John W. Kuluz

Endothelium-derived nitric oxide synthase inhibition. Effects on cerebral blood flow, pial artery diameter, and vascular morphology in rats.

Background and Purpose We determined the effects of inhibiting the production of cerebral endothelium- derived nitric oxide on pial artery diameter, cortical blood flow, and vascular morphology. Methods An inhibitor of endothelium-derived nitric oxide synthesis, NG-nitro-L-arginine methyl ester hydrochloride (L-NAME), or an equivalent volume of 0.9% saline was infused into rats intra-arterially in a retrograde fashion via the right external carotid artery at a rate of 3 mg/kg/min to a total dose of 190 mg/kg or intravenously at 1 mg/kg/min to a total dose of 15 mg/kg. Large pial arteries were continuously visualized through an operating microscope, and cortical cerebral blood flow was monitored by laser-Doppler flowmetry. To localize areas of morphological interest, the protein tracer horseradish peroxidase was injected 15 minutes before termination of the L-NAME infusion and the rats were perfusion-fixed 15 minutes later for light and electron microscopic analysis. Results Infusion of L-NAME significantly raised arterial blood pressure at both doses (for 190 mg/kg, from 103.2±3.4 to 135±3.4 mm Hg; for 15 mg/kg, from 125±2.8 to 144.4±4.0 mm Hg). Pial arteries constricted within 10 minutes after the start of the intracarotid infusion to 40% of the preinfusion diameter, while cortical cerebral blood flow decreased to an average of 72.5% of that at baseline. Morphological abnormalities in the experimental rats included microvascular stasis and focal areas of blood–brain barrier disruption to protein. Ultrastructural examination of cortical leaky sites revealed constricted arterioles with many endothelial pinocytotic vesicles and microvilli. Conclusions These observations suggest that inhibition of endothelium-derived nitric oxide synthesis affects the relation between cerebral arterial diameter and cerebral blood flow and can lead to subtle cerebral vascular pathological changes consistent with focal brain ischemia.

The effect of nitric oxide synthase inhibition on infarct volume after reversible focal cerebral ischemia in conscious rats.

Background and Purpose Previous in vitro and in vivo studies of the effects of nitric oxide synthase inhibition in the central nervous system have yielded conflicting results concerning the role of nitric oxide in the events that lead to ischemic injury. In this study, we tested the hypothesis that preischemic inhibition of nitric oxide synthase increases infarct volume after reversible focal cerebral ischemia in rats. Methods NG-nitro-L-arginine methyl ester hydrochloride 15 mg/kg IV or an equivalent volume of saline was administered to adult Wistar rats 15 minutes before middle cerebral artery occlusion by the intraluminal suture method. After 2 hours of ischemia, the suture was withdrawn, and rats were allowed to survive for 3 days. Areas of infarction in 10 hematoxylin-eosin-stained sections were measured and used to determine infarct volume. Results Administration of NG-nitro-L-arginine methyl ester hydrochloride increased hemispheric infarct volume by 137% over control (60.9±30.5 to 144.3±19.6 mm3, P<.05; mean±SEM). Cortical and subcortical infarct volumes were increased by 176% (33.8±21.9 to 93.3±15.2 mm3, P<.05) and 103% (25.1±9.4 to 51.0±5.5 mm3, P<.03), respectively. Conclusions Nitric oxide synthase inhibition increases infarct volume and decreases the variability of the response to middle cerebral artery occlusion in Wistar rats, a strain that is normally resistant to focal cerebral ischemic injury owing to extensive collateralization. The mechanism of the deleterious effect of nitric oxide synthase inhibition likely involves a more severe degree of blood flow reduction during and after middle cerebral artery occlusion, primarily by preventing the vasodilatory response of collateral vessels to proximal middle cerebral artery occlusion. Maintenance of nitric oxide synthase activity during and after focal cerebral ischemia appears to minimize ischemic injury.

Selective brain cooling during and after prolonged global ischemia reduces cortical damage in rats.

Background and Purpose: Studies of the cerebroprotectlve effects of selective brain cooling have failed to show amelioration of ischemic injury in the cerebral cortex. This study was designed to test the hypothesis that mild-to-moderate selective brain cooling initiated after the onset of global brain ischemia in rats protects the cerebral cortex and improves neurological outcome. Methods: Global forebrain ischemia for 30 minutes in 27 fasted adult male Wistar rats was achieved by bilateral carotid occlusion and hypotension. In group 1, brain temperature, measured in the temporalis muscle, was maintained at 37-38°C throughout the experiment In group 2, brain temperature fell spontaneously during ischemia to 34.7±0.1°C and rose spontaneously to 36-37°C after 10 minutes of recirculation. In group 3, brain temperature was lowered with ice packs placed around the head after 15 minutes of ischemia to 24.1±0.9°C by the end of ischemia, maintained at 30.0±1.0°C for the first hour of recirculation, then allowed to rise to 36-37°C. Results: Seven-day survival was 0% (0 of 6) in group 1, 73% (8 of 11) in group 2, and 100% (6 of 6) in group 3. Severity of neuronal damage was less in group 2 than in group 1 in the cortex (p<0.05) and hippocampal CA1 (p<0.05) and CA3 regions (p<0.05). Group 3 had less neuronal damage than group 2 in both cortex (p<0.02) and striatum (p<0.02). Furthermore, postischemic weight loss was less and neurobehavioral scores were significantly higher in group 3. Conclusions: This study shows that selective brain cooling increases survival from prolonged global ischemia and reduces neuronal injury in the cerebral cortex as well as the striatum and hippocampus.

Delayed hemorrhagic hypotension exacerbates the hemodynamic and histopathologic consequences of traumatic brain injury in rats.

Alterations in cerebral autoregulation and cerebrovascular reactivity after traumatic brain injury (TBI) may increase the susceptibility of the brain to secondary insults, including arterial hypotension. The purpose of this study was to evaluate the consequences of mild hemorrhagic hypotension on hemodynamic and histopathologic outcome after TBI. Intubated, anesthetized male rats were subjected to moderate (1.94 to 2.18 atm) parasagittal fluid–percussion (FP) brain injury. After TBI, animals were exposed to either normotension (group 1: TBI alone, n = 6) or hypotension (group 2: TBI + hypotension, n = 6). Moderate hypotension (60 mm Hg/30 min) was induced 5 minutes after TBI or sham procedures by hemorrhage. Sham-operated controls (group 3, n = 7) underwent an induced hypotensive period, whereas normotensive controls (group 4, n = 4) did not. For measuring regional cerebral blood flow (rCBF), radiolabeled microspheres were injected before, 20 minutes after, and 60 minutes after TBI (n = 23). For quantitative histopathologic evaluation, separate groups of animals were perfusion-fixed 3 days after TBI (n = 22). At 20 minutes after TBI, rCBF was bilaterally reduced by 57% ± 6% and 48% ± 11% in cortical and subcortical brain regions, respectively, under normotensive conditions. Compared with normotensive TBI rats, hemodynamic depression was significantly greater with induced hypotension in the histopathologically vulnerable (P1) posterior parietal cortex (P < 0.01). Secondary hypotension also increased contusion area at specific bregma levels compared with normotensive TBI rats (P < 0.05), as well as overall contusion volume (0.96 ± 0.46 mm3 vs. 2.02 ± 0.51 mm3, mean ± SD, P < 0.05). These findings demonstrate that mild hemorrhagic hypotension after FP injury worsens local histopathologic outcome, possibly through vascular mechanisms.

Fructose-1,6-bisphosphate reduces infarct volume after reversible middle cerebral artery occlusion in rats.

Background and Purpose We tested the hypothesis that fructose-1,6-bisphosphate, when administered 10 minutes before the end of 2 hours of reversible middle cerebral artery occlusion, reduces ischemia-reperfusion injury and infarct volume measured after a 3-day survival period in rats. Methods After 1 hour and 50 minutes of middle cerebral artery occlusion by the intraluminal suture method, fructose-1,6-bisphosphate, 500 mg/kg in group 1 and 350 mg/kg in group 2 (or an equivalent volume of 1.8% saline as placebo in each group), was given intravenously for a period of 15 minutes to fasted adult Sprague-Dawley rats. After 2 hours of ischemia, the suture was withdrawn and the rats allowed to survive for 3 days. The areas of infarction in 10 hematoxylin-eosin-stained coronal sections of the brain were measured and used to calculate infarct volume. Results In group 1, fructose-1,6-bisphosphate decreased total cerebral hemispheric infarct volume by 43% (from 199.6 ± 11.2 to 114.2 ± 35.8 mm3, P < .04; mean ± SEM). Cerebral cortical and subcortical infarct volumes were decreased by 46% (from 137.3 ± 7.5 to 74.1 ± 28.6 mm3, P < .04) and 36% (from 62.3 ± 5.1 to 40.0 ± 8.3 mm3, P < .04), respectively. In group 2, fructose-1,6-bisphosphate had no effect on infarct volume in rats that developed mild intraischemic hyperthermia, but in rats kept normothermic during ischemia, fructose-1,6-bisphosphate reduced subcortical infarct volume from 53.7±8.1 to 18.4±8.0 mm3 (P < .03). Conclusions Fructose-1,6-bisphosphate improves functional neurological outcome and reduces infarct volume after reversible middle cerebral artery occlusion in rats.

Snoezelen: A controlled multi-sensory stimulation therapy for children recovering from severe brain injury

Objective: To investigate the effects of Snoezelen therapy on physiological, cognitive and behavioural changes in children recovering from severe traumatic brain injury (TBI). Methods: An observational study was conducted to assess the physiological, cognitive and behavioural changes of children recovering from severe TBI while receiving Snoezelen therapy. Fifteen subjects completed the pre- and post-Snoezelen treatment measurements computed over 10 consecutive sessions. Physiological, cognitive and behavioural measures were administered. Data was collected prospectively on each session in the Snoezelen room and were analysed by calculating the difference between pre- and post-treatment measurements for each Snoezelen session. Results: Results revealed significant changes on physiological measures. Heart rates decreased for each subject in each treatment session and were found to be significant (p = 0.032). Muscle tone was decreased in all the affected extremities (right upper extremity p = 0.009, left upper extremity p = 0.020, right lower extremity p = 0.036 and left lower extremity p = 0.018). Agitation levels decreased over time and the overall cognitive outcome measures showed significant improvement when comparing the beginning of treatment with the end. Conclusion: This study revealed a beneficial use of Snoezelen therapy with children recovering from severe brain injury. However, there continues to be a critical need for evidenced-based research for this patient population and others in this multi-sensory environment.

Early endothelial damage and leukocyte accumulation in piglet brains following cardiac arrest

This study examined the early microvascular and neuronal consequences of cardiac arrest and resuscitation in piglets. We hypothesized that early morphological changes occur after cardiac arrest and reperfusion, and that these findings are partly caused by post-resuscitation hypertension. Three groups of normothermic piglets (37.5°–38.5°C) were investigated: group 1, non-ischemic time controls; group 2, piglets undergoing 8 min of cardiac arrest by ventricular fibrillation, 6 min of cardiopulmonary resuscitation (CPR) and 4 h of reperfusion; and group 3, non-ischemic hypertensive controls, receiving 6 min of CPR after only 10 s of cardiac arrest followed by 4-h survival. Immediately following resuscitation, acute hypertension occurred with peak systolic pressure equal to 197 ±15 mm Hg usually lasting less than 10 min. In reacted vibratome sections, isolated foci of extravasated horseradish peroxidase were noted throughout the brain within surface cortical layers and around penetrating vessels in group 2. Stained plastic sections of leaky sites demonstrated variable degrees of tissue injury. While many sections were unremarkable except for luminal red blood cells and leukocytes, other specimens contained abnormal neurons, some appearing irreversibly injured. The number of vessels containing leukocytes was higher in group 2 than in controls (3.8±0.6% vs 1.4±0.4% of vessels, P<0.05). Evidence for irreversible neuronal injury was only seen in group 2. Endothelial vacuolization was higher in groups 2 and 3 than in group 1 (P<0.05). Ultrastructural examination of leaky sites identified mononuclear and polymorphonuclear leukocytes adhering to the endothelium of venules and capillaries only in group 2. The early appearance of luminal leukocytes in ischemic animals indicates that these cells may contribute to the genesis of ischemia reperfusion injury in this model. In both groups 2 and 3 endothelial cells demonstrated vacuolation and luminal discontinuities with evidence of perivascular astrocytic swelling. Widespread microvascular and neuronal damage is present as early as 4 h after cardiac arrest in infant piglets. Hypertension appears to play a role in the production of some of the endothelial changes.

New Pediatric Model of Ischemic Stroke in Infant Piglets by Photothrombosis Acute Changes in Cerebral Blood Flow, Microvasculature, and Early Histopathology

Background and Purpose— The etiology and pathophysiology of acute ischemic stroke in children differ greatly from those in adults. The purpose of this study was to establish a new pediatric model of ischemic stroke in infant piglets for use in future studies of the response of the developing brain to focal ischemic injury. Methods— Ischemic stroke was produced in male infant piglets (2 to 4 weeks old) by photothrombotic occlusion of the middle cerebral artery. Regional cerebral blood flow was measured with radiolabeled microspheres up to 4 hours after occlusion. Early histopathology, including caspase-3 immunohistochemistry for apoptosis, was examined 4 hours after ischemia. The nature of the thrombus and its interaction with vascular endothelium were assessed by electron microscopy. Results— Severe ischemia (0 to 15 mL/100 g per min) occurred rapidly in 1.4±0.2 g of tissue at 15 minutes and increased to 2.4±0.7 g at 4 hours. Similarly, moderate ischemia (16 to 30 mL/100 g per min) was measured in 1.2±0.3 g of tissue at 15 minutes and increased to 2.0±0.6 g at 4 hours. These regional cerebral blood flow values represent ischemic levels of blood flow in 20% to 25% of the volume of the ischemic hemisphere at 4 hours after ischemia. Ischemic infarction occurred in both gray and white matter, and cerebral microvessels in the ischemic hemisphere contained large numbers of inflammatory leukocytes. Caspase-3–positive cells were few in number and were found in the periphery of the infarct; cell death appeared to occur primarily by necrosis rather than apoptosis at 4 hours. Electron microscopy revealed a pure platelet thrombus firmly attached to the vascular endothelium, which in some areas appeared to be detached from the basement membrane. Conclusions— Ischemic stroke can be produced in infant piglets by middle cerebral artery photothrombosis. The stroke involved both gray and white matter and exhibited a robust inflammatory component. The mean infarct volume determined histopathologically amounted to 9.6±2.4% of the affected (ipsilateral) hemisphere, which was correlated well with the mass equivalent of tissue (12.0±3.5%), in which severe declines in regional cerebral blood flow were observed at 4 hours.

New Research in the Field of Stroke: Therapeutic Hypothermia after Cardiac Arrest

Therapeutic hypothermia as a potential treatment for stroke, cerebral ischemia, and other neurological diseases has gained momentum since the initial discovery that relatively small differences in intraischemic brain temperature critically determine ischemic neuronal vulnerability.1 Since that time, laboratories throughout the world have investigated the potential use of mild-to-moderate hypothermia in many ischemia models.2,3 Various studies have also investigated potential mechanisms contributing to hypothermic protection. Pathomechanisms sensitive to intra- and postischemic temperature reductions and elevations include glutamate release, stabilization of the blood-brain barrier, oxygen radical production, intracellular signal conduction, protein synthesis, ischemic depolarization, reduced cerebral metabolism, membrane stabilization, inflammation, activation of protein kinases, cytoskeletal breakdown, and early gene expression.2,4 Because the pathophysiology of ischemic brain injury is complex, the fact that many injury mechanisms have been reported to be temperature sensitive may account for the dramatic effects of temperature on ischemic outcome. Thus, therapeutic hypothermia has the necessary support from preclinical data to initiate well-designed clinical studies targeting various patient populations. In a recent issue of The New England Journal of Medicine , the results of 2 randomized clinical trials showed clearly that mild hypothermia improves neurologic outcome and reduces overall mortality in survivors of out-of-hospital–witnessed cardiac arrest.5,6 In the study from Australia, a total of 77 patients who remained comatose after the return of spontaneous circulation (ROSC) were randomized to receive 12 hours of hypothermia or standard normothermic temperature management.5 In that study, surface cooling was begun in the field and the target temperature of 32°C to 34°C was reached within 2 hours of ROSC. Forty-nine percent of those treated with hypothermia were discharged home or to a rehabilitation facility, as compared with 26% of those not treated with hypothermia ( P =0.046). In the second study from Europe, 9 centers in 5 countries participated, with …

Alterations of CaMKII after hypoxia-ischemia during brain development.

Transient brain hypoxia‐ischemia (HI) in neonates leads to delayed neuronal death and long‐term neurological deficits. However, the underlying mechanisms are incompletely understood. Calcium‐calmodulin‐dependent protein kinase II (CaMKII) is one of the most abundant protein kinases in neurons and plays crucial roles in synaptic development and plasticity. This study used a neonatal brain HI model to investigate whether and how CaMKII was altered after HI and how the changes were affected by brain development. Expression of CaMKII was markedly up‐regulated during brain development. After HI, CaMKII was totally and permanently depleted from the cytosol and concomitantly deposited into a Triton‐insoluble fraction in neurons that were undergoing delayed neuronal death. Autophosphorylation of CaMKII‐Thr286 transiently increased at 30 min of reperfusion and declined thereafter. All these changes were mild in P7 pups but more dramatic in P26 rats, consistent with the development‐dependent CaMKII expression in neurons. The results suggest that long‐term CaMKII depletion from the cytosolic fraction and deposition into the Triton‐insoluble fraction may disable synaptic development, damage synaptic plasticity, and contribute to delayed neuronal death and long‐term synaptic deficits after transient HI.