Mild intraischemic hypothermia suppresses consumption of endogenous antioxidants after temporary focal ischemia in rats

Oxidative damage by free radicals has been proposed as a mechanism of cerebral injury due to ischemia and reperfusion. Hypothermia protects against ischemic necrosis; however, its effect on oxidative stress has not been investigated. In this study, the effects of hypothermia on oxidative stress were studied by determining consumption of endogenous antioxidants after temporary focal ischemia in rats. Thirty-two Sprague-Dawley rats anesthetized with 1.5% isoflurane underwent 3 h of middle cerebral artery occlusion under hypothermic (33°C) or normothermic (37°C) conditions followed by 3 h of normothermic reperfusion. In the first study (n = 8per group), intraischemic hypothermia suppressed the reduction of tissue concentrations of endogenous antioxidants, ascorbate (P≤ 0.05), and glutathione (P≤ 0.05) in ischemic cortex but not in caudoputamen. In a parallel study (n = 8per group), hypothermia reduced tissue damage in ischemic frontoparietal cortex (P ≤ 0.05), but not in caudoputamen. Laser-Doppler estimates of cortical blood flow showed that intraischemic hypothermia significantly attenuated early postischemic hyperperfusion (P ≤ 0.01) and delayed postischemic hypoperfusion (P ≤ 0.01). These results demonstrate that intraischemic mild hypothermia reduces oxidative stress and cell injury after prolonged focal ischemia followed by reperfusion. The reduction of oxidative stress by hypothermia may be related indirectly to attenuation of postischemic blood flow changes.     

Mild hypothermia on anoxic depolarization and subsequent cortical injury following transient ischemia.

Anoxic depolarization (AD) is one of the major physiological characteristics in the ischemic core. The effect of mild hypothermia on the appearance of AD and subsequent brain injury following profound ischemia is studied to evaluate the protective mechanism of hypothermia against severe ischemia. Sprague-Dawley rats were subjected to transient ischemia by hypotension (50-20 mmHg) and bilateral carotid artery occlusion (BCA-O) for 20 min in normothermia and 30 min in hypothermia. The temperature of body and temporal muscles was maintained at 37.5 degrees C and 36.5 degrees C in normothermia and 33.0 degrees C and 31.0 degrees C in hypothermia, respectively. Recording of the DC potential shift and electrocorticogram and monitoring of the cortical blood flow (CoBF) with a laser Doppler flowmeter were done epidurally on the right parietal cortex. The right parietal cortex pathology was examined 24 h after ischemia in normothermia and after 30 days in hypothermia. AD appeared in all seven normothermic rats with a fall in the CoBF to 9%-10% of the control flow. However, in spite of CoBF reduction to 8%-9% of the control flow, it did not appear in five hypothermic rats. Intra-ischemic CoBF was not statistically different between these two groups. AD appeared with the CoBF decreasing to 4%-5% of the control flow in seven hypothermic rats. Intra-ischemic CoBF in hypothermic rats exhibiting AD was significantly lower than the other two groups. The interval between BCA-O and the appearance of AD in hypothermic rats was 5.1 +/- 0.3 min (mean +/- SE), which was significantly longer than the 2.2 +/- 0.5 min observed in normothermia (p < 0.0005). Of seven normothermic rats exhibiting AD, two died within 24 h and four revealed massive neuronal injury. Of seven hypothermic rats with AD, four died between day 2 and day 13, and one revealed diffuse cerebral infarction. However, no severe ischemic injury or ischemic death was observed in all five hypothermic rats without AD. The incidence of severe neuronal injury or ischemic death was significantly lower in hypothermic rats without AD compared with normothermic rats with AD (p < 0.02) or hypothermic rats with AD (p < 0.05). Although mild hypothermia delays AD, it is suggested that raising the cerebral blood flow threshold for AD appearance has a key role in the hypothermic protection of a severely ischemic area such as the ischemic core.     

Mild postischemic hypothermia limits cerebral injury following transient focal ischemia in rat neocortex

Intraischemic mild hypothermia has been shown to attenuate cerebral infarction occurring after transient focal ischemia. In contrast, the capacity of mild hypothermia to provide a protective effect when administered postischemically has not been clearly defined for transient focal events such as occur in many types of stroke. The present study addressed this issue by investigating the influence of timing and duration of mild hypothermia on cerebral infarction in a rat model of reversible focal ischemia. Sprague-Dawley rats (n = 45) were subjected to 3 h of focal neocortical ischemia by occluding reversibly one middle cerebral artery and both carotid arteries. Mild hypothermia was established after reperfusion and maintained for brief (1 h) or prolonged (21 h) periods. Animals were sacrificed 24 or 48 h after ischemia. A significant reduction (32%) in the volume of infarction was obtained when hypothermia was established immediately after reperfusion and maintained for a prolonged (21 h) period. In contrast, immediate but brief (1 h) hypothermia did not reduce infarction volume. Delaying hypothermia until 30 min post reperfusion and maintaining it for 21 h reduced infarction volume by 22%; however, this effect did not achieve statistical significance. These findings demonstrate that mild postischemic hypothermia is capable of protecting against cerebral injury following transient focal ischemia but that prolonged hypothermia is required to achieve this effect. These findings are consistent with increasing evidence that the window of therapeutic opportunity after transient focal ischemia is rather brief and that critical mechanisms involved in this form of ischemic injury remain activated over a rather lengthy postischemic interval.     

Neuroprotective effects of female gonadal steroids in reproductively senescent female rats

BACKGROUND AND PURPOSE: Young adult female rats sustain smaller infarcts after experimental stroke than age-matched males. This sex difference in ischemic brain injury in young animals disappears after surgical ovariectomy and can be restored by estrogen replacement. We sought to determine whether ischemic brain injury continues to be smaller in middle-aged, reproductively senescent female rats compared with age-matched males and to test the effect of ovarian steroids on brain injury after experimental stroke in females. METHODS: Four groups of 16-month old Wistar rats (males [n=9], untreated females [n=9], and females pretreated with 17beta-estradiol [25-microgram pellets administered subcutaneously for 7 days; n=9] or progesterone [10-mg pellets administered subcutaneously for 7 days; n=9] were subjected to 2 hours of middle cerebral artery occlusion with the intraluminal filament technique, followed by 22 hours of reperfusion. Physiological variables and laser-Doppler cerebral cortical perfusion were monitored throughout ischemia and early reperfusion. In a separate cohort of males (n=3), untreated females (n=3), females pretreated with 17beta-estradiol (n=3), and females pretreated with progesterone (n=3), end-ischemic regional cerebral blood flow was measured by [(14)C]iodoantipyrine autoradiography. RESULTS: As predicted, infarct size was not different between middle-aged male and female rats. Cortical infarcts were 21+/-5% and 31+/-6% of ipsilateral cerebral cortex, and striatal infarcts were 44+/-7% and 43+/-5% of ipsilateral striatum in males and females, respectively. Both estrogen and progesterone reduced cortical infarct in reproductively senescent females (5+/-2% and 16+/-4% in estrogen- and progesterone-treated groups, respectively, compared with 31+/-6% in untreated group). Striatal infarct was smaller in the estrogen- but not in the progesterone-treated group. Relative change in laser-Doppler cerebral cortical perfusion from preischemic baseline and absolute end-ischemic regional cerebral blood flow were not affected by hormonal treatments. CONCLUSIONS: We conclude that the protection against ischemic brain injury found in young adult female rats disappears after reproductive senescence in middle-aged females and that ovarian hormones alleviate stroke injury in reproductively senescent female rats by a blood flow-independent mechanism. These findings support a role for hormone replacement therapy in stroke injury prevention in postmenopausal women.     

Failure of delayed and prolonged hypothermia to favorably affect hemorrhagic stroke in rats

Prolonged hypothermia reduces global and focal cerebral ischemic injury in rodents even when delayed for hours. However, it is not known whether hypothermia can reduce injury following intracerebral hemorrhage (ICH). Accordingly, we studied striatal injury and concomitant motor deficits after 2 days of hypothermia, induced 1 h after creation of an ICH by infusion of bacterial collagenase. Rats were first trained to retrieve food pellets in the Montoya staircase task. They were then implanted with core temperature telemetry probes and later subjected to normothermic ICH or sham operation (vehicle injection). Half self-regulated temperature after surgery; others were cooled to 33 degrees C (24 h) and then 35 degrees C (24 h). Hypothermia did not affect behavioral scores of sham animals (89.8% of baseline in staircase test) or histology. Untreated (normothermic) ICH rats lost 23.1 mm(3) of tissue at a 1-month survival, which significantly impaired food pellet retrieval (66.0% retrieval) with the contralateral limb (tested on days 21-25). Contrary to our hypothesis, hypothermia failed to lessen either the reaching impairment (62.8%) or the lesion (22.2 mm(3)). While other hemorrhagic insults or complications may be improved with hypothermia, our data suggest that it will not salvage tissue that is quickly lost after ICH. We also assessed walking across a horizontal ladder and spontaneous paw usage in a cylinder test at 1-4 weeks after ICH, but neither test was sufficiently sensitive to this mild insult. This indicates that skilled reaching is more severely disrupted than spontaneous paw usage or walking after a striatal hemorrhage.     

Topographically graded postischemic presence of metalloproteinases is inhibited by hypothermia

To test the hypothesis that presence of metalloproteases (MMPs), their inhibitors (TIMPs) and their substrate laminin-5 differs between the ischemic core and the surrounding tissue, we examined the impact of middle cerebral artery occlusion/reperfusion (MCA:O/R) on these molecules in different regions of the infarct. We also investigated the influence of hypothermia on the progression of the ischemic lesion and MMP activity. Brain sections from 64 Wistar rats subjected to MCA:O/R were examined by means of cytohistochemistry and zymography. The artery was occluded for 2 h followed by 3, 5, 8 and 12 h of reperfusion. Well characterized antibodies against laminin-5, MMPs and TIMP-2 were used. A total of 32 rats were treated with hypothermia. The presence of each antigen was related to the following regions of interest: ischemic core with BBB breakdown (I(c)), surrounding ischemic tissue without BBB breakdown (I(r)), and the contralateral non-ischemic region (N). Regions of interest were defined by MRI. The I(c) increased over time at the cost of the I(r). BBB breakdown occurred early in the ischemic core and increased over time. Hypothermia reduced the BBB breakdown at all time points. A graded decreased presence of laminin-5 was observed with 16.5+/-3.7(N)>10+/-2.8(I(r))>4+/-1.4(I(c)) immunopositive microvessels/mm(2) at 3 h of reperfusion. MMP-9 showed a reverse pattern with 0 (N)<4+/-0.8(I(r))<10+/-1.5(I(c)) immunopositive microvessels/mm(2). Hypothermia decreased the MMP activity measured by zymography. Laminin-5 and MMP presence relate directly to the degree of postischemic injury. Hypothermia reduces the conversion from the I(r) to ischemic core and the degree of BBB as well as MMP abundance.     

Therapeutic hypothermia influences cell genesis and survival in the rat hippocampus following global ischemia

Delayed hypothermia salvages CA1 neurons from global ischemic injury. However, the effects of this potent neuroprotectant on endogenous repair mechanisms, such as neurogenesis, have not been clearly examined. In this study, we quantified and phenotyped newly generated cells within the hippocampus following untreated and hypothermia-treated ischemia. We first show that CA1 pyramidal neurons did not spontaneously regenerate after ischemia. We then compared the level of neuroprotection when hypothermia was initiated either during or after ischemia. Treatment efficacy decreased with longer delays, but hypothermia delayed for up to 12 hours was neuroprotective. Although bromodeoxyuridine (BrdU) incorporation was elevated in ischemic groups, CA1 neurogenesis did not occur as the BrdU label did not colocalize with neuronal nuclei (NeuN) in any of the groups. Instead, the majority of BrdU-labeled cells were Iba-positive microglia, and neuroprotective hypothermia decreased the delayed generation of microglia during the third postischemic week. Conversely, hypothermia delayed for 12 hours significantly increased the survival of newly generated dentate granule cells at 4 weeks after ischemia. Thus, our findings show that CA1 neurogenesis does not contribute to hypothermic neuroprotection. Importantly, we also show that prolonged hypothermia positively interacts with postischemic repair processes, such as neurogenesis, resulting in improved functional outcome.     

Induced cerebral hypothermia reduces post-hypoxic loss of phenotypic striatal neurons in preterm fetal sheep

Perinatal hypoxic-ischemic injury of the basal ganglia is a significant cause of disability in premature infants. Prolonged, moderate cerebral hypothermia has been shown to be neuroprotective after experimental hypoxia-ischemia; however, it has not been tested in the preterm brain. We therefore examined the effects of severe hypoxia and the potential neuroprotective effects of delayed hypothermia on phenotypic striatal neurons. Preterm (0.7 gestation) fetal sheep received complete umbilical cord occlusion for 25 min followed by cerebral hypothermia (fetal extradural temperature reduced from 39.4+/-0.3 degrees C to 29.5+/-2.6 degrees C) from 90 min to 70 h after the end of occlusion. Hypothermia was associated with a significant overall reduction in striatal neuronal loss compared with normothermia-occlusion fetuses (mean+/-SEM, 5.5+/-1.2% vs. 38.1+/-6.5%, P<0.01). Immunohistochemical studies showed that occlusion resulted in a significant loss of calbindin-28 kd, glutamic acid decarboxylase isoform 67 and neuronal nitric oxide synthase-immunopositive neurons (n=7, P<0.05), but not choline acetyltransferase-positive neurons, compared with sham controls (n=7). Hypothermia (n=7) significantly reduced the loss of calbindin-28 kd and neuronal nitric oxide synthase, but not glutamic acid decarboxylase-immunopositive neurons. In conclusion, delayed, prolonged moderate head cooling was associated with selective protection of particular phenotypic striatal projection neurons after severe hypoxia in the preterm fetus. These findings suggest that head cooling may help reduce basal ganglia injury in some premature babies.     

MRI of combination treatment of embolic stroke in rat with rtPA and atorvastatin

To test the hypothesis that combination treatment of embolic stroke with rtPA and statins improves the efficacy of thrombolytic therapy in rats. Rats subjected to embolic MCA occlusion (MCAo) were randomized into control (n = 10) and treatment (n = 9) groups. Four hours after MCAo, a combination of rtPA and atorvastatin (treatment) or saline (control) was administered. MRI measurements were performed on all animals at 2 h, 24 h and 48 h after MCAo. The patency of cerebral microvessels was examined using fluorescent microscopy. MRI images showed complete blockage of the right MCA and a reduction of CBF in the territory supplied by the MCA 2 h after MCAo for all animals. By 48 h after stroke, MRI showed that the decreased lesion size, elevated CBF and increased incidence of recanalization were found in treated rats compared with the control rats. The combination treatment significantly increased microvascular patency (16.3 +/- 5.5% vs. 12.4 +/- 3.5%, of field-of-view) and reduced the infarct volume (23.1 +/- 9.6% vs. 38.8 +/- 13.3%, of hemisphere). These data demonstrate that the co-administration of rtPA and atorvastatin 4 h after ischemia is efficacious and is reflected by the MRI indices of recanalization of the MCA, reduction of secondary microvascular perfusion deficits and reduction of the ischemic lesion.     

Indefatigable CA1 sector neuroprotection with mild hypothermia induced 6 hours after severe forebrain ischemia in rats.

Considerable controversy exists about whether postischemic hypothermia can permanently salvage hippocampal CA1 neurons or just postpone injury. Studies of very brief cooling in rat have found transient benefit, whereas experiments in gerbil using protracted hypothermia report lasting protection. This discrepancy might be because of the greater efficacy of longer cooling or it might, for example, represent an important species difference. In the present study, a 48-hour period of mild hypothermia was induced starting 6 hours after 10 minutes of severe four-vessel occlusion ischemia in rats. Untreated normothermic ischemia resulted in total CA1 cell loss (99%), whereas delayed hypothermia treatment reduced neuronal loss to 14% at a 28-day survival. In unregulated rats, brain temperature spontaneously fell during ischemia, and stayed subnormal for an extended period after ischemia. This mild cooling resulted in more variable and less severe CA1 injury (75%). Finally, vertebral artery cauterization under halothane anesthesia caused an approximately 2 degrees C drop in brain temperature for 1 hour, but prevention of this hypothermia did not significantly affect CA1 damage. In summary, protracted postischemic hypothermia provided robust and long-term CA1 protection in rat. These results encourage the clinical assessment of prolonged hypothermia and its use as a model to understand ischemic CA1 injury.     

Effects of ionotropic glutamate receptor blockade and 5-HT1A receptor activation on spreading depression in rat neocortical slices

The effect of the AMPA antagonist NBQX (10 microM), NMDA antagonist ketamine (100 microM) and 5-HT1A agonist 8-OH-DPAT (1, 10 and 100 microM) on the properties of a KCl-induced spreading depression (SD) was studied in parietal cortical slices of adult rats. Whereas NBQX did not significantly affect the SD, ketamine significantly (p < 0.01) reduced the amplitude of the first SD peak (12.8 +/- 4.6 mV) and blocked the second SD peak when compared with the controls (19.8 +/- 5.2 mV and 25 +/- 5 mV, respectively). Ketamine also decreased the SD duration at half maximal amplitude from 34.9 +/- 12.4 s to 22.2 +/- 12 s (p < 0.05). 8-OH-DPAT attenuated the duration of the SD from 42 +/- 15.6 s to 21.2 +/- 10.6 s (p < 0.05, 100 microM). These data indicate that not only NMDA receptor blockade, but also activation of the 5-HT1A receptor attenuates the SD and may be beneficial in the reduction of ischemic injury following focal cerebral ischemia.     

Prolonged but delayed postischemic hypothermia: a long-term outcome study in the rat middle cerebral artery occlusion model.

Delayed but prolonged hypothermia persistently decreases cell death and functional deficits after global cerebral ischemia in rodents. Postischemic hypothermia also reduces infarction after middle cerebral artery occlusion (MCAO) in rat. Because initial neuroprotection is sometimes transient and may not subserve functional recovery, especially on demanding tasks, the authors examined whether postischemic cooling would persistently reduce infarction and forelimb reaching deficits after MCAO. Male spontaneously hypertensive rats were trained to retrieve food pellets in a staircase test that measures independent forelimb reaching ability. Later, rats underwent 90 minutes of normothermic MCAO, through a microclip, or sham operation. In some rats, prolonged cooling (33 degrees C for 24 hours and then 35 degrees C for 24 hours) began 2.5 hours after the onset of ischemia (60 minutes after the start of reperfusion; n = 17 with subsequently 1 death) or sham procedures (n = 4), whereas untreated sham (n = 4) and ischemic (n = 16 with subsequently 1 death) rats maintained normothermia. An indwelling abdominal probe continually measured core temperature, and an automated fan and water spray system was used to produce hypothermia. One month later rats were reassessed in the staircase test over five days and then killed. The contralateral limb impairment in food pellet retrieval was completely prevented by hypothermia (P = 0.0001). Hypothermia reduced an infarct volume of 67.5 mm3 after untreated ischemia to 35.8 mm3 (P < 0.0001). These findings of persistent benefit encourage the clinical assessment of hypothermia.     

MRZ 2/579, a novel uncompetitive N-methyl-D-aspartate antagonist, reduces infarct volume and brain swelling and improves neurological deficit after focal cerebral ischemia in rats

The purpose of this study was to evaluate the effects of MRZ 2/579, an uncompetitive N-methyl-D-aspartate antagonist, on infarct size, extent of swelling and neurological deficit in a model of transient middle cerebral artery occlusion in rats. Physiologically controlled Sprague-Dawley rats received 2 h MCAo by retrograde insertion of an intraluminal suture coated with poly-L-lysine. The agent (MRZ 2/579) or vehicle (sodium chloride 0.9%) was administered i.v. immediately after suture removal following a 2-h period of MCAo. Two experimental groups were studied: group A was treated by vehicle (bolus infusion:1 ml/kg for 10 min followed by infusion of 6 ml/kg/h over 6 h). Group B was treated by MRZ 2/579 (bolus infusion:10 mg/kg for 10 min followed by infusion of 6 mg/kg/h over 6 h). The neurological status was evaluated during occlusion (at 60 min) and daily for 3 days after MCAo. Brains were then perfusion-fixed, and infarct volumes and brain swelling were determined. MRZ 2/579 significantly improved the neurological score compared to vehicle-treated rats at 48 h (6.2+/-0.6 and 8.7+/-0.5, respectively; P<0.004) and 72 h after MCAo (5.2+/-0.6 and 8.4+/-0.5, respectively; P<0.001). Treatment with MRZ 2/579 also significantly reduced total infarct volume (29.3+/-11.1 and 83.2+/-16.5 mm(3), respectively; P<0. 01), cortical infarct volume (24.8+/-11.2 and 70.0+/-18.0 mm(3), respectively; P<0.04) and subcortical infarction (21.2+/-4.1 and 49. 6+/-4.5 mm(3), respectively; P<0.0002). Brain swelling was also markedly reduced compared with vehicle-treated rats (4.7+/-1.3 and 10.8+/-2.1%, respectively; P<0.02). These results demonstrate that treatment with MRZ 2/579, when administered promptly after reperfusion, confers neuroprotective effects on infarct volume, brain swelling, and neurological score compared to the vehicle group.     

Neuroprotective effect of hypothermia at defined intraischemic time courses in cortical cultures

Many experimental and clinical studies have shown that hypothermia confers cerebroprotective benefits against ischemic insults. Because of the many conflicting reports on hypothermic neuroprotection, we undertook this cellular study to identify the optimal temperature or a range of temperatures for maximal neuroprotection at different times (6-24 hr) during ischemic insults. Cultured Wistar rat cortical neurons were exposed to oxygen deprivation at defined times and temperatures (37 degrees C normothermia, 32 degrees C mild hypothermia, 27 degrees C moderate hypothermia, 22 degrees C deep hypothermia, and 17 degrees C profound hypothermia). The survival rate of neurons was evaluated by assessing viable neurons on photomicrographs. The normothermic group demonstrated a significantly lower survival rate of cultured neurons (6 hr, 80.3% +/- 2.7%; 12 hr, 56.1% +/- 2.1%; 18 hr, 34.2% +/- 1%; 24 hr, 18.1% +/- 2.2%) compared to hypothermic groups (P < 0.001). The survival rate for the profound hypothermic group was significantly reduced (P < 0.01) compared to other hypothermic groups (at 17 degrees C: 12 hr, 85.9% +/- 2.5%, 18 hr, 74.7% +/- 3.7%, 24 hr, 58.7% +/- 2.7%). Almost equal survival rates were observed among mild, moderate, and deep hypothermic groups following <18 hr exposure to hypoxia, but the deep hypothermic group showed a significantly higher survival rate (84.1% +/- 1.6%; P < 0.001) when subjected to hypoxia for 24 hr. In conclusion, hypothermia offers marked neuroprotection against hypoxia, but attenuation of neuronal cell death was less with profound hypothermia compared to mild, moderate, and deep hypothermia. Deep hypothermia affords maximal protection of neurons compared to mild and moderate hypothermia during long-lasting hypoxia (>18 hr).     

Protective effects of brief intra- and delayed postischemic hypothermia in a transient focal ischemia model in the neonatal rat

Hypothermia provides neuroprotection in virtually all animal models of ischemia, including adult stroke models and the neonatal hypoxic-ischemic (HI) model. In these studies, brief periods of hypothermia are examined in a neonatal model employing transient focal ischemia in a 7-day-old rat pup. Pups underwent permanent middle cerebral artery (MCA) occlusion coupled with a temporary (1 h) occlusion of the ipsilateral common carotid artery (CCA). This study included five treatment groups: (1) normothermic (Normo)-brain temperature was maintained at 37 degrees C; (2) intraischemic hypothermia (IntraH)-28 degrees C during the 1-h ischemic period only; (3) postischemic hypothermia (PostH)-28 degrees C for the second hour of reperfusion only; (4) late-onset postischemic hypothermia (LPostH) cooled to 28 degrees C for the fifth and sixth hours of reperfusion only; and (5) Shams. After various times (3 days-6 weeks), the lesion was assessed using 2,3,5-triphenyltetrazolium chloride (TTC) or hematoxylin and eosin (H&E) stains. Intraischemic hypothermia resulted in significant protection in terms of survival, lesion size, and histology. Postischemic hypothermia was not effective in reducing lesion size early after ischemia, but significantly reduced the eventual long-term damage (2-6 weeks). Late-onset postischemic hypothermia did not reduce infarct volume. Therefore, both intraischemic and postischemic hypothermia provided neuroprotection in the neonatal rat, but with different effects on the degenerative time course. While there were no observable differences in simple behaviors or growth, all hypothermic conditions significantly reduced mortality rates. While the protection resulting from intraischemic hypothermia is similar to what is observed in other models, the degree of long-term ischemic protection observed after 1 h of postischemic hypothermia was remarkable and distinct from what has been observed in other adult or neonatal models.     

Mild hypothermia on anoxic depolarization and subsequent cortical injury following transient ischemia

Abstract

Anoxic depolarization (AD) is one of the major physiological characteristics in the ischemic core. The effect of mild hypothermia on the appearance of AD and subsequent brain injury following profound ischemia is studied to evaluate the protective mechanism of hypothermia against severe ischemia. Sprague-Dawley rats were subjected to transient ischemia by hypotension (50-20 mmHg) and bilateral carotid artery occlusion (BCA-O) for 20 min in normothermia and 30 min in hypothermia. The temperature of body and temporal muscles was maintained at 37.5°C and 36.5°C in normothermia and 33.0°C and 31.0°C in hypothermia, respectively. Recording of the DC potential shift and electrocorticogram and monitoring of the cortical blood flow (CoBF) with a laser Doppler flowmeter were done epidurally on the right parietal cortex. The right parietal cortex pathology was examined 24 h after ischemia in normothermia and after 30 days in hypothermia. AD appeared in all seven normothermic rats with a fall in the CoBF to 9%-10% of the control flow. However, in spite of CoBF reduction to 8%-9% of the control flow, it did not appear in five hypothermic rats. Intra-ischemic CoBF was not statistically different between these two groups. AD appeared with the CoBF decreasing to 4%-5% of the control flow in seven hypothermic rats. Intra-ischemic CoBF in hypothermic rats exhibiting AD was significantly lower than the other two groups. The interval between BCA-O and the appearance of AD in hypothermic rats was 5.1 ± 0.3 min (mean ± SE), which was significantly longer than the 2.2 ± 0.5 min observed in normothermia (p < 0.0005). Of seven normothermic rats exhibiting AD, two died within 24 h and four revealed massive neuronal injury. Of seven hypothermic rats with AD, four died between day 2 and day 13, and one revealed diffuse cerebral infarction. However, no severe ischemic injury or ischemic death was observed in all five hypothermic rats without AD. The incidence of severe neuronal injury or ischemic death was significantly lower in hypothermic rats without AD compared with normothermic rats with AD (p < 0.02) or hypothermic rats with AD (p < 0.05). Although mild hypothermia delays AD, it is suggested that raising the cerebral blood flow threshold for AD appearance has a key role in the hypothermic protection of a severely ischemic area such as the ischemic core. [Neurol Res 1999; 21: 670-676]     

Importance of posttraumatic hypothermia and hyperthermia on the inflammatory response after fluid percussion brain injury: biochemical and immunocytochemical studies.

The purpose of this study was to investigate: 1) the temporal and regional profile of polymorphonuclear leukocyte (PMNL) infiltration after moderate traumatic brain injury using the parasagittal fluid percussion model and 2) the effects of posttraumatic hypothermia (30 degrees C) and hyperthermia (39 degrees C) on the acute and subacute inflammatory response. We hypothesized that posttraumatic hypothermia would reduce the degree of PMNL accumulation whereas hyperthermia would exacerbate this response to injury. In the first series of experiments we quantitated the temporal profile of altered myeloperoxidase activity under normothermic (37 degrees C) conditions (n = 20). The rats were allowed to survive for 3 hours, 24 hours, 3 days, or 7 days after trauma, and brains were dissected into cortical and subcortical regions ipsilateral and contralateral to injury. Additional animals were perfused and fixed for the immunocytochemical visualization of myeloperoxidase (n = 15). In the second series of experiments, rats (n = 25) were killed 3 hours or 3 days after the 3-hour monitoring period of normothermia (36.5 degrees C), hypothermia (30 degrees C), or hyperthermia (39 degrees C) (n = 4 to 5 per group), and myeloperoxidase activity was again quantitated. In normothermic rats, the enzymatic activity of myeloperoxidase was significantly increased (P < 0.05) at 3 hours within the anterior cortical segment (213.97 +/- 56.2 versus control 65.5 +/- 52.3 U/g of wet tissue; mean +/- SD) and posterior (injured) cortical and subcortical segments compared to sham-operated rats (305.76 +/- 27.8 and 258.67 +/- 101.4 U/g of wet tissue versus control 62.8 +/- 24.8 and 37.28 +/- 35.6 U/g of wet tissue; P < 0.0001, P < 0.05, respectively). At 24 hours and 7-days after trauma only the posterior cortical region (P < 0.005, P < 0.05, respectively) exhibited increased myeloperoxidase activity. However, 3 days after trauma, myeloperoxidase activity was also significantly increased within the anterior cortical segment (P < 0.05) and in posterior cortical and subcortical regions compared to sham-operated cortex (P < 0.0001, P < 0.05, respectively). Immunocytochemical analysis of myeloperoxidase reactivity at 3 hours, 24 hours, 3- and 7-days demonstrated large numbers of immunoreactive leukocytes within and associated with blood vessels, damaged tissues, and subarachnoid spaces. Posttraumatic hypothermia and hyperthermia had significant effects on myeloperoxidase activity at both 3 hours and 3 days after traumatic brain injury. Posttraumatic hypothermia reduced myeloperoxidase activity in the injured and noninjured cortical and subcortical segments compared to normothermic values (P < 0.05). In contrast, posttraumatic hyperthermia significantly elevated myeloperoxidase activity in the posterior cortical region compared to normothermic values at both 3 hours and 3 days (473.5 +/- 258.4 and 100.11 +/- 27.58 U/g of wet tissue, respectively, P < 0.05 versus controls). These results indicate that posttraumatic hypothermia decreases early and more prolonged myeloperoxidase activation whereas hyperthermia increases myeloperoxidase activity. Temperature-dependent alterations in PMNL accumulation appear to be a potential mechanism by which posttraumatic temperature manipulations may influence traumatic outcome.     

Neuroprotective effects of NS-7, voltage-gated Na+/Ca2+ channel blocker in a rodent model of transient focal ischaemia

The aim of the present study was to characterize neuroprotective activity of NS- 7, a mixed voltage-gated sodium and calcium channel blocker in a model of transient focal ischaemia in rats. Ischaemia was induced by a 75 min reversible occlusion of middle cerebral artery (MCAo) using a nylon filament. NS-7 (0.5 mg/kg i.v.) or 0.9% NaCl (1 ml/kg i.v.) were infused over 3 min. starting 30 min after the MCAo. Infarct analysis was performed 72 h after ischaemia. Application of NS- 7 produced significant protection seen in neurological tests and diminished brain damage by 37% in total infarct (17.7+/- 3.0% vs. 27.9 +/- 3.2% control; [p < 0.01]; t-test), 47.8% in cortical infarct size by (8.5 +/- 2.4% vs. 16.2 +/- 2.4% control; [p < 0.01]), and by 21.5% in striatal infarction (9.2 +/- 0.8% vs. 11.7 +/- 0.9% control; [p < 0.05]). The results indicate that NS- 7 has potential for neuroprotection against transient ischaemic insult.