Embolic occlusion of internal carotid artery in conscious rats: Immediate effects of cerebral ischemia.

In most preclinical models of focal ischemic stroke, vascular occlusion is performed under general anesthesia. However, anesthetic agents exert confounding effects on mean arterial blood pressure (MABP), cerebrovascular tone, oxygen demand, and neurotransmitter receptor transduction. Moreover, the majority of studies do not use a blood clot, which more fully models embolic stroke. Here, we developed a blood clot injection model to produce large cerebral artery ischemia in unanesthetized rats. Under isoflurane anesthesia, an indwelling catheter was implanted in the internal carotid artery via a common carotid arteriotomy and preloaded with a 0.38-mm-diameter clot of 1.5, 3, or 6 cm length. After discontinuing anesthesia, the rat was returned to a home cage where it regained normal mobility, grooming, eating activity, and a stable recovery of MABP. One hour later, the clot was injected over a 10-s period and the rats were observed for 24 h. Clot injection produced a brief period of irritability, then 15-20 min of complete inactivity, followed by lethargic activity at 20-40 min, ipsilateral deviation of the head and neck at 1-2 h, and limb weakness and circling at 2-4 h. Neurologic deficits, elevated MABP, infarct volume, and increased hemisphere water content varied directly with clot size. Mortality after 6-cm clot injection (53%) was greater than that after 1.5-cm (10%) or 3-cm (20%) injection. Combined non-survivor groups had the greatest MABP, infarct volume, and water content. Among all groups, the pressor response correlated with infarct volume. The coefficient of variation of infarct volume with the 3-cm clot was less than that in published studies with the filament or standard clot models, and therefore may provide stronger statistical power for stroke translational studies. The more severe outcomes from the 6-cm clot model may be useful for the study of malignant stroke.

Brain water as a function of age and weight in normal rats.

Rats are frequently used for studying water content of normal and injured brain, as well as changes in response to various osmotherapeutic regimens. Magnetic resonance imaging in humans has shown that brain water content declines with age as a result of progressive myelination and other processes. The purpose of this study was to quantify changes in brain water content during rat development and aging. Brain water content was measured by standard techniques in 129 normal male Sprague-Dawley rats that ranged in age (weight) from 13 to 149 days (18 to 759 g). Overall, the results demonstrated a decrease in water content from 85.59% to 76.56% with increasing age (weight). Nonlinear allometric functions relating brain water to age and weight were determined. These findings provide age-related context for prior rat studies of brain water, emphasize the importance of using similarly aged controls in studies of brain water, and indicate that age-related changes in brain water content are not specific to humans.

Effects of Volume Replacement for Urinary Losses from Mannitol Diuresis on Brain Water in Normal Rats.

BACKGROUND/OBJECTIVE: It is frequently recommended that urine output following perioperative mannitol administration be replaced 1:1 with an isotonic crystalloid solution. It is possible that this strategy could increase brain water by reducing the serum osmolality achieved with prior mannitol administration. Therefore, brain water content of rats treated with mannitol alone or mannitol plus normal saline (NS) was studied over a range of urinary replacement ratios. METHODS: Male Wister rats received mannitol 3.2 gm/100 gm infused over 45 min followed by hourly determinations of urine output (UO). Control animals received no additional therapy, whereas animals undergoing intervention received hourly replacement of their urinary losses with 0.9% NS in decreasing NS:UO ratios (1:1, 1:2, 1:3). Three hours after completion of the mannitol infusion, a final tally of UO was made. At that time in all animals, blood was obtained for determination of hemoglobin and electrolyte concentrations and plasma osmolality. Following that, the animals were sacrificed to determine brain water content. Additional groups underwent the same protocol but for 5 h with 1:1 urinary replacement, or received a volume of NS equal to that of the mannitol administered to all other control and intervention animals. RESULTS: 1:1 replacement of urinary loss with NS following mannitol administration was associated with brain water content indistinguishable from control animals receiving only a volume of NS equal to that of the mannitol administered to all other groups. Regression analysis demonstrated a decrease in the final brain water content of 0.67% (CI95 0.43-0.92, p < 0.001) per replacement level as NS:UO replacement ratios were decreased from 1:1 to 1:2 and, finally to 1:3. At the final NS:UO replacement ratio of 1:3, brain water content was indistinguishable from the control group receiving mannitol without NS replacement (p = 0.48) For 1:1 replacement following mannitol, brain water did not differ between experiments of 3 or 5 h duration (p = 0.52). CONCLUSIONS: In rats, NS replacement of UO 1:1 following mannitol administration leads to brain water content no different than if NS had been given in place of mannitol. Only when the NS:UO replacement ratio was 1:3, brain water was similar to that of control animals receiving mannitol alone. The recommendation to replace UO 1:1 with an equal volume of isotonic crystalloid following perioperative mannitol administration must recognize how this strategy could elevate brain water content compared to less vigorous replacement of UO.

Comparison of equivolume, equiosmolar solutions of mannitol and hypertonic saline with or without furosemide on brain water content in normal rats.

BACKGROUND: Mannitol and hypertonic saline (HS) are used by clinicians to reduce brain water and intracranial pressure and have been evaluated in a variety of experimental and clinical protocols. Administering equivolume, equiosmolar solutions in healthy animals could help produce fundamental data on water translocation in uninjured tissue. Furthermore, the role of furosemide as an adjunct to osmotherapy remains unclear. METHODS: Two hundred twenty isoflurane-anesthetized rats were assigned randomly to receive equivolume normal saline, 4.2% HS (1,368 mOsm/L 25% mannitol (1,375 mOsm/L), normal saline plus furosemide (8 mg/kg), or 4.2% HS plus furosemide (8 mg/kg) over 45 min. Rats were killed at 1, 2, 3, and 5 h after completion of the primary infusion. Outcome measurements included body weight; urinary output; serum and urinary osmolarity and electrolytes; and brain, lung, skeletal muscle, and small bowel water content. RESULTS: In the mannitol group, the mean water content of brain tissue during the experiment was 78.0% (99.3% CI, 77.9-78.2%), compared to results from the normal saline (79.3% [99.3% CI, 79.1-79.5%]) and HS (78.8% [99.3% CI, 78.6-78.9%]) groups (P < 0.001), whereas HS plus furosemide yielded 78.0% (99.3% CI, 77.8-78.2%) (P = 0.917). After reaching a nadir at 1 h, brain water content increased at similar rates for mannitol (0.27%/h [99.3% CI, 0.14-0.40%/h]) and HS (0.27%/h [99.3% CI, 0.17-0.37%/h]) groups (P = 0.968). CONCLUSIONS: When compared to equivolume, equiosmolar administration of HS, mannitol reduced brain water content to a greater extent over the entire course of the 5-h experiment. When furosemide was added to HS, the brain-dehydrating effect could not be distinguished from that of mannitol.

Infarct volume after hyperacute infusion of hypertonic saline in a rat model of acute embolic stroke.

INTRODUCTION: Hypertonic saline (HS) can treat cerebral edema arising from a number of pathologic conditions. However, physicians are reluctant to use it during the first 24 h after stroke because of experimental evidence that it increases infarct volume when administered early after reperfusion. Here, we determined the effect of HS on infarct size in an embolic clot model without planned reperfusion. METHODS: A clot was injected into the internal carotid artery of male Wistar rats to reduce perfusion in the middle cerebral artery territory to less than 40 % of baseline, as monitored by laser-Doppler flowmetry. After 25 min, rats were randomized to receive 10 mL/kg of 7.5 % HS (50:50 chloride:acetate) or normal saline (NS) followed by a 0.5 mL/h infusion of the same solution for 22 h. RESULTS: Infarct volume was similar between NS and HS groups (in mm(3): cortex 102 ± 65 mm(3) vs. 93 ± 49 mm(3), p = 0.72; caudoputamenal complex 15 ± 9 mm(3) vs. 21 ± 14, p = 0.22; total hemisphere 119 ± 76 mm(3) vs. 114 ± 62, p = 0.88, respectively). Percent water content was unchanged in the infarcted hemisphere (NS 81.6 ± 1.5 %; HS 80.7 ± 1.3 %, p = 0.16), whereas the HS-treated contralateral hemisphere was significantly dehydrated (NS 79.4 ± 0.8 %; HS 77.5 ± 0.8 %, p < 0.01). CONCLUSIONS: HS reduced contralateral hemispheric water content but did not affect ipsilateral brain water content when compared to NS. Infarct volume was unaffected by HS administration at all evaluated locations.

Age-dependent effects of testosterone in experimental stroke.

Although male sex is a well-recognized risk factor for stroke, the role of androgens in cerebral ischemia remains unclear. Therefore, we evaluated effects of testosterone on infarct size in both young adult and middle-aged rats (Wistar, 3-month versus 14-month old) and mice (C57/BL6, 3-month versus 12-month old) subjected to middle cerebral artery occlusion. In young adult groups, castrates displayed less ischemic damage as compared with intact males and castrates with testosterone replacement (Cortex: 24% in castrates versus 42% in intact versus 40% with testosterone; Striatum: 45% versus 73% versus 70%) at 22 h reperfusion. Surprisingly, supplementing testosterone in middle-aged rats to the physiologic levels ordinarily seen in young males reduced infarction (Cortex: 2% with testosterone versus 31%; Striatum: 38% with testosterone versus 68%). Testosterone effects on infarct size were blocked by the androgen receptor (AR) antagonist flutamide and further confirmed in young versus middle-aged mice. Baseline cerebral aromatase mRNA levels and activity were not different between young and middle-aged rats. Aromatase activity increased in ischemic tissue, but only in young males. Lastly, stroke damage was not different in aging aromatase knockout mice versus wild-type controls. Our findings indicate that testosterone's effects in experimental stroke are age dependent, mediated via AR, but not cerebral aromatase.

Effect of hypertonic saline concentration on cerebral and visceral organ water in an uninjured rodent model.

OBJECTIVE: Hypertonic saline has been shown to be an effective osmotic agent to reduce brain water and hence brain volume and intracranial pressure. A direct correlation between dose and effect has been demonstrated, but no studies have compared the effects of different concentrations of the same osmotic load of hypertonic saline over time. We compared the effects of different tonicity of infused hypertonic saline on cerebral, lung, and small bowel water extraction over time under controlled conditions. DESIGN: Laboratory study. SETTING: Medical school. SUBJECTS: Male Wistar rats (280-450 g). INTERVENTIONS: Anesthetized rats were randomized to a 15-min intravenous bolus infusion of 0.9% NaCl or five equisodium but different concentrations of hypertonic saline: 4.2%, 7.5%, 10%, 23.4%, and 30%. Following infusion, animals remained anesthetized for 60, 180, or 300 mins without additional fluids given (n > or = 6 per group). Blood was sampled, total urine output was measured, and the animal was then killed under deep anesthesia. Cerebral, lung, and small bowel water contents were derived by wet/dry weight measures. MEASUREMENTS AND MAIN RESULTS: After 60 mins, hypertonic saline administered at 50 mosm/kg resulted in an increase in serum osmolarity in all hypertonic saline groups (p < .05 vs. normal saline), with a significantly greater increase measured using 23.4% or 30% hypertonic saline (23.4%, 365.0 +/- 8.8 mosm/L, p < .05 vs. other lesser hypertonic saline doses). The durable effect was present throughout the 300-min period by all but the lowest hypertonic saline (4.2% NaCl). Lung but not small bowel organ water was diminished by hypertonic saline. Brain water content (79.1 +/- 0.2% in normal saline controls) was, however, significantly reduced. CONCLUSIONS: Hypertonic saline is effective in reducing organ water content in a setting of preserved blood-brain barrier but is not as effective in visceral organs. At equiosmotic doses of hypertonic saline, concentration plays no substantial role in altering serum osmolarity but appears to benefit duration of action. At very high concentrations, such as 23.4% NaCl, additional water extraction is also manifested. At such high concentration of NaCl, tonicity, indeed, matters, especially in water shifts across the blood-brain barrier.

Hypertonic saline: first-line therapy for cerebral edema?

This article highlights the experimental and clinical data, controversies and postulated mechanisms surrounding osmotherapy with hypertonic saline (HS) solutions in the neurocritical care arena and builds on previous reviews on the subject. Special attention is focused on HS therapy on commonly encountered clinical paradigms of acute brain injury including traumatic brain injury (TBI), post-operative "retraction edema", intracranial hemorrhage (ICH), tumor-associated cerebral edema, and ischemia associated with ischemic stroke.

Osmotherapy with hypertonic saline attenuates water content in brain and extracerebral organs.

OBJECTIVE: Because of their beneficial effects in patients with hemorrhagic shock and multiple-system trauma, hypertonic saline solutions are increasingly being used perioperatively for volume resuscitation. Although the anti-edema effects of hypertonic saline on brain are well documented in a variety of brain injury paradigms, its effects on the water content on other organs has not been studied rigorously. In this study, we tested the hypothesis that a) hypertonic saline when given as an intravenous bolus and continuous infusion attenuates water content of small bowel, lung, and brain in rats without neuro-injury; and b) attenuation of stroke-associated increases in lung water is dependent on achieving a target serum osmolality. DESIGN: Prospective laboratory animal study. SETTING: Research laboratory in a teaching hospital. SUBJECTS: Adult male Wistar rats. INTERVENTIONS: In the first series of experiments, under controlled conditions of normoxia, normocarbia, and normothermia, spontaneously breathing, halothane-anesthetized (1.0-1.5%) adult male Wistar rats (280-320 g) were treated in a blinded randomized fashion with 7.5% hypertonic saline or 0.9% normal saline in a 8-mL/kg intravenous infusion for 3 hrs followed by a continuous intravenous infusion (1 mL/kg/hr) of 5% hypertonic saline or normal saline, respectively (n=10 each), for 48 hrs. A second group of rats were treated with continuous infusion only for 48 hrs of either 7.5% hypertonic saline or normal saline (1 mL/kg/hr) (n=10 each) without an intravenous bolus. Naïve rats served as controls (n=10). Tissue water content of small bowel, lung, and brain was determined by comparing the wet-to-dry ratios at the end of the experiment. In a second series of experiments, rats (n=94) were subjected to 2 hrs of transient middle cerebral artery occlusion by the intraluminal occlusion technique. At 6 hrs following middle cerebral artery occlusion, rats were treated in a blinded randomized fashion with a continuous intravenous infusion of normal saline, 3% hypertonic saline, or 7.5% hypertonic saline for 24, 48, 72, and 96 hrs. Surgical shams served as controls (n=7). Hypertonic saline was instituted as chloride/acetate mixture (50:50) in all experiments. Serum osmolality was determined at the end of the experiment in all animals. MEASUREMENTS AND MAIN RESULTS: In rats without neuro-injury that received intravenous bolus followed by a continuous infusion, lung water content was significantly reduced with hypertonic saline (73.9+/-1.1%; 359+/-10 mOsm/L) (mean+/-sd) compared with normal saline treatment (76.1+/-0.53%; 298+/-4 mOsm/L) as was water content of small bowel (hypertonic saline, 69.1+/-5.8%; normal saline, 74.7+/-0.71%) and brain (hypertonic saline, 78.1+/-0.87%; normal saline, 79.2+/-0.38%) at 48 hrs. Stroke-associated increases in lung water content were attenuated with 7.5% hypertonic saline at all time points. There was a strong correlation between serum osmolality and attenuation of stroke-associated increases in lung water content (r=-.647) CONCLUSIONS: Bowel, lung, and brain water content is attenuated with hypertonic saline when serum osmolality is >350 mOsm/L without adverse effect on mortality in animals with and without neuro-injury. Attenuation of water content of extracerebral organs with hypertonic saline treatment may have therapeutic implications in perioperative fluid management in patients with and without brain injury.

Diagnosis and treatment of vascular air embolism.

Vascular air embolism is a potentially life-threatening event that is now encountered routinely in the operating room and other patient care areas. The circumstances under which physicians and nurses may encounter air embolism are no longer limited to neurosurgical procedures conducted in the "sitting position" and occur in such diverse areas as the interventional radiology suite or laparoscopic surgical center. Advances in monitoring devices coupled with an understanding of the pathophysiology of vascular air embolism will enable the physician to successfully manage these potentially challenging clinical scenarios. A comprehensive review of the etiology and diagnosis of vascular air embolism, including approaches to prevention and management based on experimental and clinical data, is presented. This compendium of information will permit the healthcare professional to rapidly assess the relative risk of vascular air embolism and implement monitoring and treatment strategies appropriate for the planned invasive procedure.

Adjuvant bupivacaine scalp block facilitates stabilization of hemodynamics in patients undergoing craniotomy with general anesthesia: a preliminary report.

STUDY OBJECTIVE: To evaluate the effect of 0.25% bupivacaine scalp block on alterations in hemodynamics and plasma catecholamine metabolites during general anesthesia in patients undergoing frontotemporal craniotomy. DESIGN: Prospective, clinical study. SETTING: Operating room of a university hospital. PATIENTS: 16 ASA physical status II and III patients who were scheduled for frontotemporal craniotomy. INTERVENTIONS: Patients were prospectively randomized to receive a saline control (C group) or bupivacaine scalp block (SB group) as an adjuvant to general anesthesia using isoflurane in 50% N(2)O-O(2). MEASUREMENTS: Routine monitoring of electrocardiogram, heart rate (HR), and mean arterial blood pressure (MAP) were recorded at two-minute intervals from the beginning of anesthesia until 10 minutes after incision, followed by 5-minute intervals throughout the remaining course of the surgery. By prospective design, increases in MAP or HR by 20% above the mean baseline values were treated with 2.5 mg/kg of thiopental combined with 2 mug/kg of fentanyl. Arterial blood was sampled at 5 minutes before and after skin incision and at the start of dural opening for measuring serum catecholamine metabolites by high-performance liquid chromatography. MAIN RESULTS: Only two patients in the SB group needed additional anesthetics for stabilizing their hemodynamics during the course of anesthesia. In contrast, all C group patients required supplemental anesthesia for controlling the abrupt rise in hemodynamic parameters. In addition, absolute MAP and HR values were significantly higher in the C group than in the SB group during the surgical period between incision and dural opening. The differences in hemodynamics observed between the two groups were, however, not accompanied with a significant change in plasma catecholamine metabolites at each predetermined time interval measured. CONCLUSIONS: Pretreatment with 0.25% bupivacaine scalp block appeared to be an effective adjuvant treatment for maintaining stable hemodynamics for patients undergoing craniotomy during general anesthesia especially at the time of skin incision and dural opening. This study design was unable to discern any correlation between elevation in hemodynamic parameters and a rise in serum catecholamine levels.

Plasma arginine-vasopressin following experimental stroke: effect of osmotherapy.

Neurohumoral responses have been implicated in the pathogenesis of ischemia-evoked cerebral edema. In a well-characterized animal model of ischemic stroke, the present study was undertaken to 1) study the profile of plasma arginine-vasopressin (AVP), and 2) determine whether osmotherapy with mannitol and various concentrations of hypertonic saline (HS) solutions influence plasma AVP levels. Halothane-anesthetized adult male Wistar rats were subjected to 2 h of middle cerebral artery occlusion with the intraluminal filament technique. Plasma AVP levels (means +/- SD) were significantly elevated at 24 h (42 +/- 21 pg/ml), 48 h (50 +/- 28 pg/ml), and 72 h (110 +/- 47 pg/ml), and returned to baseline at 96 h (22 +/- 15 pg/ml) following middle cerebral artery occlusion compared with sham-operated controls (14 +/- 7 pg/ml). Plasma AVP levels at 72 h were significantly attenuated with 7.5% HS (37 +/- 8 pg/ml; 360 +/- 11 osmol/l) compared with 0.9% saline (73 +/- 6; 292 +/- 6 osmol/l), 3% HS (66 +/- 8 pg/ml; 303 +/- 12 osmol/l), or mannitol (74 +/- 9 pg/ml; 313 +/- 14 osmol/l) treatment. HS (7.5%) significantly attenuated water content in the ipsilateral and contralateral hemispheres compared with surgical shams, 0.9% saline, 3% HS, and mannitol treatments. Peak plasma AVP levels were not associated with direct histopathological injury to the anterior hypothalamus. Attenuation of brain water content with 7.5% HS treatment coincides with attenuated serum AVP levels, and we speculate that this may represent one additional mechanism by which osmotherapy attenuates edema associated with ischemic stroke.

Effect of duration of osmotherapy on blood-brain barrier disruption and regional cerebral edema after experimental stroke.

Osmotherapy is the cornerstone of medical management for cerebral edema associated with large ischemic strokes. We determined the effect of duration of graded increases in serum osmolality with mannitol and hypertonic saline (HS) on blood-brain barrier (BBB) disruption and regional cerebral edema in a well-characterized rat model of large ischemic stroke. Halothane-anesthetized adult male Wistar rats were subjected to transient (2-h) middle cerebral artery occlusion (MCAO) by the intraluminal occlusion technique. Beginning at 6 h after MCAO, rats were treated with either no intravenous fluids or a continuous intravenous infusion (0.3 mL/h) of 0.9% saline, 20% mannitol, 3% HS, or 7.5% HS for 24, 48, 72, and 96 h. In the first series of experiments, BBB permeability was quantified by the Evans blue (EB) extravasation method. In the second series of experiments, water content was assessed by comparing wet-to-dry weight ratios in six predetermined brain regions. Blood-brain barrier disruption was maximal in rats treated with 0.9% saline for 48 h, but did not correlate with increases in serum osmolality or treatment duration with osmotic agents. Treatment with 7.5% HS attenuated water content in the periinfarct regions and all subregions of the contralateral nonischemic hemisphere to a greater extent than mannitol did with no adverse effect on survival rates. These data show that (1) BBB integrity is not affected by the duration and degree of serum osmolality with osmotic agents, and (2) attenuation of increases in brain water content with HS to target levels >350 mOsm/L may have therapeutic implications in the treatment of cerebral edema associated with ischemic stroke.

Ischemic neuroprotection with selective kappa-opioid receptor agonist is gender specific.

BACKGROUND AND PURPOSE: We demonstrated previously that treatment with selective kappa-opioid receptor (KOR) agonist BRL 52537 hydrochloride [(+/-)-1-(3,4-dichlorophenyl) acetyl-2-(1-pyrrolidinyl) methylpiperidine] (1) has a long therapeutic window for providing ischemic neuroprotection, and (2) attenuates ischemia-evoked NO production in vivo in rats. Neuronally derived NO has been shown to be deleterious in the male but not in the female rodent model of focal ischemic stroke. We tested the hypothesis that BRL provides significant neuroprotection from transient focal ischemia in male but not in female rats. METHODS: Halothane-anesthetized adult male and female Wistar rats (250 to 275 g) were subjected to 2 hours of middle cerebral artery occlusion (MCAO) by the intraluminal suture technique. Adequacy of MCAO and reperfusion was monitored with laser-Doppler flowmetry over the ipsilateral parietal cortex. In the first experiment, male and female rats were treated in a blinded randomized fashion with vehicle saline or 1 mg/kg per hour BRL infusion started at the onset of reperfusion and continued for 22 hours. In the second experiment, ovariectomized (OVX) female rats were treated with vehicle or BRL. Infarct volume in the cortex and caudoputamen (CP) complex was assessed by triphenyl tetrazolium chloride staining at 72 hours after MCAO. RESULTS: Infarct volume (percentage of ipsilateral structure; mean+/-SEM) was attenuated significantly in male rats with BRL treatment (cortex 23+/-5%; CP 44+/-6%; n=15) compared with vehicle-treated male rats (cortex 38+/-4%; CP 66+/-4%; n=15) but not in female rats (BRL-cortex 26+/-6; CP 55+/-8%; vehicle-cortex 26+/-5; CP 62+/-5%; n=10 each). Neurologic deficit score was improved in BRL-treated male rats but not in female rats. Infarct volume was not different in OVX female rats treated with vehicle or BRL. CONCLUSIONS: These data: (1) demonstrate that this dose of selective KOR agonist provides ischemic neuroprotection in male but not female rats, (2) demonstrate that the lack of protection by BRL is not attributable to circulating ovarian hormones, and (3) highlight the importance of using animal models of both sexes in preclinical studies of experimental ischemia.

Increases in lung and brain water following experimental stroke: effect of mannitol and hypertonic saline.

OBJECTIVE: Pulmonary edema is a serious condition following brain injury of diverse etiologies, including large hemispheric infarctions. We have previously shown that treatment with hypertonic saline attenuates cerebral edema associated with experimental ischemic stroke. In a well-characterized animal model of large ischemic stroke, we tested the hypotheses that lung water increases following cerebral ischemia and determined the effects of osmotherapy with hypertonic saline and mannitol on total lung water, as well as on cerebral edema. DESIGN: Prospective laboratory animal study. SETTING: Research laboratory in a university teaching hospital. SUBJECTS: Adult male Wistar rats (300-450 g, n = 103). INTERVENTIONS: Under controlled conditions of normoxia, normocarbia, and normothermia, spontaneously breathing, halothane-anesthetized (1.0-1.5%) rats were subjected to permanent middle cerebral artery occlusion by the intraluminal occlusion technique. MEASUREMENTS AND MAIN RESULTS: Cerebral perfusion was monitored by laser-Doppler flowmetry over ipsilateral parietal cortex to ensure adequate vascular occlusion. At 6 hrs following middle cerebral artery occlusion, rats were treated in a blinded randomized fashion with no intravenous fluids (n = 24), a continuous intravenous infusion (0.3 mL/hr) of 0.9% saline (n = 21), 20% mannitol (2 g/Kg) (n = 20), 5% hypertonic saline (n = 20), or 7.5% hypertonic saline (n = 18) as a chloride/acetate mixture (50:50) until the end of the experiment. Brains and lungs were harvested, and tissue water content was estimated by comparing wet-to-dry weight ratios of ipsilateral and contralateral cerebral hemispheres at 48 hrs postischemia. Sham-operated rats served as controls (n = 20). Serum osmolality was determined at the end of the experiment in all animals. Lung water content was increased significantly in rats subjected to middle cerebral artery occlusion and treated with no intravenous fluids (76.7 +/- 0.7%, 317 +/- 7 mOsm/L) (mean +/- sd) and saline (76.8 +/- 1.2%, 311 +/- 10 mOsm/L), compared with sham-operated controls (74.5 +/- 0.9%, 302 +/- 4 mOsm/L). Treatment with 20% mannitol (74.4 +/- 1.2%, 352 +/- 15 mOsm/L), 5% hypertonic saline (75.6 +/- 1.3%, 339 +/- 16 mOsm/L), and 7.5% hypertonic saline (74.9 +/- 0.7%, 360 +/- 23 mOsm/L) significantly attenuated lung water content. Hemispheric brain water content increased both in the ipsilateral ischemic and contralateral hemispheres treated with saline (ipsilateral, 85.1 +/- 1.7%; contralateral, 80.7 +/- 0.7%), compared with sham-operated controls (ipsilateral, 79.6 +/- 0.9%; contralateral, 79.5 +/- 0.9%), as well as in rats that received no fluids (ipsilateral, 84.6 +/- 1.8%; contralateral, 80.4 +/- 0.9%). Treatment with 5% hypertonic saline (ipsilateral, 83.8 +/- 1.0%; contralateral, 79.7 +/- 0.6%) and 7.5% hypertonic saline (ipsilateral, 82.3 +/- 1.3%; contralateral, 78.6 +/- 0.7%) resulted in attenuation of stroke-associated increases in brain water content to a greater extent than mannitol (ipsilateral, 83.6 +/- 1.6%; contralateral, 79.1 +/- 1.0%). CONCLUSIONS: In a well-characterized animal model of large ischemic stroke, total lung water content increases, which is likely neurogenic in origin. Attenuation of stroke-associated increases in lung and brain water content with continuous infusion of hypertonic saline may have therapeutic implication in the treatment of cerebral and pulmonary edema following ischemic stroke.

Effects of combined estrogen and progesterone on brain infarction in reproductively senescent female rats.

Recent data from the Women's Health Initiative have highlighted many fundamental issues about the utility and safety of long-term estrogen use in women. Current hormone replacement therapy for postmenopausal women incorporates progestin with estrogen, but it is uncertain if combined therapy provides major cerebrovascular risks or benefits to these women. No experimental animal stroke studies have examined combined hormone administration. The authors tested the hypothesis that combined hormone treatment reduces ischemic injury in middle-aged female rat brain. Reproductively senescent female rats underwent 2-hour middle cerebral artery occlusion (MCAO) followed by 22 hours reperfusion. Estrogen implants were placed subcutaneously at least 7 days before MCAO, and progesterone intraperitoneal injections were given 30 minutes before MCAO, at initiation, and at 6 hours of reperfusion. Rats received no hormone, a 25-microg estrogen implant, a 25-microg estrogen implant plus 5 mg/kg intraperitoneal progesterone, or 5 mg/kg intraperitoneal progesterone. Cortical, caudoputamen, and total infarct volumes were assessed by 2,3,5-triphenyltetrazolium chloride staining and digital image analysis at 22 hours reperfusion. Cortical and total infarct volumes, except in the acute progesterone-treated group, were significantly attenuated in all estrogen-alone and combined hormone-treated groups. There were no significant differences in caudoputamen infarct volumes in all hormone-treated groups as compared with untreated rats. These data have potential clinical implications relative to stroke for postmenopausal women taking combined hormone replacement therapy.

Experimental spinal cord ischemia: model characterization and improved outcome with arterial hypertension.

OBJECTIVE: Paraplegia from spinal cord ischemia is a devastating complication of thoracoabdominal aortic aneurysm repair. Perioperative hypoperfusion of the spinal cord is a critical determinant of residual neurologic deficits. We determined if functional and histologic outcome is dependent on systemic blood pressure in a rat model of spinal cord ischemia. DESIGN: Randomized, controlled, prospective study. SETTING: Research laboratory at a university teaching hospital. SUBJECTS: Adult male Wistar rats. INTERVENTIONS: Endotracheally intubated adult male Wistar rats (300-450 g) anesthetized with halothane underwent a thoracotomy and placement of a clip across the descending aorta for 27 mins. Mean proximal arterial blood pressure (MPABP) was monitored with a cannula placed in the left common carotid artery. Halothane was adjusted (1.25-1.5%) to maintain MPABP between 70 and 90 mm Hg (n = 20) or 140 and 150 mm Hg (n = 20). Shamoperated rats (n = 10) had a thoracotomy without aortic clamping at an MPABP of 70-90 mm Hg. Following 1, 24, 48, and 72 hrs of recovery from anesthesia, motor function of the hind paws was scored as follows: 0, no evidence of deficit; 1, toes flat under body when walking but with ataxia; 2, knuckle walks; 3, movements in hind limbs but unable to knuckle walk; 4, no movement, drags hind limbs. Body temperature was maintained between 37 and 38 degrees C throughout the experiment. MEASUREMENTS AND MAIN RESULTS: All sham operated rats with MPABP 70-90 mm Hg recovered without neurologic deficits, whereas those that underwent aortic occlusion with MPABP between 70 and 90 mm Hg emerged from anesthesia with grade 3 and 4 deficits and remained in this condition without improvement at 72 hrs. Histopathology at 72 hrs demonstrated moderate to severe neuronal loss with involvement of dorsal, intermediate, and ventral horns. Only eight of 20 rats that underwent aortic occlusion with MPABP between 140 and 150 mm Hg had grade 1 and 2 deficits on emergence but had no neurologic deficit after 1 hr. Most of the surviving neurons in these animals appeared normal histologically, particularly motor neurons around the periphery of the ventral horn. CONCLUSIONS: Systemic blood pressure is a critical determinant of outcome following spinal cord ischemia, and controlled peri-operative blood pressure augmentation may ameliorate neurologic deficits in patients who undergo thoracoabdominal vascular procedures and are at risk for spinal cord hypoperfusion.

Prolonged opportunity for ischemic neuroprotection with selective kappa-opioid receptor agonist in rats.

BACKGROUND AND PURPOSE: We have previously demonstrated that pretreatment with selective kappa-opioid agonist BRL 52537 hydrochloride [(+/-)-1-(3,4-dichlorophenyl) acetyl-2-(1-pyrrolidinyl) methylpiperidine], provides ischemic neuroprotection following transient focal ischemia in rats. The present study was undertaken to a) define "therapeutic opportunity" for ischemic neuroprotection with BRL 52537, and b) determine if BRL 52537 attenuates ischemia-evoked efflux of dopamine and its metabolites in the striatum in vivo following transient focal ischemia. METHODS: Using the intraluminal filament technique, halothane-anesthetized male Wistar rats were subjected to 2 hours of middle cerebral artery occlusion (MCAO). In a blinded, randomized fashion, rats were treated with saline (vehicle) or 1 mg/Kg/hr BRL 52537 infusion for 22 hours, initiated at onset, 2, 4, or 6 hours of reperfusion (Rep). In a separate set of experiments utilizing in vivo microdialysis, extracellular levels of dopamine and its metabolites were determined in the striatum during 2 hours of MCAO and 3 hours of reperfusion. RESULTS: Infarct volume (% of contralateral structure; mean +/-SEM) in cortex was significantly attenuated when BRL 52537 was administered at reperfusion (22+/-6%), 2 hours (21+/-6%), and 4 hours (18+/-5%) compared with controls (39+/-5%). In striatum, infarct volume was significantly attenuated when BRL 52537 was administered at reperfusion (38+/-9%), 2 hours (40+/-8%), 4 hours (50+/-8%), and 6 hours (46+/-9%) as compared with controls (70+/-4%). A 6- to 8-fold increase in dopamine in microdialysates occurred within 40 minutes of MCAO. Pretreatment with BRL 52537 did not alter microdialysate levels of dopamine or its metabolites in the striatum during MCAO and early reperfusion, as compared with saline controls. CONCLUSIONS: These data demonstrate that BRL 52537 provides robust ischemic neurprotection with a long therapeutic opportunity (at least 6 hours) without altering ischemia-evoked efflux of dopamine (DA) and its metabolites in striatum during ischemia and early reperfusion.

Kappa-opioid receptor selectivity for ischemic neuroprotection with BRL 52537 in rats.

UNLABELLED: Kappa-opioid receptors (KOR) have been implicated in neuroprotection from ischemic neuronal injury, but less work has been performed with transient focal cerebral ischemia to determine the role of KOR during reperfusion. We tested the effects of a selective and specific KOR agonist, BRL 52537 hydrochloride [(+/-)-1-(3,4-dichlorophenyl)acetyl-2-(1-pyrrolidinyl)methylpiperidine], and the standard KOR antagonist, nor-binaltorphimine dihydrochloride [nor-BNI; 17,17'-(dicyclopropylmethyl)-6,6',7,7'-6,6'-imino-7,7'-binorphinan-3,4',14,14'-tetrol], on functional and histological outcome after transient focal ischemia in the rat. By use of the intraluminal filament technique, halothane-anesthetized adult male Wistar rats were subjected to 2 h of middle cerebral artery occlusion confirmed by laser Doppler flowmetry. In a blinded, randomized fashion, rats were treated with 1). saline (vehicle) 15 min before reperfusion followed by saline at reperfusion for 22 h, 2). saline 15 min before reperfusion followed by BRL 52537 (1 mg x kg(-1) x h(-1)) at reperfusion for 22 h, 3). saline 15 min before reperfusion followed by nor-BNI (1 mg x kg(-1) x h(-1)) at reperfusion for 22 h, or 4) nor-BNI (1 mg/kg) 15 min before reperfusion followed by BRL 52537 (1 mgx kg(-1)x h(-1)) and nor-BNI (1 mg x kg(-1) x h(-1)) at reperfusion for 22 h. Infarct volume (percentage of ipsilateral structure) analyzed at 4 days of reperfusion was significantly attenuated in saline/BRL 52537 rats (n = 8; cortex, 10.2% +/- 4.3%; caudoputamen [CP], 23.8% +/- 6.7%) (mean +/- SEM) compared with saline/saline treatment (n = 8; cortex, 28.6% +/- 4.9%; CP, 53.3% +/- 5.8%). Addition of the specific KOR antagonist nor-BNI to BRL 52537 completely prevented the neuroprotection (n = 7; cortex, 28.6% +/- 5.3%; CP, 40.9% +/- 6.2%) conferred by BRL 52537. BRL 52537 did not produce postischemic hypothermia. These data demonstrate that KORs may provide a therapeutic target during early reperfusion after ischemic stroke. IMPLICATIONS: The neuroprotective effect of selective kappa-opioid agonists in transient focal ischemia is via a selective action at the kappa-opioid receptors.

Neuroprotective kappa-opioid receptor agonist BRL 52537 attenuates ischemia-evoked nitric oxide production in vivo in rats.

BACKGROUND AND PURPOSE: Kappa-opioid receptors (KOR) have been implicated in neuroprotection from ischemic neuronal injury. We tested the effects of a selective and specific KOR agonist, BRL 52537 hydrochloride [(+/-)-1-(3,4-dichlorophenyl)acetyl-2-(1-pyrrolidinyl) methylpiperidine], on infarct volume and nitric oxide production after transient focal ischemia in the rat. METHODS: With the use of the intraluminal filament technique, halothane-anesthetized male Wistar rats (weight, 250 to 300 g) were subjected to 2 hours of focal cerebral ischemia confirmed by Doppler flowmetry. In a blinded randomized fashion, rats were treated with intravenous saline or 1 mg/kg per hour BRL 52537 infusion, initiated 15 minutes before occlusion and maintained until 2 hours of reperfusion. In a second experiment, rats were treated during reperfusion with saline or 1 mg/kg per hour BRL 52537, initiated at onset of reperfusion and continued for 22 hours. In a final experiment, in vivo striatal nitric oxide production was estimated via microdialysis by quantification of citrulline recovery after labeled arginine infusion in striatum of intravenous BRL 52537- or saline-treated rats. RESULTS: In rats treated with BRL 52537 during ischemia and early reperfusion, infarct volume was significantly attenuated in cortex (16+/-6% versus 40+/-7% of ipsilateral cortex in saline group) and in caudoputamen (30+/-8% versus 66+/-6% of ipsilateral caudoputamen in saline group). Infarct volume was also reduced by treatment administered only during reperfusion in cortex (19+/-8% in BRL 52537 group [n=10] versus 38+/-6% in saline group) and in caudoputamen (35+/-9% versus 66+/-4% in saline group). BRL 52537 treatment markedly attenuated NO production in ischemic striatum compared with saline-treated controls. CONCLUSIONS: These data demonstrate that (1) the selective KOR agonist BRL 52537 provides significant neuroprotection from focal cerebral ischemia when given as a pretreatment or as a posttreatment and (2) attenuation of ischemia-evoked nitric oxide production in vivo may represent one mechanism of ischemic neuroprotection.