Albumin therapy augments the effect of thrombolysis on local vascular dynamics in a rat model of arteriolar thrombosis: a two-photon laser-scanning microscopy study.

BACKGROUND AND PURPOSE: Results of our recent pilot clinical trial suggest that the efficacy of thrombolytic therapy in acute ischemic stroke may be enhanced by the coadministration of high-dose albumin. Here, we explored the microvascular hemodynamic effects of this combined therapy in a laboratory model of cortical arteriolar thrombosis. METHODS: We studied the cortical microcirculation of physiologically monitored rats in vivo by two-photon laser-scanning microscopy after plasma-labeling with fluorescein-dextran. We induced focal thrombosis in 30- to 50-microm cortical arterioles by laser irradiation and measured arteriolar flow velocity by repeated line-scanning. At 30 minutes post-thrombosis, we treated animals with the thrombolytic agent, reteplase, which was coadministered with either human albumin, 2 g/kg, or with saline control. RESULTS: Baseline arteriolar flow velocity averaged 3.8+/-0.7 mm/s, was immediately reduced by thrombosis to 22% to 25% of control values, and remained unchanged before treatment. Subthrombolytic doses of reteplase combined with saline led to a median increase in flow velocity to 37% of control distal to the thrombus (P=nonsignificant versus pretreatment). By contrast, reteplase combined with albumin therapy resulted in a prompt, highly significant increase of median flow velocity to 58% of control levels (P=0.013 versus reteplase+saline), which remained significantly higher than the reteplase+saline group at multiple time-points over the subsequent hour. CONCLUSIONS: The beneficial effect of subthrombolytic doses of reteplase on microvascular hemodynamics distal to a cortical arteriolar thrombosis is markedly enhanced by the coadministration of high-dose albumin therapy; these results have important clinical implications for the management of patients with acute ischemic stroke.

Experimental intracerebral hematoma in the rat: characterization by sequential magnetic resonance imaging, behavior, and histopathology. Effect of albumin therapy.

We characterized acute intracerebral hemorrhage (ICH) in the rat by sequential magnetic resonance imaging (MRI) and correlated MRI findings with neurobehavior and histopathology. In addition, we investigated whether albumin treatment would reduce ICH-induced brain injury. ICH was produced in rats by a double-injection method in which 45 microl of fresh arterial blood was injected into the right striatum. Susceptibility-weighted (SWI) and T2-weighted (T2WI) MRI was carried out on a 4.7T magnet at 0-1 h, 6 h, 24 h, 72 h, and 7 days after ICH. Animals were treated with either 25% human albumin, 1.25 g/kg, or saline vehicle i.v. at 90 min after ICH. Neurological status was evaluated before ICH and after treatment (at 4 h, 24 h, 48 h, 72 h, and 7 days). Brains were then perfusion-fixed, re-imaged on an 11.7T magnet, and studied by histopathology and immunochemistry. MRI revealed a consistent hematoma involving the striatum and overlying corpus callosum, with significant volume changes over time. Lesion volumes computed from T2WI images and by histopathology agreed closely with one another and were highly correlated (p=0.002). SWI lesion volumes were also highly correlated to histological volumes (p<0.001) but overestimated histological hematoma volume by approximately 5-fold. Albumin treatment significantly improved neurological scores compared to saline at 72 h (3.8+/-0.6 vs. 1.5+/-0.7) and 7 days (3.8+/-0.4 vs. 1.3+/-0.5, respectively, p<0.05), but did not affect histological or MRI lesion volumes. Taken together, sequential MRI plus histopathology provides a comprehensive characterization of experimental ICH. Albumin treatment improves neurological deficit after ICH but does not affect MRI or histological hematoma size.

Remote organ ischemic preconditioning protect brain from ischemic damage following asphyxial cardiac arrest.

Ischemic preconditioning (IPC) is a phenomenon whereby an organ's adaptive transient resistance to a lethal ischemic insult occurs by preconditioning this organ with a sub-lethal/mild ischemic insult of short duration. Besides IPC, recent studies reported that a short sub-lethal ischemia and reperfusion in various organs can induce ischemic tolerance in another organ as well. This phenomenon is known as remote ischemic preconditioning (RPC). In the present study we tested the hypothesis that tolerance for ischemia can be induced in brain by RPC and IPC in a rat model of asphyxial cardiac arrest (ACA). RPC was induced by tightening the upper two-thirds of both hind limbs using a tourniquet for 15 or 30 min and IPC was induced by tightening bilateral carotid artery ligatures for 2 min. Eight minutes of ACA was induced 48 h after RPC or IPC. After 7 day of resuscitation, brains were extracted and examined for histopathological changes. In CA1 hippocampus, the number of normal neurons was 63% lower in cardiac-arrested rats as compared to the control group. The number of normal neurons in the 15 min RPC, 30 min RPC, and IPC groups was higher than the ACA group by 54, 70, and 67%, respectively. This study demonstrates that RPC and IPC are able to provide neuroprotection in a rat model of ACA. Besides direct application of RPC or IPC paradigms, the exploration of the mechanisms of observed neuroprotection by RPC and IPC may also lead to a possible therapy for CA patients.

Neuroprotective effect of darbepoetin alfa, a novel recombinant erythropoietic protein, in focal cerebral ischemia in rats.

BACKGROUND AND PURPOSE: Darbepoetin alfa is a novel erythropoiesis-stimulating protein developed for treating anemia. In animal models, exogenous recombinant human erythropoietin has been reported to be beneficial in treating experimental cerebral ischemia. In this study, we determined whether darbepoetin alfa would protect in a rat model of transient focal cerebral ischemia. METHODS: Rats received 2-hour middle cerebral artery suture-occlusion. The drug (darbepoetin alfa, 10 microg/kg) or vehicle was administered intraperitoneally 2 hours after onset of middle cerebral artery occlusion. Animals were allowed to survive for 3 or 14 days. Behavioral tests were performed sequentially. Infarct volumes and brain swelling were determined. RESULTS: Darbepoetin alfa-treated rats showed improved neuroscores relative to vehicle-treated animals beginning within 1 hour of treatment and persisting throughout the 14-day survival period. Darbepoetin alfa significantly reduced corrected total (cortical + subcortical) infarct volume (56.3+/-20.6 and 110.8+/-6.8 mm3, respectively) and total infarct areas at multiple levels compared with vehicle in the 14-day survival group. Brain swelling was not affected by treatment. CONCLUSIONS: Darbepoetin alfa confers behavioral and histological neuroprotection after focal ischemia in rats.

Albumin treatment reduces neurological deficit and protects blood-brain barrier integrity after acute intracortical hematoma in the rat.

BACKGROUND AND PURPOSE: Acute intracerebral hemorrhage (ICH) is a common and severe form of stroke. To date, medical management of ICH has had scant impact on morbidity and mortality. Because albumin therapy is markedly neuroprotective in preclinical models of ischemic stroke, and because ischemic and hemorrhagic stroke share several common injury mechanisms, we hypothesized that albumin therapy might also benefit ICH. METHODS: Acute intracortical hematoma was produced in anesthetized, normothermic rats by the single stereotaxic injection of 50 muL of autologous, nonheparinized whole blood over 5 minutes. Separate animal groups were treated either with 25% human albumin, 1.25 g/kg, or with intravenous saline vehicle at 60 minutes after ICH. Neurobehavior was quantified sequentially over the next 2 to 7 days. Damage to the blood-brain barrier was assessed at 2 days after ICH by fluorometric measurement of Evans blue extravasation in dissected brain regions. RESULTS: High-grade neurological deficits were present in all rats at 50 minutes after ICH (score 10.3+/-0.2, mean+/-SEM [maximal score 12]). Albumin-treated rats showed improved neuroscores relative to saline-treated animals beginning within hours of treatment and persisting throughout the 7-day survival period. At 3 and 7 days, mean total neuroscores of the albumin group were 38% to 43% lower than in saline-treated animals. Perihematomal Evans blue discoloration was readily evident in saline-treated ICH rats but was reduced by albumin treatment. Hemispheric Evans blue content ipsilateral to the hematoma was reduced by 49% by albumin treatment (albumin 93.9+/-13.3 versus saline 184.7+/-33.7 mg/g, P<0.05). Hematoma volume and brain swelling were not affected by albumin treatment. CONCLUSIONS: Prompt albumin therapy improves neurological function and blood-brain barrier integrity after acute intracortical hematoma. These observations have important potential clinical implications.

Docosahexaenoic acid complexed to albumin elicits high-grade ischemic neuroprotection.

BACKGROUND AND PURPOSE: High-dose human albumin therapy is strongly neuroprotective in models of brain ischemia and trauma and is currently being studied in a pilot-phase clinical stroke trial. Among its actions in ischemia, albumin induces the systemic mobilization of n-3 polyunsaturated fatty acids and may help to replenish polyunsaturated fatty acids lost from neural membranes. METHODS: We complexed 25% human albumin to docosahexaenoic acid (DHA; 22:6n-3) and compared its neuroprotective efficacy with that of native albumin in rats with 2-hour focal ischemia produced by intraluminal suture-occlusion of the middle cerebral artery. RESULTS: In animals treated with DHA-albumin, 0.63 g/kg, the improvement in neurobehavioral scores at 72 hours significantly exceeded that of other treatment groups, and the extent of histological protection (86% reduction in cortical infarction) was highly significant and tended to surpass the degree of cortical protection produced by native albumin at 1.25 g/kg (65%). DHA-albumin 0.63 g/kg, but not native albumin, also significantly reduced subcortical infarction and markedly diminished brain swelling. Lipidomic analysis of DHA-albumin-treated postischemic brains revealed a large accumulation of the neuroprotective DHA metabolite, 10,17S-docosatriene, in the ipsilateral hemisphere. CONCLUSIONS: The high-grade neuroprotection afforded by the DHA-albumin complex at relatively low albumin doses is clinically advantageous in that it might reduce the likelihood of acute intravascular volume overload and congestive heart failure sometimes induced when patients with compromised cardiovascular function are treated with high-dose albumin.

Protein kinase C delta cleavage initiates an aberrant signal transduction pathway after cardiac arrest and oxygen glucose deprivation.

Protein kinase C (PKC) isozymes have been known to mediate a variety of complex and diverse cellular functions. deltaPKC has been implicated in mediating apoptosis. Using two models of cerebral ischemia, cardiac arrest in rats and oxygen glucose deprivation (OGD) in organotypic hippocampal slices, we tested whether an ischemic insult promoted deltaPKC cleavage during the reperfusion and whether the upstream pathway involved release of cytochrome c and caspase 3 cleavage. We showed that cardiac arrest/OGD significantly enhanced deltaPKC translocation and increased its cleavage at 3 h of reperfusion. Since deltaPKC is one of the substrates for caspase 3, we next determined caspase 3 activation after cardiac arrest and OGD. The maximum decrease in levels of procaspase 3 was observed at 3 h of reperfusion after cardiac arrest and OGD. We also determined cytochrome c release, since it is upstream of caspase 3 activation. Cytochrome c in cytosol increased at 1 h of reperfusion after cardiac arrest/OGD. Inhibition of either deltaPKC/caspase 3 during OGD and early reperfusion resulted in neuroprotection in CA1 region of hippocampus. Our results support the deleterious role of deltaPKC in reperfusion injury. We propose that early cytochrome c release and caspase 3 activation promote deltaPKC translocation/cleavage.

Astrocytes react to oligemia in the forebrain induced by chronic bilateral common carotid artery occlusion in rats.

The effects of oligemia (moderate ischemia) on the brain need to be explored because of the potential role of subtle microvascular changes in vascular cognitive impairment and dementia. Chronic bilateral common carotid artery occlusion (BCCAO) in adult rats has been used to study effects of oligemia (hypoperfusion) using neuropathological and neurochemical analysis as well as behavioral tests. In this study, BCCAO was induced for 1 week, or 2, 4, and 6 months. Sensitive immunohistochemistry with marker proteins was used to study reactions of astrocytes (GFAP, nestin), and lectin binding to study microglial cells during BCCAO. Overt neuronal loss was visualized with NeuN antibodies. Astrocytes reacted to changes in the optic tract at all time points, and strong glial reactions also occurred in the target areas of retinal fibers, indicating damage to the retina and optic nerve. Astrocytes indicated a change in the corpus callosum from early to late time points. Diffuse increases in GFAP labeling occurred in parts of the neocortex after 1 week of BCCAO, in the absence of focal changes of neuronal marker proteins. No significant differences emerged in the cortex at longer time points. Nestin labeling was elevated in the optic tract. Reactions of microglia cells were seen in the cortex after 1 week. Measurements of the basilar artery indicated a considerable hypertrophy, indicative of macrovascular compensation in the chronic occlusion model. These results indicate that chronic BCCAO and, by inference, oligemia have a transient effect on the neocortex and a long-lasting effect on white matter structures.

A selective endothelin ET(A) receptor antagonist, SB 234551, improves cerebral perfusion following permanent focal cerebral ischemia in rats.

In recent experimental studies, a selective antagonist of endothelin ET(A) receptors, SB 234551, improved neurological and histological outcome in both head trauma and transient focal cerebral ischemia. The present study was conducted to ascertain the degree to which hemodynamic alterations are responsible for this therapeutic effect in a model of permanent middle cerebral artery occlusion (MCAo) in rats. Anesthetized Sprague-Dawley rats were subjected to permanent MCAo by insertion of an intraluminal nylon suture coated with poly-L-lysine. The agent (SB 234551, 30 microg/kg/min = 1.8 mg/kg/h) or vehicle (PBS; 0.6 ml/h) was administered by i.v. infusion beginning 15 min after onset of MCAo and lasting for 23.75 h. Autoradiographic measurement of local cerebral blood flow (lCBF) was performed at 24 h. Physiological data were similar among groups. SB 234551 augmented perfusion by 1.7- to 1.8-fold in both the ischemic hemisphere and in the contralateral (non-ischemic) hemisphere when compared to vehicle-treated ischemic animals. In the ischemic hemisphere, the brain regions significantly benefited were those lying outside the zone of most dense ischemia (i.e., paramedian cortex and thalamus), while in the non-ischemic hemisphere all regions measured showed significant lCBF augmentation. This study demonstrates that SB 234551 therapy results in significant improvement of local cerebral perfusion in the ischemic as well as in the non-ischemic hemispheres after permanent MCAo.

Albumin therapy of transient focal cerebral ischemia: in vivo analysis of dynamic microvascular responses.

BACKGROUND AND PURPOSE: To study whether intravascular or hemodynamic factors contribute to the marked neuroprotective effect of albumin therapy in focal cerebral ischemia, 2 complementary methods were applied: laser-scanning confocal microscopy (LSCM) and laser-Doppler perfusion imaging (LDPI). METHODS: In the LSCM study, Sprague-Dawley rats were anesthetized with halothane/nitrous oxide, and a cranial window was placed over the dorsolateral frontoparietal cortex. Rats received 2-hour middle cerebral artery occlusion (MCAO) by an intraluminal suture and were treated with human albumin (1.25 g/kg; n=4) or saline (n=3) after 30 minutes of recirculation. Video images of cortical vessels were continually acquired and were digitized offline to measure diameters and fluorescent erythrocyte velocities. In the LDPI study, cortical perfusion was measured in anesthetized Sprague-Dawley rats that received 2-hour MCAO and were treated with albumin (2.5 g/kg; n=6) or saline (n=5) at 30 minutes after recirculation. RESULTS: In the LSCM study, MCAO was associated with arteriolar dilation and slowing of capillary and venular erythrocyte perfusion. During the first 15 to 30 minutes of postischemic recirculation, prominent foci of vascular stagnation developed within cortical venules, associated with thrombuslike foci and adherent corpuscular structures consistent in size with neutrophils. Saline administration failed to affect these phenomena, while albumin therapy was followed by significant increases in arteriolar diameter ( approximately 12%; P=0.007) and by a prompt improvement of venular and capillary erythrocyte perfusion and a partial disappearance of adherent thrombotic material. Albumin therapy increased erythrocyte flow velocity in both capillaries (288+/-73% versus 76+/-18% in the saline group; P=0.023) and venules (2.7-fold [P=0.001] versus 1.0-fold in the saline group [P=NS]). In the LDPI study, cortical perfusion declined during MCAO and rose initially with recirculation (to approximately 135% of baseline) in both groups. Mean cortical perfusion improved slightly (approximately 14%; P=NS) in albumin-treated animals. CONCLUSIONS: These results reveal a beneficial effect of albumin therapy in reversing stagnation, thrombosis, and corpuscular adherence within cortical venules in the reperfusion phase after focal ischemia and support its utility in the treatment of acute ischemic stroke.

Neuroprotective effect of SolCD39, a novel platelet aggregation inhibitor, on transient middle cerebral artery occlusion in rats.

BACKGROUND AND PURPOSE: SolCD39 is a soluble form of recombinant human ecto-ATP/ADPase (NTPDase1) and represents a new class of antithrombotic agents. SolCD39 blocks and reverses platelet activation, preventing recruitment of additional platelets into a growing thrombus. The purpose of this study was to examine the effect of solCD39 on neurological deficit, infarct size, and extent of edema after transient middle cerebral artery occlusion (MCAO) in rats. METHODS: Physiologically controlled Sprague-Dawley rats underwent 2-hour MCAO by retrograde insertion of an intraluminal suture coated with poly-l-lysine. The agent (solCD39) was administered intravenously before MCAO or at 1-hour or 3-hour recirculation. Other groups received vehicle (Tris-buffered saline) or human albumin (as a "positive" neuroprotective control; 25%, 0.5% of body weight) at 1-hour recirculation. Neurological status was evaluated during occlusion (at 60 minutes) and daily for 3 days after MCAO. Brains were perfusion-fixed at 72 hours, and infarct volumes and brain swelling were determined. RESULTS: Pretreatment with solCD39 significantly improved the neurological score at 72 hours compared with the vehicle group (4.4+/-0.6 versus 7.6+/-0.6, respectively; P=0.008). Cortical infarct areas were significantly reduced at multiple levels by pretreatment with solCD39. Total striatal infarct area was also significantly reduced compared with vehicle by both solCD39 pretreatment (48% mean reduction) and solCD39 treatment at 3-hour recirculation (51% mean reduction). Treatment with SolCD39 significantly reduced total infarct volume (corrected for brain swelling) by an average of 71% to 72% when administered either before ischemia or at 3 hours of recirculation compared with vehicle. Treatment with albumin significantly reduced neurological score and total, cortical, and subcortical infarction at multiple levels, as expected. CONCLUSIONS: Treatment with SolCD39, administered either before or at 3 hours after MCAO, improves neurological score and reduces infarct size compared with vehicle. A pharmacological agent of this type appears to have potential for the treatment of focal ischemic stroke.

Experimental intracerebral hemorrhage in the mouse: histological, behavioral, and hemodynamic characterization of a double-injection model.

BACKGROUND AND PURPOSE: A major limitation of intracerebral hemorrhage (ICH) research is the lack of reproducible animal models. The present study was conducted to validate in the mouse the double-injection method of ICH initially developed in the rat. We investigated the effect of intrastriatal injection of blood or cerebrospinal fluid (CSF) on cerebral blood flow (CBF), neurological score, hematoma volume, and brain swelling. METHODS: Male C57BL/6 mice were anesthetized with halothane/nitrous oxide delivered by face mask. Rectal and cranial temperatures were regulated at 37 degrees C to 37.5 degrees C. Mice were placed in a stereotactic frame, and a 30-gauge stainless steel cannula was introduced through a burr hole into the left striatum. Each mouse received a 5-microL injection of either whole blood or CSF (over 3 minutes), followed 7 minutes later by 10 microL injected over 5 minutes. The injection cannula was slowly withdrawn 10 minutes after the second injection. Control mice had only cannula insertion. CBF was studied by laser Doppler perfusion imaging. Neurological status was evaluated on days 1 and 2. After 2 days, hematoma volume and brain swelling were calculated. RESULTS: Physiological values were stable. Mice with ICH but not those with CSF or cannula alone had a marked, persistent neurological deficit and a highly reproducible hematoma, whose mean+/-SEM volume was 2.0+/-0.2 mm3 compared with a lesion size of 0.2+/-0.1 mm3 in mice with CSF. Residual swelling of the ipsilateral hemisphere at 48 hours was 5.7% in the hematoma and 1.5% in the CSF groups. Relative CBF in the neocortex ipsilateral to the injection site declined by approximately 45% to 60% during the first 20 minutes after cannula insertion/injection in all groups but began to renormalize at approximately 25 to 30 minutes in the CSF and cannula-only groups; in the hematoma group, cortical hypoperfusion of approximately 35% to 50% persisted during the 90-minute measurement period. CONCLUSIONS: The present ICH model in mice produces a consistent neurological deficit, hypoperfusion, hematoma volume, and brain swelling. This model closely mimics human hypertensive basal ganglionic ICH and should be useful for the evaluation of pharmaceutical therapies. Laser Doppler perfusion imaging is a useful new technique to quantify relative CBF changes and can be used for studies of dynamic changes of CBF in this in vivo model of ICH in mice.

Effect of ischemic preconditioning on the expression of putative neuroprotective genes in the rat brain.

Previous studies have demonstrated that sublethal ischemic insults protect from subsequent ischemia in the intact brain. There are two windows for the induction of tolerance by ischemic preconditioning (IPC). One occurs within 1 h following IPC, and the other one develops from 1 to 3 days after IPC. The goal of this study was to determine whether IPC neuroprotection may be mediated by expression of known neuroprotective genes and to characterize the temporal and spatial expression patterns of these genes. IPC was produced by bilateral carotid artery occlusions and hypotension (50 mmHg) for 2 min. After various survival times, the expression of MAP-2, brain-derived neurotrophic factor (BDNF), c-jun, c-fos, nerve growth factor (NGF) and HSP70 was assessed by in situ hybridization of coronal brain sections with 35S labeled probes. BDNF, NGF, and c-jun were significantly upregulated in the hippocampus. c-fos was detected in the hippocampus, cortex and striatum. HSP70 mRNA was induced in the cortex, hippocampus, striatum, and thalamus. MAP-2 showed no change in expression, confirming previous studies that no cell death occurs following IPC. The increase in expression of these stress-related, neurotrophic and immediate early genes in response to a mild preconditioning insult may help mediate the protection of vulnerable neurons to subsequent lethal ischemic insults.

DNA microarray analysis of cortical gene expression during early recirculation after focal brain ischemia in rat.

Focal brain ischemia is followed by changes in gene expression as reflected by altered mRNA levels. DNA microarray analysis can be used to survey thousands of genes for differential expression triggered by ischemic metabolic stress. In this study, Sprague-Dawley rats were subjected to 2 h of middle cerebral artery occlusion (MCAO) using an intravascular poly-L-lysine-coated filament, and brains were removed after 3 h of recirculation for mRNA isolation. A differential measurement of mRNAs from post-ischemic and sham control animals was performed using the Mouse UniGene 1 microarray. Established values for differential expression were used (> or =1.7 or < or =-1.7 fold), and hits (n=2-3 arrays) divided into known 'ischemia-hypoxia response' genes and 'newly connected' annotated genes. n=28 ischemia-hypoxia response genes were up-regulated and n=6 were down-regulated. Regulated genes comprised immediate early genes, heat shock proteins, anti-oxidative enzymes, trophic factors, and genes involved in RNA metabolism, inflammation and cell signaling. Based on the ability of the microarray to replicate known changes in gene expression, n=35 newly connected genes were found up-regulated and n=41 down-regulated. DNA microarray analysis allows one to develop novel working hypotheses for responses to brain ischemia based on the regulation of annotated genes.

Effect of the first window of ischemic preconditioning on mitochondrial dysfunction following global cerebral ischemia.

Rats may develop sustained tolerance against lethal cerebral ischemia after exposure to a sublethal ischemic insult (ischemic preconditioning (IPC)). Two windows for the induction of tolerance by IPC have been proposed, one that occurs within 1h following IPC, and the other one that occurs 1-3 days after IPC. An important difference between these two windows is that in contrast to the second window, neuroprotection against lethal ischemia is transient in the first window. We tested the hypothesis that rapid IPC would reduce or prevent ischemia-induced changes in mitochondrial function. IPC and ischemia were produced by bilateral carotid occlusions and systemic hypotension (50 mmHg) for 2 and 10 min, respectively. The non-synaptosomal mitochondria were harvested 30 min following the 10 min 'test' ischemia. Mitochondrial rate of respiration decreased by 10% when the substrates were pyruvate and malate, and 29% when the substrates were ascorbic acid and N,N,N',N'-tetramethyl-p-phenylenediamine ( P< 0.01). The activities of complex I-III decreased in ischemic group by 16, 23 (P < 0.05) and 24%, respectively. IPC was unable to prevent decreases in the rate of respiration and activities of different complexes. These data suggest that rapidly induced IPC is unable to protect the integrity of mitochondrial oxidative phosphorylation following cerebral ischemia, perhaps explaining why IPC only provides transitory protection in the 'first window'.

Automated registration of laser Doppler perfusion images by an adaptive correlation approach: application to focal cerebral ischemia in the rat.

Hemodynamic changes are extremely important in analyzing responses from a brain subjected to a stimulus or treatment. The Laser Doppler technique has emerged as an important tool in neuroscience research. This non-invasive method scans a low-power laser beam in a raster pattern over a tissue surface to generate the time course of images in unit of relative flux changes. Laser Doppler imager (LDI) records cerebral perfusion not only in the temporal but also in the spatial domain. The traditional analysis of LD images has been focused on the region-of-interest (ROI) approach, in which the analytical accuracy in an experiment that necessitates a relative repositioning between the LDI and the scanned tissue area will be weakened due to the operator's subjective decision in data collecting. This report describes a robust image registration method designed to obviate this problem, which is based on the adaptive correlation approach. The assumption in mapping corresponding pixels in two images is to correlate the regions in which these pixels are centered. Based on this assumption, correlation coefficients are calculated between two regions by a method in which one region is moved around over the other in all possible combinations. To avoid ambiguity in distinguishing maximum correlation coefficients, an adaptive algorithm is adopted. Correspondences are then used to estimate the transformation by linear regression. We used a pair of phantom LD images to test this algorithm. A reliability test was also performed on each of the 15 sequential LD images derived from an actual experiment by imposing rotation and translation. The result shows that the calculated transformation parameters (rotation: theta =7.7+/-0.5 degrees; translation: Delta x =2.8+/-0.3, Deltay=4.7+/-0.4) are very close to the prior-set parameters (rotation: theta=8 degrees; translation: Delta x=3, Delta y=5). This result indicates that this approach is a valuable adjunct to LD perfusion monitoring. An original sequence of LD images that recorded cerebral perfusion through a cranial window before, during and after middle cerebral artery occlusion (MCAo) is presented, together with the registered image sequence. Cerebral perfusion data acquired in a pixel-based manner from different anatomic locations of the registered LD image sequence are also presented over the whole time-course of the experiment.

Caffeinol confers cortical but not subcortical neuroprotection after transient focal cerebral ischemia in rats.

The combination of low-dose ethanol and caffeine (caffeinol) protects cortical areas of the brain from damage produced by distal focal ischemia in rats. There are no data, however, as to whether caffeinol influences injury in subcortical brain regions. Rats were anesthetized with halothane and subjected to 2 h of MCAo by poly-l-lysine-coated intraluminal suture. Caffeinol [a combination of ethanol, 0.33 g/kg, and caffeine, 10 mg/kg (n=5)] or vehicle (0.9% NaCl; n=7) was administered by i.v. infusion over a 2.5-h period beginning 15 min after reperfusion. Neurological status was evaluated daily, and histopathology was quantified at 3 days. Caffeinol therapy significantly improved the neurological score, reduced the total infarct volume (by 52%) and cortical infarct areas at multiple coronal levels, but subcortical infarction and brain swelling were not affected.

Nuclear localization of the hypoxia-regulated pro-apoptotic protein BNIP3 after global brain ischemia in the rat hippocampus.

The 19 kD interacting protein 3, Nip3/BNIP3, is a pro-apoptotic member of the Bcl-2 family induced during hypoxia via the hypoxia-inducible factor (HIF) 1. BNIP3 has been linked to both apoptotic and necrotic cell death involving mitochondrial permeability transition. Since apoptotic and necrotic mechanisms may occur in brain ischemia, immunohistochemical changes of BNIP3 were studied at 1, 2, 3 and 7 days after transient global brain ischemia (12.5 min) in ventilated normothermic rats. In control brains, BNIP3-like immunoreactivity was moderately strong in neuronal processes or cytoplasm and absent in the nucleus. In the ischemia-vulnerable CA1 neurons, BNIP3-positive granules were seen in the nucleus at 1 and 2 days, and these neurons were damaged at 3 and 7 days. The resistant CA3 neurons showed nuclear BNIP3 labeling by 1 day and then returned to the normal state. BNIP3-positive granules did not overlap with the nucleolus. Constitutively expressed BNIP3 may participate in apoptotic and necrotic processes after brain ischemia. Nuclear location of BNIP3 after brain ischemia indicates a novel role for the regulation of cell survival in neurons or a general disturbance of the nuclear envelope.

Delayed post-ischemic albumin treatment neither improves nor worsens the outcome of transient focal cerebral ischemia in rats.

Human albumin therapy within the first 4 h is highly neuroprotective in focal ischemia, but it is unknown whether delayed albumin therapy is deleterious. Rats received 2 h middle cerebral artery suture-occlusion. Human albumin (25%, 2.5 mg/kg; n=12) or vehicle (0.9% saline, 5 ml/kg; n=9) were administered at 19 h. Neurological status was evaluated daily, and histopathology and brain swelling were quantified at 3 days. Delayed albumin treatment, while ineffective, failed to show adverse effects.

Enriched environment delays the onset of hippocampal damage after global cerebral ischemia in rats.

An enriched environment has been shown to improve cognitive, behavioral and histopathological outcome after focal cerebral ischemia and head trauma. The purpose of this study was to determine the effect of an enriched environment on histopathology following global cerebral ischemia. Wistar rats (21 weeks of age) were placed in different environments [standard cages (SC) or enriched environment (EE) cages] for 2 months before and either 6 days or 2 months after ischemia. Rats underwent 10 min of global ischemia by bilateral carotid artery occlusions plus hypotension. Five groups (n=4-5 in each group) were studied: (1) rats kept in SC before and 2 months after ischemia; (2) rats kept in SC before ischemia but transferred to an EE for 2 months after ischemia; (3) rats kept in EE before and after ischemia for 2 months; (4) rats kept in SC before and 6 days after ischemia; (5) rats kept in EE before and 6 days after ischemia. At 7 days or 2 months after ischemia, brains were perfusion-fixed, and ischemic injury was assessed by counting numbers of normal neurons in the hippocampal CA1 sector. Physiological variables showed no inter-group differences. Rats housed in EE for 2 months before and for 6 days (but not 2 months) after global ischemia showed significantly better preservation of pyramidal neurons in the hippocampal CA1 area when compared to control animals (middle CA1, 20.5+/-5.4 vs. 2.8+/-0.6; lateral CA1, 31.5+/-7.2 vs. 2.6+/-0.6, respectively). The present data suggest that housing in EE for 2 months before and 6 days after ischemia can delay the onset of damage to hippocampal pyramidal neurons, which eventually occurs despite 2-month EE.