Myron D. Ginsberg

High-dose human ALB (albumin) therapy is highly neuroprotective in animal models of ischaemic stroke. A recently completed 82-subject pilot-phase dose-escalation trial has shown that ALB is safe, with strong preliminary suggestions of possible efficacy. We are now proceeding to a large randomized, double-blinded, placebo-controlled multicentre trial funded by the NIH (National Institutes of Health), the ALIAS (Albumin In Acute Stroke) Phase III Trial, which is designed to ascertain definitively whether high-dose ALB therapy confers neuroprotection in subjects with acute ischaemic stroke treated within 5 h of stroke onset. The primary efficacy outcome measure is a favourable outcome, defined as an NIHSS (NIH Stroke Scale) score of 0–1 or a modified Rankin Scale score of 0–1 at 3 months post-randomization. Separate randomization (1:1) to ALB or placebo therapy will be carried out in two cohorts of 900 subjects each, one that receives standard-of-care thrombolytic therapy and the other that does not. Approx. 60 North American clinical sites will participate. Subject enrolment is expected to commence in July 2006.

Background and Purpose— In preclinical stroke models, high-dose human albumin confers robust neuroprotection. We investigated the safety and tolerability of this therapy in patients with acute ischemic stroke. Methods— The ALIAS ( Al bumin i n A cute S troke) Pilot Clinical Trial used a multiple-tier, open-label, dose-escalation design. Subjects with acute ischemic stroke (NIH Stroke Scale [NIHSS] of 6 or above) received a 2-hour infusion of 25% human albumin (ALB) beginning within 16 hours of stroke onset. Six successive ALB dose tiers were assessed ranging from 0.34 to 2.05 g/kg. Neurologic and cardiac function was sequentially monitored. At 3 months, the NIHSS, modified Rankin Scale, and Barthel Index were measured. Results— Eighty-two subjects (mean age, 65 years) received ALB at 7.8±3.4 hours after stroke onset (mean±standard deviation). Forty-two patients also received standard-of-care intravenous tissue plasminogen activator (tPA). Vital signs were unaltered by ALB treatment. Dose-related increases in plasma albumin and mild hemodilution were maximal at 4 to 12 hours. Age-related plasma brain natriuretic peptide levels increased at 24 hours after ALB but did not predict cardiac adverse events. The sole ALB-related adverse event was mild or moderate pulmonary edema in 13.4% of subjects, which was readily managed with diuretics. In the tPA-treated subgroup, symptomatic intracranial hemorrhage occurred in only one of 42 subjects. Conclusions— Twenty-five percent human albumin in doses ranging up to 2.05 g/kg was tolerated by patients with acute ischemic stroke without major dose-limiting complications. tPA therapy did not affect the safety profile of ALB. The companion article presents neurologic outcome data and efficacy analysis in these subjects.

Azulenyl nitrones are novel chain‐breaking antioxidants with low oxidation potentials and high lipophilicity—properties favoring their efficacy as neuroprotectants. We tested the second‐generation azulenyl nitrone, stilbazunenlyl nitrone (STAZN), in focal ischemic stroke. Physiologically monitored rats received 2 hours of middle cerebral artery occlusion by intraluminal suture, resulting in substantial cortical and striatal infarcation. Neurobehavior was quantified on a standard battery, and brains were perfusion‐fixed for quantitative histopathology at 3 days. In 3 independent series, rats were treated at either 2h + 4h, or 2h + 4h + 24h + 48h, after onset of ischemia; vehicle‐treated rats received dimethylsulfoxide or saline. All animals (n = 52) developed high‐grade neurological deficits (score 11 of 12) during ischemia, which improved, in STAZN‐treated rats, within 1–1.5 h of the initial dose and fell to a median score of 3 at 72 h, compared to 8 in vehicle rats. STAZN treatment reduced mean cortical infarct volume by 64–97%, and total infarct volume by 42–72%. In over one‐half of STAZN‐treated animals, cortical infarction was virtually abolished. Regression analysis predicted that STAZN would confer ∼50% cortical neuroprotection even in the most severely affected cases. The potency of STAZN was orders‐of‐magnitude greater than other nitrones such as NXY‐059. These results suggest that STAZN has great promise for ischemic stroke. Ann Neurol 2003

Background— The pathophysiology of cerebral ischemia is well studied in small-animal models, which offer reproducibility and control of confounding variables—factors essential to hypothesis-testing. This presentation first highlights insights into the ischemic penumbra enabled by a multimodal experimental approach; second, discusses gene expression in ischemia; and third, confronts the challenges of neuroprotectant therapy. Summary of Review— The ischemic penumbra : Transient (2-hour) middle cerebral artery suture-occlusion in anesthetized rats gives rise to highly consistent neurological and histopathological sequelae. Autoradiographic local cerebral blood flow (LCBF) studies at 2 hours of occlusion define the penumbra as a region of intermediate CBF depression (20% to 40% of control) surrounding the densely ischemic core (5% to 20% of control) and constituting one half of the entire lesion. Local glucose metabolic rate in the acute penumbra is not reduced despite the critical CBF reduction, so that the penumbral metabolism/blood flow ratio is markedly elevated. In contrast, following 1 hour of recirculation, glucose metabolism throughout the previously ischemic hemisphere has become markedly depressed, and the metabolism/flow ratio has pseudonormalized. By correlating these data with histopathology using multimodal image analysis, the probability of infarction is shown to be highly determined by the degree of antecedent CBF reduction. These animal data agree strikingly with published results in patients with acute stroke studied by positron emission tomography. This remarkable correspondence belies the assertion that data from lower species may not be relevant to human stroke. Gene expression : Perfusion gradients also determine differential patterns of gene expression in ischemia. This can be demonstrated by correlating in situ hybridization autoradiographs for gene expression with autoradiographic LCBF data and histological infarct maps derived from replicate series. In other studies, DNA microarray technology is used to screen for thousands of expressed genes. In the 2-hour middle cerebral artery occlusion model with 3-hour recirculation, we have identified 28 known ischemia-hypoxia response genes that are upregulated and 6 that are downregulated, together with 35 upregulated and 41 downregulated genes newly connected with ischemia. These findings underscore the enormous complexity of ischemic biology and suggest possible novel mechanisms for future exploration. Neuroprotection : A desirable neuroprotectant would, in theory, antagonize multiple injury mechanisms. We have explored 2 such therapies of particular promise. Mild brain hypothermia (32°C target temperature, for 5 hours) is highly neuroprotective even when initiated at the onset of recirculation. Another highly protective agent is human albumin, administered in doses of 1.25 to 2.5 g/kg—a therapy that reduces infarct volume in this ischemia model by 60% to 65%, markedly diminishes brain swelling, and has a therapeutic window extending to 4 hours. Conclusion— The careful study of rodent ischemia models can yield valuable, clinically relevant insights into the pathophysiology of ischemic stroke.

Human albumin therapy is highly neuroprotective in focal cerebral ischemia. Because albumin is the main carrier of free fatty acids (FFA) in plasma, we investigated the content and composition of plasma FFA in jugular vein (JV), femoral artery (FA) and femoral vein (FV) of rats given intravenous human albumin (1.25 g/kg) or saline vehicle (5 mL/kg) 1 h after a 2 h middle cerebral artery occlusion (MCAo) or sham surgery. Arachidonic acid was the only FFA significantly increased by MCAo in all plasma samples prior to albumin administration, remaining at the same level regardless of subsequent treatments. Albumin treatment induced in both MCAo‐ and sham‐groups a 1.7‐fold increase in total plasma FFA (mainly 16:0, 18:1, 18:2n‐6) during 90‐min reperfusion. MCAo selectively stimulated the albumin‐mediated mobilization of n‐3 polyunsaturated fatty acids (PUFA), with an early increase in 22:5n‐3 and 22:6n‐3 in the FA prior to detectable changes in the JV. In the MCAo‐albumin group, the lower level of FFA in JV as compared with FA and FV suggests an albumin‐mediated systemic mobilization and supply of FFA to the brain, which may favor the replenishment of PUFA lost from cellular membranes during ischemia and/or to serve as an alternative source of energy, thus contributing to albumin neuroprotection.

The authors have recently demonstrated that high-dose human albumin is markedly neuroprotective in experimental traumatic brain injury (TBI) and cerebral ischemia. The pathophysiology of TBI involves acute uncoupling of cerebral glucose utilization and blood flow. The intent of this study was to establish whether the use of human albumin therapy in a model of acute TBI would influence this phenomenon. Anesthetized, physiologically regulated rats received moderate (1.5-2 atm) fluid-percussion injury to the parietal lobe. Fifteen minutes after trauma or sham injury, rats in one group received human albumin (2.5 g/kg) administered intravenously and those in another group received 0.9% saline vehicle. At 60 minutes and 24 hours posttrauma, autoradiographic studies of local cerebral blood flow (LCBF) and local cerebral glucose utilization (LCMRglu) were conducted, and the LCMRglu/LCBF ratio was determined. Sham-injured rats had normal levels of LCBF and LCMRglu, and no differences between vehicle- and albumin-treated rats were evident. Sixty minutes after TBI, LCBF was moderately reduced bilaterally in vehicle-treated rats, whereas in albumin-treated animals, the LCBF contralateral to the side of injury was generally normal. Despite acutely depressed LCBF, LCMRglu in vehicle-treated rats at 60 minutes was paradoxically normal bilaterally, and foci of elevated LCMRglu were noted in the ipsilateral hippocampus and thalamus. By contrast, in albumin-treated rats studied 60 minutes post-TBI, reduced LCMRglu values were measured in the ipsilateral caudoputamen and parietal cortex, whereas LCMRglu in other ipsilateral and contralateral sites did not differ from that measured in sham-injured animals. The metabolism/blood flow ratio was normal in sham-injured rats, but became markedly elevated in vehicle-treated rats 60 minutes post-TBI (on average, by threefold ipsilaterally and 2.1-fold contralaterally). By contrast, the mean metabolism/blood flow ratio in albumin-treated animals was elevated by only 1.6-fold ipsilaterally and was normal contralaterally. Twenty-four hours after TBI, LCBF contralateral to the side of injury had generally returned to normal levels in the albumin-treated group. These results demonstrate that human albumin therapy benefits the posttraumatic brain by diminishing the pronounced metabolism > blood flow dissociation that would otherwise occur within the 1st hour after injury. Viewed together with our previous evidence of histological neuroprotection, these findings indicate that human albumin therapy may represent a desirable treatment modality for acute TBI.

In addition to producing acute neuronal necrosis within selectively vulnerable brain regions, our recent studies have shown that global cerebral ischemia may also be followed by protracted degenerative changes occurring over the course of 10 weeks. Chronic brain pathology may be associated with the abnormal deposition of β-amyloid precursor protein (βAPP). In the present study, we used a monoclonal antibody to the N-terminal portion of βAPP to characterize the brains of rats surviving 1–10 weeks following 10 min of global brain ischemia produced by bilateral carotid artery occlusions plus systemic hypotension. After ischemia, increased βAPP immunolabeling emerged in several brain regions. In the hippocampus, granular deposits appeared in the damaged CA1 area by 2 weeks, and by 4–10 weeks the remnants of necrotic CA1 neurons were also immunolabeled. In striatum and thalamus, regions with necrotic cell death also revealed granular βAPP deposits. The neocortex was devoid of overt ischemic neuronal damage but revealed prominent βAPP immunoreactivity. Large ovoid deposits of low-density βAPP immunostaining occurred in cortical neurons at 1–2 weeks. At 4–10 weeks, large round or oval deposits immunoreactive for βAPP appeared in several cortical regions. The highest density of deposits was seen in the temporal and piriform cortices. Our results indicate that abnormal βAPP deposition may result from ischemic as well as chronic neurodegenerative processes.

We undertook a detailed characterization of the cellular responses to acute global cerebral ischemia complicated by hyperglycemia. Anesthetized, physiologically monitored male Wistar rats received 12.5 min of global forebrain ischemia by bilateral common carotid artery occlusions plus hemorrhagic hypotension to 45 mm Hg. Cranial temperature was maintained at normothermic levels. Hyperglycemic animals received dextrose (2.5 ml of a 25% solution, intraperitoneally) prior to ischemia; this doubled the mean plasma glucose concentration to 296 mg/100 ml. At 3 days (n = 10) or 24 h (n = 4) after ischemia, brains were perfusion-fixed and paraffin-embedded for light microscopic histopathology and for the histochemical visualization of activated microglia and the immunocytochemical visualization of glial fibrillary acid protein. Normal-neuron counts in the vulnerable hippocampal CA1 sector of hyperglycemic-ischemic (HI) rats were reduced to one-third the number observed in normoglycemic-ischemic (NI) animals. Ischemic cell counts in the striatum were increased fivefold or more in HI compared to NI rats, and normal small-neuron counts were reduced by two-thirds. The neocortex and striatum of NI rats showed only mild damage, while the majority of HI rats had extensive lesions, and several showed large cortical, striatal or thalamic infarcts. In addition, widespread cortical ischemic neuronal changes were evident in HI animals. No endothelial alterations were present in NI rats. By contrast, HI rats showed prominent peri- and intravascular polymorphonuclear and monocytic accumulation evident at 24 h; frequent white cell thrombi in pial arterioles on day 3; and thickening of vascular endothelium, with foci of parenchymal rarefaction or microinfarction adjacent to occluded vessels. Prominent microglial activation, often along the course of penetrating blood vessels, was common in the striatum and neocortex of HI animals but was much less extensive in the NI group. Activated microglia in HI rats were typically hypertrophic and amoeboid. These results suggest that the detrimental influence of hyperglycemia in ischemia is initially mediated by an action on vascular endothelium, which in turn leads to widespread foci of infarction and neuronal loss.

Background —Moderate elevations of brain temperature, when present during or after ischemia or trauma, may markedly worsen the resulting injury. We review these provocative findings, which form the rationale for our recommendation that physicians treating acute cerebral ischemia or traumatic brain injury diligently monitor their patients for incipient fever and take prompt measures to maintain core-body temperature at normothermic levels. Summary of Review —In standardized models of transient forebrain ischemia, intraischemic brain temperature elevations to 39°C enhance and accelerate severe neuropathological alterations in vulnerable brain regions and induce damage to structures not ordinarily affected. Conversely, the blunting of even mild spontaneous postischemic hyperthermia confers neuroprotection. Mild hyperthermia is also deleterious in focal ischemia, particularly in reversible vascular occlusion. The action of otherwise neuroprotective drugs in ischemia may be nullified by mild hyperthermia. Even when delayed by 24 hours after an acute insult, moderate hyperthermia can still worsen the pathological and neurobehavioral outcome. Hyperthermia acts through several mechanisms to worsen cerebral ischemia. These include (1) enhanced release of neurotransmitters; (2) exaggerated oxygen radical production; (3) more extensive blood-brain barrier breakdown; (4) increased numbers of potentially damaging ischemic depolarizations in the focal ischemic penumbra; (5) impaired recovery of energy metabolism and enhanced inhibition of protein kinases; and (6) worsening of cytoskeletal proteolysis. Recent studies demonstrate the feasibility of direct brain temperature monitoring in patients with traumatic and ischemic injury. Moderate to severe brain temperature elevations, exceeding core-body temperature, may occur in the injured brain. Cerebral hyperthermia also occurs during rewarming after hypothermic cardiopulmonary bypass procedures. Several studies have now shown that elevated temperature is associated with poor outcome in patients with acute stroke. Finally, recent clinical trials in severe closed head injury have shown a beneficial effect of moderate therapeutic hypothermia. Conclusions —The acutely ischemic or traumatized brain is inordinately susceptible to the damaging influence of even modest brain temperature elevations. While controlled clinical investigations will be required to establish the therapeutic efficacy and safety of frank hypothermia in patients with acute stroke, the available evidence is sufficiently compelling to justify the recommendation, at this time, that fever be combated assiduously in acute stroke and trauma patients, even if “minor” in degree and even when delayed in onset. We suggest that body temperature be maintained in a safe normothermic range (eg, 36.7°C to 37.0°C [98.0°F to 98.6°F]) for at least the first several days after acute stroke or head injury.