ADH activity of the ADH- strain was barely detectable in a conventional ADH assay using 15 mM ethanol as substrate; however, it increased markedly with high concentrations of ethanol (up to 3 M) or hexenol (7 mM). Furthermore, in a hydrophobic reaction medium containing 1.0 M t-butanol, liver ADH activity of this strain at low concentrations of ethanol (< 100 mM) greatly increased (about sevenfold), to more than 50% that of ADH+ deermouse. These results were attributable to the presence of Class III ADH and the absence of Class I ADH in the liver of ADH- deermouse. It was also found that even the ADH+ strain has low liver ADH activity (< 40% that of the ddY mouse) with 15 mM ethanol as substrate, probably due to low activity in Class I ADH. Consequently, liver ADH activity of this strain was lower than its stomach ADH activity, in contrast with the ddY mouse, whose ADH activity was much higher in the liver than in the stomach, as well as other mammals. Thus, the ADH systems in both ADH- and ADH+ deermouse were different not only from each other but also from that in the ddY mouse; the ADH- strain was deficient in only Class I ADH, and the ADH+ strain was deficient in Class II ADH and down-regulated in Class I ADH activity. Therefore, Class III ADH, which was found in both strains and activated allosterically, may participate in alcohol metabolism in deermouse, especially in the ADH- strain.

Transgenic mice and knockout mutants in the study of oxidative stress in brain injury.

A rapid increase in the need to explore the molecular basis of cellular function and injury in the central nervous system has led neuroscientists to employ transgenic mouse technology. The successful making of transgenic mice (Tg) overexpressing human CuZn-superoxide dismutase (SOD-1) activity has made it possible to investigate the role of oxygen free radicals in ischemic and traumatic brain injury in a molecular fashion. It has been demonstrated that the 3-fold increase in SOD-1 transgene activity in SOD-1 Tg mice offers protection against cerebral ischemia and reperfusion in two different models of focal cerebral ischemia, as compared to nontransgenic wild-type littermates. Studies involving traumatic brain injury have also demonstrated that acute injuries, including brain edema and blood-brain barrier permeability, are significantly reduced in SOD-1 Tg mice. Furthermore, chronic neurological deficits, such as beam walking, beam balance, and body weight, are significantly improved in these transgenic animals following traumatic brain injury. In addition to the SOD-1 Tg mice being a useful tool for the study of CNS injury, targeted disruption of the mouse gene for mitochondrial manganese SOD (SOD-2) has been successful. These SOD-2 knockout mutant mice, in addition to the recently developed knockout mutants of neuronal nitric oxide synthase (NOS), are believed to offer a unique opportunity to elucidate the oxidative mechanisms in brain injury following stroke and trauma.

Expression of c-fos mRNA after a mild focal cerebral ischemia in SOD-1 transgenic mice.

To clarify the role of oxygen free radicals in expression of the c-fos protooncogene, the distribution of c-fos mRNA was investigated in CuZn-superoxide dismutase (SOD-1) transgenic (Tg) mice compared to control nontransgenic (nTg) littermates after a mild (i.e. 10 min) focal cerebral ischemia. c-fos mRNA expression occurred at 1 to 6 h after reperfusion in the ipsilateral hippocampus and thalamus in Tg mice, whereas it did only at 1 h in the same regions in nTg mice. In the ipsilateral cortex, there were no significant differences in the pattern of the expression between nTg and Tg mice. These results suggest that oxygen radicals may suppress the expression of c-fos in the hippocampus and thalamus, the areas known to be without blood supply from the middle cerebral artery, following a mild focal cerebral ischemia and reperfusion.

MK-801 inhibits the induction of immediate early genes in cerebral cortex, thalamus, and hippocampus, but not in substantia nigra following middle cerebral artery occlusion.

Middle cerebral artery (MCA) occlusion in rats induced c-fos and junB mRNA 4h later in all ipsilateral cortex outside the MCA distribution and in many subcortical structures: medial striatum; most of thalamus including medial and lateral geniculate nuclei: substantia nigra; and hippocampus. The N-methyl-D-aspartate (NMDA) antagonist, MK-801 (4 mg/kg, i.p.) inhibited c-fos and junB mRNA induction in the cortex, striatum, thalamus, and hippocampus but not in the substantia nigra. These data show that c-fos and junB mRNA induction in cortex, striatum, thalamus, hippocampus involves the activation of NMDA receptors whereas different receptors must be implicated in the induction in substantia nigra.

Prolonged expression of hsp70 mRNA following transient focal cerebral ischemia in transgenic mice overexpressing CuZn-superoxide dismutase.

The distribution of heat shock protein hsp70 mRNA after 10 min of middle cerebral artery (MCA) occlusion was investigated through in situ hybridization in transgenic (Tg) mice overexpressing CuZn-superoxide dismutase (CuZn-SOD) and in control nontransgenic (nTg) littermates. In the ischemic cortex of nTg mice, hsp70 mRNA was detected 1 h after reperfusion and was observed for up to 6 h. In Tg mice, however, it was still detectable within the cortex even at 24 h. In the caudate putamen, hsp70 mRNA appeared at 1 h and was present for up to 6 h in both nTg and Tg mice. Although hsp70 mRNA was detected in the thalamus only at 1 h in nTg mice, it was observed for up to 6 h in Tg mice. Similarly, hsp70 mRNA was detected in the hippocampus of nTg mice only at 1 h, whereas it was detected in Tg mice at 1 h and continued up to 24 h, with high intensity in the CA1 subfield. Despite the significant amounts of hsp70 mRNA in both Tg and nTg mice following ischemia, there was no observable neuronal necrosis (as assessed using hematoxylin and eosin staining) for up to 7 days. Cortical cerebral blood flow (CBF), measured by laser-Doppler flowmetry, did not differ between nTg and Tg mice during ischemia and reperfusion, despite exhibiting hyperemia following hypoperfusion. These results suggest that oxidative stress affects the expression of hsp70 following temporary focal ischemia. An alteration in oxidation stress, which resulted from reduced levels of superoxide radicals in the presence of the CuZn-SOD transgenes, may permit the prolonged expression of hsp70.(ABSTRACT TRUNCATED AT 250 WORDS)

Human copper-zinc superoxide dismutase transgenic mice are highly resistant to reperfusion injury after focal cerebral ischemia.

BACKGROUND AND PURPOSE: We have demonstrated in a previous study that superoxide radicals play a role in the pathogenesis of cerebral infarction, using a transgenic mouse model of distal middle cerebral artery occlusion, permanent ipsilateral cerebral carotid artery occlusion, and 1-hour contralateral cerebral carotid artery occlusion that produced infarction only in the cortex. However, the role of superoxide radicals in reperfusion injury in transgenic mice overexpressing superoxide dismutase (SOD) is unknown. Using a mouse model of intraluminal blockade of middle cerebral artery that produced both cortical and striatal infarction, we now further examined the role of superoxide radicals in ischemic cerebral infarction after reperfusion in transgenic mice overexpressing human CuZn-SOD activity. METHODS: Transgenic mice of strain Tg HS/SF-218, carrying human SOD-1 genes, and nontransgenic littermates were anesthetized with chloral hydrate (350 mg/kg IP) and xylazine (4 mg/kg IP). Physiological parameters were maintained at a normal range using a 30% O2/70% N2O gas mixture inserted via an inhalation mask. Body temperature was maintained at 37 +/- 0.5 degrees C by using a heating pad throughout the studies. The middle cerebral artery occlusion was achieved with a 5-0 rounded nylon suture placed within the internal cerebral artery for 3 hours followed by the removal of the suture to allow reperfusion for another 3 hours. Cerebral infarct size in brain slices and infarct volume, neurological deficit, cortical blood flow, and glutathione levels were measured in both transgenic and nontransgenic mice. RESULTS: Compared with the nontransgenic mice, the infarcted areas were significantly decreased in coronal slices from transgenic mice. The infarct volume (in cubic millimeters) was reduced by 26% in transgenic mice after ischemia and reperfusion. This decrease in the infarct volume in transgenic mice closely paralleled the reduced neurological deficits. Introduction of the suture to block blood supply to the middle cerebral artery territory produced a rapid decrease in the relative surface blood flow in the ipsilateral core and the peri-ischemic (penumbra) areas. There were no significant differences in the local cerebral blood flow in the ischemic core or the penumbra areas between the transgenic and nontransgenic groups. However, the level of reduced glutathione in the penumbra area was significantly higher in transgenic mice than in nontransgenic mice, whereas there was no difference in the reduced glutathione levels in the ischemic core between these two groups. CONCLUSIONS: Our study demonstrated that superoxide radicals play a major role in the pathogenesis of cerebral infarction in reperfusion injury after a focal stroke. The reduction in infarct volume and neurological deficits is not dependent on the changes in cerebral blood flow but rather correlate with reduced oxidative stress in the ischemic brain tissue, which was indicated by the relatively high levels of endogenous reduced glutathione in transgenic mice.

Brain infarction is not reduced in SOD-1 transgenic mice after a permanent focal cerebral ischemia.

Using a mouse model with intraluminal blockade of the middle cerebral artery (MCA) which produced both cortical and striatal infarction, the effect that superoxide radicals have on cerebral infarction, local cerebral blood flow, and neurological deficits after 24 h of permanent focal cerebral ischemia in transgenic mice (Tg) overexpressing human CuZn-superoxide dismutase (SOD-1) was examined. There were no difference between SOD-1 Tg mice and non-Tg littermates observed in the infarct areas of brain slices, the infarct volume, the local cerebral blood flow, or the neurological deficits. These data suggest that pre-existing high levels of antioxidant enzyme failed to provide neuronal protection against permanent focal cerebral ischemia.

Induction of heat shock hsp70 mRNA and HSP70 kDa protein in neurons in the 'penumbra' following focal cerebral ischemia in the rat.

Induction of hsp70 heat shock protein (HSP70) and hsp70 mRNA was examined using adjacent sections in the same rat brain following permanent middle cerebral artery (MCA) occlusions, hsp70 mRNA was induced within 4 h of MCA occlusion and persisted for at least 24 h. Cellular resolution autoradiographs suggested that hsp70 mRNA was induced primarily in neurons in the periphery of ischemia both outside and inside of the infarction, with small amounts of hsp70 mRNA being induced in the core of the infarction. HSP70 protein was localized in neurons outside the infarction and in endothelial cells within the infarction at 24 h but not at 4 h following permanent MCA occlusions. It is proposed that the penumbra, one of the areas that can be rescued by pharmacological agents, can be defined anatomically as the volume of tissue outside the area of infarction in which HSP70 protein is expressed primarily in neurons.