Robert D. Pearlstein

Exposure of the dorsal root ganglion in rats to pulsed radiofrequency currents activates dorsal horn lamina I and II neurons.

OBJECTIVE Application of pulsed radiofrequency (RF) currents to the dorsal ganglion has been reported to produce long-term relief of spinal pain without causing thermal ablation. The present study was undertaken to identify spinal cord neurons activated by exposure of the dorsal ganglion to pulsed RF currents in rats. METHODS Left-sided hemilaminectomy was performed in adult Sprague-Dawley rats to expose the C6 dorsal root ganglion. An RF electrode (0.5 mm diameter) with a thermocouple for temperature monitoring was positioned on the exposed ganglion, and rats were assigned to one of three treatment groups: pulsed RF treatment (20 ms of 500-kHz RF pulses delivered at a rate of 2 Hz for 120 s to produce tissue heated to 38°C), continuous RF (continuous RF currents for 120 s to produce tissue heated to 38°C), or sham treatment (no RF current; electrode maintained in contact with ganglion for 120 s). RESULTS Treatment with pulsed RF but not continuous RF was associated with a significant increase in the number of cFOS-immunoreactive neurons in the superficial laminae of the dorsal horn as observed 3 hours after treatment. CONCLUSION Exposure of the dorsal ganglion to pulsed RF currents activates pain-processing neurons in the dorsal horn. This effect is not mediated by tissue heating.

Simvastatin Increases Endothelial Nitric Oxide Synthase and Ameliorates Cerebral Vasospasm Resulting From Subarachnoid Hemorrhage

Background and Purpose— Endothelial nitric oxide synthase (eNOS) activity is decreased after subarachnoid hemorrhage (SAH). Simvastatin increases eNOS activity. We hypothesized that simvastatin would increase eNOS protein and ameliorate SAH-induced cerebral vasospasm. Methods— Mice were treated with subcutaneous simvastatin or vehicle for 14 days and then subjected to endovascular perforation of the right anterior cerebral artery or sham surgery. Three days later, neurological deficits were scored (5 to 27; 27=normal), and middle cerebral artery diameter and eNOS protein were measured. The study was repeated, but simvastatin treatment was started after SAH or sham surgery. Results— In SAH mice, simvastatin pretreatment increased middle cerebral artery diameter (SAH-simvastatin=74±22 &mgr;m, SAH-vehicle=52±18 &mgr;m, P =0.03; sham-simvastatin=102±8 &mgr;m, sham-vehicle=105±6 &mgr;m). Pretreatment reduced neurological deficits (SAH-simvastatin=25±2, SAH-vehicle=20±2, P =0.005; sham-simvastatin and sham-vehicle=27±0). Simvastatin pretreatment also increased eNOS protein. Simvastatin posttreatment caused a modest increase in middle cerebral artery diameter in SAH mice (SAH-simvastatin=56±12 &mgr;m, SAH-vehicle=45±4 &mgr;m, P =0.03; sham-simvastatin=92±13 &mgr;m, sham-vehicle=99±10 &mgr;m) and reduced neurological deficits (SAH-simvastatin=21±1, SAH-vehicle=19±2, P =0.009). Simvastatin posttreatment did not significantly increase eNOS protein. Conclusions— Simvastatin treatment before or after SAH attenuated cerebral vasospasm and neurological deficits in mice. The mechanism may be attributable in part to eNOS upregulation.

Apolipoprotein E Isoform-Specific Differences in Outcome from Focal Ischemia in Transgenic Mice:

Apolipoprotein E (apoE), a 34-kD glycosylated lipid-binding protein, is expressed as three common isoforms in humans (E2, E3, or E4). Clinical evidence suggests that the apoE genotype (APOE) may be a risk factor for poor outcome after acute central nervous system injury. This was examined further in transgenic mice constructed with the human APOE3 or APOE4 gene under the control of human promoter and tissue expression elements. Presence of human apoE3 and apoE4 proteins in brains of human APOE homozygous transgenic mice was confirmed by Western blotting. APOE3 (n = 12) and APOE4 (n = 10) mice underwent 60 minutes of middle cerebral artery occlusion. After 24-hour recovery, infarct size was measured. Infarct volumes (mean ± standard deviation) were smaller in the APOE3 group (cortex: APOE3 = 18 ± 4 mm3; APOE4 = 30 ± 11 mm3, P = 0.04; subcortex: APOE3 = 12 ± 4 mm3; APOE4 = 18 ± 4 mm3, P = 0.003). Hemiparesis was less severe in APOE3 mice (P = 0.02). These data indicate that human isoform-specific effects of apoE are relevant to acute pathomechanisms of focal ischemic brain damage when examined in the mouse. APOE transgenic mice may provide an appropriate model to examine the mechanistic basis for the differential effects of human apoE isoforms in acute central nervous system injury.

Differential Effects of Anesthetic Agents on Outcome from Near-complete but Not Incomplete Global Ischemia in the Rat

BackgroundIt has been postulated that anesthetic agents that reduce cerebral metabolic rate will protect the brain against ischemia when electroencephalographic (EEG) activity is persistent, but will provide no protection when ischemia is severe enough to cause EEG isoelectricity. No outcome studies

Mice overexpressing extracellular superoxide dismutase have increased resistance to focal cerebral ischemia.

Transgenic mice, which had been transfected with the human extracellular superoxide dismutase gene, causing an approximate five-fold increase in brain parenchymal extracellular superoxide dismutase activity, were used to investigate the role of extracellular superoxide dismutase in ischemic brain injury. Transgenic (n = 21) and wild-type (n = 19) mice underwent 90 min of intraluminal middle cerebral artery occlusion and 24 h of reperfusion. Severity of resultant hemiparesis and cerebral infarct size were measured. Wild-type mice had larger infarcts (cortex: wild type =37+/-14 mm3, transgenic = 27+/-13 mm3, P=0.03; subcortex: wild type = 33+/-14 mm3, transgenic = 23+/-10 mm3, P = 0.02). Neurological scores, however, were similar (P = 0.29). Other mice underwent autoradiographic determination of intra-ischemic cerebral blood flow. The volume of tissue at risk of infarction (defined as volume of tissue where blood flow was <25 ml/100g/min) was similar between groups (cortex: wild type = 51+/-15 mm3, transgenic = 47+/-9 mm3, P=0.65; subcortex: wild type = 39+/-16 mm3, transgenic= 37+/-17 mm3, P=0.81). These results indicate that antioxidant scavenging of free radicals by extracellular superoxide dismutase plays an important role in the histological response to a focal ischemic brain insult.

Isoflurane provides long-term protection against focal cerebral ischemia in the rat.

Background:Long-term neuroprotection by isoflurane has been questioned. The authors examined factors in experimental models potentially critical to definition of enduring isoflurane neuroprotection. Methods:Rats were prepared for temporary middle cerebral artery occlusion (MCAO). Pericranial normothermia was maintained. Neurologic deficits (range, 0–48; 0 = no deficit) and cerebral infarct volumes were measured. In experiment 1, rats underwent 50 or 80 min MCAO while awake or anesthetized with 1.8% isoflurane. Blood pressure was controlled with phenylephrine. Outcome was evaluated 2 weeks later. In experiment 2, rats underwent 50 min MCAO while awake or anesthetized with isoflurane, with outcome evaluated 8 weeks later. In experiment 3, rats underwent 50 min MCAO while awake or anesthetized with isoflurane and 2 weeks recovery. Effects of phenylephrine and the mitochondrial adenosine triphosphate–sensitive K+ channel antagonist 5-hydroxydecanoate were studied. In experiment 4, isoflurane-anesthetized rats underwent 50 min MCAO with permanent or temporary common carotid artery occlusion, with outcome evaluated 2 weeks later. Results:In experiment 1, isoflurane reduced neurologic deficit (median ± interquartile range; awake vs. isoflurane: 11 ± 12 vs. 8 ± 6 for 80 min and 13 ± 4 vs. 3 ± 9 for 50 min; P = 0.0006) and infarct size (160 ± 97 vs. 84 ± 62 mm3 for 80 min and 169 ± 78 vs. 68 ± 61 mm3 for 50 min; P < 0.0001). In experiment 2, isoflurane protection persisted at 8 weeks after ischemia. In experiment 3, there was no effect of phenylephrine or 5-hydroxydecanoate. In experiment 4, permanent common carotid ligation increased infarct size threefold versus temporary occlusion. Conclusions:Isoflurane repeatedly improved long-term neurologic and histologic outcome from focal ischemia independent of ischemia duration, perfusion pressure, or pretreatment with 5-hydroxydecanoate.

Halothane reduces focal ischemic injury in the rat when brain temperature is controlled.

Background Previous work has demonstrated that rats anesthetized with halothane during focal cerebral ischemia have better histologic and neurologic outcome than do rats undergoing the same insult when awake. The purpose of this experiment was to determine whether this difference persists when brain temperature is held similar in halothane‐ anesthetized and awake experimental groups. Methods Two ischemia experiments were performed. In both, the middle cerebral artery was occluded for 90 min. Temperature was monitored from a radiotelemetered thermistor implanted in the cerebral cortex. Four days after ischemia, infarct volume and neurologic function were assessed. In experiment 1, brain temperature was not controlled in awake rats. Temperature in rats anesthetized with halothane, approximately 1 minimum alveolar concentration, was regulated by servomechanism by surface heating or cooling to replicate the temperature profiles generated by awake animals. To address methodologic issues regarding infarct volume analysis, a subset of nine rats was examined for the effect of the histologic staining technique and the mathematical modeling algorithms used for computation of infarct volume values. In experiment 2, the brain temperature of awake and halothane‐ anesthetized rats was maintained normothermic (38.0 degrees Celsius) throughout ischemia and early recirculation. Results In experiment 1 no difference between groups was observed for cortical (halothane 146 plus/minus 95 mm3 and awake 126 plus/minus 108 mm3; P = 0.64) or subcortical (halothane 110 plus/minus 48 mm3 and awake 100 plus/minus 66 mm3; P 0.66) infarct volume. Neurologic function was also similar between groups. Total infarct volume was approximately 11% greater when histologic sections were stained with hematoxylin and eosin than when they were stained with nitro blue tetrazolium, although volumes correlated closely between the two techniques (r2 0.996). Analysis by orthogonal or frustum projection from two‐dimensional planimetric areas to three‐ dimensional volumes resulted in nearly identical values (r2 0.999). In experiment 2, halothane‐anesthetized rats experienced a 46% reduction in cortical infarct volume (halothane 106 plus/minus 97 mm3 and awake 197 plus/minus 103 mm3; P = 0.03). The incidence of hemiparesis was reduced in the anesthetized group (P = 0.03). Conclusions When brain temperature was maintained normothermic throughout the focal ischemic insult, a neurologic and histologic protective effect for halothane anesthesia was observed. This effect of halothane was not sufficient to persist when large variations in brain temperature were allowed. Regulation of brain temperature is a critical factor in the determination of the effects of anesthetics on focal ischemic brain damage.

Effects of metalloporphyrin catalytic antioxidants in experimental brain ischemia

Reactive oxygen species play a role in the response of brain to ischemia. The effects of metalloporphyrin catalytic antioxidants (AEOL 10113 and AEOL 10150) were examined after murine middle cerebral artery occlusion (MCAO). Ninety minutes after reperfusion from 90 min MCAO in the rat, AEOL 10113, AEOL 10150, or vehicle were given intracerebroventricularly. AEOL 10113 and AEOL 10150 similarly reduced infarct size (35%) and neurologic deficit. AEOL 10113 caused behavioral side effects at twice the neuroprotective dose while AEOL 10150 required a 15-fold increase from the neuroprotective dose to cause behavioral changes. AEOL 10150, given 6 h after 90 min MCAO, reduced total infarct size by 43% without temperature effects. Brain AEOL 10150 elimination t(1/2) was 10 h. In the mouse, intravenous AEOL 10150 infusion post-MCAO reduced both infarct size (25%) and neurologic deficit. Brain AEOL 10150 uptake, greater in the ischemic hemisphere, was dose- and time-dependent. AEOL 10150 had direct effects on proteomic events and ameliorated changes caused by ischemia. In primary mixed neuronal/glial cultures exposed to 2 h of O(2)/glucose deprivation, AEOL 10150 reduced lactate dehydrogenase release dose-dependently and selectively preserved aconitase activity in concentrations consistent with neuroprotection in vivo. AEOL 10150 is an effective neuroprotective compound offering a wide therapeutic window with a large margin of safety against adverse behavioral side effects.

A comparison of strain-related susceptibility in two murine recovery models of global cerebral ischemia

Genetically engineered mice are increasingly important in stroke research. The strains on which these constructs are built are known to have inherent differential sensitivities to ischemic insults. This has been largely attributed to differences in vascular anatomy. This study compared the outcome from forebrain ischemia in two common murine background strains using two different types of ischemic insult. C57Bl/6 and SV129 mice were subjected to two vessel (bilateral carotid) occlusion (2VO) or 2VO plus systemic hypotension (2VO+Hypo; mean arterial pressure=30+/-2 mmHg) for 10-20 min. Ventilation and pericranial temperature were controlled. Cerebral blood flow (CBF) was determined by 14C-iodoantipyrine autoradiography. Histologic damage in forebrain structures was measured 3 days post-ischemia. During 2VO+Hypo, the EEG became isoelectric in all animals. During 2VO alone, EEG isoelectricity occurred in 73% of C57Bl/6 and 50% of SV129 mice. Forebrain CBF was reduced to a similar extent in both strains. Greater CBF variability was seen with 2VO alone versus 2VO+Hypo. CBF was less in the 2VO+Hypo model. SV129 mice had wider posterior communicating but smaller basilar artery diameters. With or without hypotension, SV129 mice had markedly less severe histologic damage than C57Bl/6 mice. A time-dependent increase in histologic damage was demonstrated in the 2VO+Hypo model but not with 2VO alone. The 2VO and 2VO+Hypo models produced similar magnitudes of histologic injury in C57Bl/6 mice subjected to 10-min ischemia. SV129 mice were resistant to ischemia in either model. The 2VO+Hypo model produced a more uniform severity of ischemia as defined by CBF and EEG examination. Despite this, the murine strain had a substantially greater impact on histologic outcome than did cerebrovascular anatomy or the type of model used to produce the ischemic insult.

Extracellular superoxide dismutase deficiency worsens outcome from focal cerebral ischemia in the mouse

The role of endogenous extracellular superoxide dismutase (EC-SOD) was examined in a murine model of transient focal cerebral ischemia. Homozygous EC-SOD deficient (EC-SOD-/-; n = 18) and wild type (EC-SOD+/+; n = 19) littermates were anesthetized with halothane and subjected to 50 min of intraluminal middle cerebral artery occlusion with pericranial temperature maintained at 37.0 degrees C. After 24 h of reperfusion, resultant hemiparesis and cerebral infarct size were measured. Total infarct volume was 81% greater (P = 0.03) and hemiparesis was more severe (P = 0.01) in EC-SOD-/- versus EC-SOD+/+ mice. The worsened ischemic outcome observed in EC-SOD-/- mice is consistent with prior work which found transgenic EC-SOD overexpressing mice to exhibit enhanced tolerance to focal ischemia. The results suggest that endogenous antioxidant activity in the extracellular compartment plays an important role in the histologic/neurologic response to focal cerebral ischemia.