138. Effects of cerebral ischemia and ischemic preconditioning on neuronal hemoglobin
Y. He, Y. Hua, W. Liu, H. Hu, R. Keep and G. Xi
Neurosurgery, University of Michigan, Ann Arbor, Michigan, USA
Objectives: Recent studies have showed hemoglobin (Hb) is expressed in neurons.
1
The presence of neuronal Hb and its high affinity for oxygen indicate it may have an important role in regulating oxygen hemostasis in neurons. On the other hand, it has been known that exogenous Hb is deleterious to the brain after brain hemorrhage.
2
The present study examined if cerebral ischemia or ischemic preconditioning (IPC) affects neuronal Hb levels in vivo and in vitro.
Methods: There were three parts to the in vivo study. In the first part, rats were subjected to 15 mins of transient middle cerebral artery occlusion, an IPC stimulus, or sham operation (n = 6, each group). In the second part, rats underwent a permanent middle cerebral artery occlusion (pMCAO) to induce brain injury or a sham operation (n = 10, each group). In the third part, rats underwent a further pMCAO three days after IPC or sham operation (n = 7, each group). All rats were killed 24 h later for immunohistochemistry, Western blot analysis and real-time polymerase chain reaction (PCR). In vitro, primary cultured neurons and astrocytes were used for Western blotting and real-time PCR. Cultured neurons were exposed to 2 h of oxygen-glucose deprivation with 22 h of reoxygenation.
Results: We found that Hb is widely expressed in rat cerebral neurons but not astrocytes. Western blot analysis showed that Hb expression was significantly upregulated in the ipsilateral caudate and the cortical core of the middle cerebral artery territory after IPC. To further confirm the synthesis of Hb within the brain, quantitative real-time PCR was performed on rat brains subjected to IPC or sham operation. There was a 1.8±0.9 fold increase in HbA mRNA in the ipsilateral caudate and 2.6±1.2 fold increase in the ipsilateral cortical core sample in IPC-treated rats compared to shams (P<0.05). HbB mRNA was also upregulated by 27.9±8.4 times in the ipsilateral caudate and 4.8±2.3 times in the ipsilateral cortical core compared to shams (P<0.05). Hb levels also increased in more penumbral cortex and the contralateral hemisphere 24 h after pMCAO, but expression in the ipsilateral caudate and cortical core area were decreased. Ischemic preconditioning modified pMCAO-induced brain Hb changes. Neuronal Hb levels in vitro were increased by 2 h of oxygen-glucose deprivation and 22 h of reoxygenation.
Conclusions: Our present study demonstrates that Hb is expressed in cerebral neurons, and that neuronal Hb is inducible after cerebral ischemia. An understanding of the mechanisms of induction of neuronal Hb after ischemia and the function of neuronal Hb should be helpful in seeking effective new treatment for stroke.
722. Beyond post-stroke depression: effects of delayed cipramil treatment on secondary exofocal postischemic degeneration and long-term stroke outcome
G. Kronenberg1,2,3, M. Balkaya1,2, V. Prinz1,2, K. Gertz1,2, S. Ji1,2, H. Hoertnagl4, C. Waebert5, R. Stumm6 and M. Endres1
1Neurology; 2Center for Stroke Research Berlin; 3Department of Psychiatry Campus Benjamin Franklin; 4Institute of Pharmacology and Toxicology; Charite—Universitaetsmedizin Berlin, Berlin, Germany; 5Stroke and Neurovascular Regulation Laboratory; Massachusetts General Hospital/Harvard Medical School, Charlestown, Massachusetts, USA; 6Institute of Pharmacology and Toxicology, Otto-v.-Guericke University, Magdeburg, Germany
Objectives: Post-stroke depression (PSD) is the most frequent neuro-psychiatric complication of stroke, affecting up to 50% of stroke survivors. Moreover, PSD has severe adverse effects on functional recovery and on longer-term survival. Despite its great clinical and therapeutic relevance, PSD remains underresearched. This may be partly due to the fact that suitable animal models are lacking and existing models are poorly characterized. Here, we set out to validate our well-defined stroke paradigm of 30 mins middle cerebral artery occlusion (MCAo) in the mouse as a potential model to study pathogenetic mechanisms underlying the development of PSD and how these are impacted by psychopharmacotherapy.
Methods: 129/SV mice were subjected to either sham operation or MCAo/reperfusion. A subset of animals was treated with selective serotonin reuptake inhibitor (SSRI) citalopram starting seven days after induction of stroke. Behavioral assessment was begun at 14 weeks after MCAo and included sucrose consumption, spontaneous locomotor activity, the novelty-suppressed feeding paradigm, the elevated plus maze, the light-dark box as well as Porsolt's test. Animals were killed at 16 weeks after MCAo and brains harvested for further histological and biochemical evaluation. The primary lesion was delineated using NeuN staining. Accumulation of microglia in the ischemic striatum was quantified using activated microglia marker Iba1. The integrity of the mesolimbic dopaminergic pathway was probed using tyrosine hydroxylase (TH) immunohistochemistry and in situ hybridization. Dopamine levels were measured with HPLC. Striatal dopamine transporter density was examined using autoradiography.
Results: Following left-sided MCAo, animals reproducibly developed an ′affective′ phenotype characterized by anhedonia, increased anxiety and despair. By contrast, right-sided MCAo was not associated with a depression-related phenotype but resulted in hyperactivity. Subacute citalopram administration reversed the behavioral alterations of left MCAo confirming pharmacological treatment validity of our model. Across groups (left MCAo, sham, treatment with either vehicle or citalopram) we observed a significant negative correlation between striatal dopamine levels and the latency to feed in the novelty-suppressed feeding test. Similarly, striatal dopamine levels showed a strong positive correlation with time in the lit compartment of the light-dark box and with the latency to float in Porsolt's test. MCAo caused significantly reduced dopamine and dopamine transporter levels in ischemic striatum in parallel with a reduced number of TH+ neurons in the ipsilateral midbrain. Delayed citalopram treatment largely attenuated all of these effects. Moreover, citalopram treatment reduced the size of the primary lesion along with the number of activated microglia in the ischemic striatum.
Conclusions: We here describe a reliable experimental model of post stroke depression. Our results highlight the significance of the mesolimbic dopaminergic pathway for the development of affective stigmata in this paradigm. Subacute treatment with SSRI citalopram not only reversed depressive behaviors, but also reduced the size of the primary ischemic lesion and prevented secondary exofocal postischemic degeneration of midbrain dopaminergic neurons. Neuroprotection against secondary neuronal injury mechanisms may also underlie the clinical benefits of antidepressant therapy after stroke regarding both functional recovery and survival.
137. Deferoxamine reduces intracerebral hemorrhage-induced brain injury in aged rats
Y. Hua1, M. Okauchi1, R. Keep1, L. Morgenstern2, T. Schallert1 and G. Xi1
1Neurosurgery; 2Neurology, University of Michigan, Ann Arbor, Michigan, USA
Objectives: Deferoxamine (DFX) reduces brain edema, neuronal death and neurological deficits after intracerebral hemorrhage (ICH) in young rats.1,2 In the present study, we investigated whether DFX is effective on brain injury after ICH in aged rats.
Methods: There are two sets of experiments in this study. In the first set, male Fischer 344 rats (18-months old) had an intracaudate injection of 100-μl autologous whole blood. Sham rats only received a needle insertion. Rats were treated with DFX (10, 50 or 100 mg/kg, i.m.; n = 9) or vehicle (n = 12) 2 and 6 h post ICH and then every 12 h up to 72 h. Rats were sacrificed at day 3 for brain edema measurement. In the second set, rats were treated with DFX or vehicle 2 and 6 h post ICH and then every 12 h for 7 days. Rats had magnetic resonance (MR) scans for T2 imaging eight weeks after ICH and were sacrificed for brain atrophy measurement. Forelimb placing score and corner turn score were assessed at four and eight weeks. Body weight and blood pressure were measured at 1, 3, 7, 14, 21, 28, 42, and 56 days after surgery. Body weight was expressed as % change in body weight using this formula: % change in body weight = (body weight on each time point-body weight before surgery)/body weight before surgery. Mean arterial blood pressure was measured using tail cuff plethysmography.
Results: ICH caused a marked increase in perihematomal water content in vehicle-treated rats (81.6±0.9 versus 77.7±0.4% in sham control, P<0.01). The systemic administration of 100 mg/kg DFX starting 2 h after ICH reduced edema (80.3±0.6 versus 81.6±0.9% in the vehicle-treated group; P<0.01). DFX at doses 50 and 10 mg/kg also reduced perihematomal brain edema (80.4±0.5%, P<0.01, and 80.7±0.9%, P<0.05, respectively). DFX treatment did not change brain water content in the sham-operated rats. MR scans showed that DFX reduced ICH-induced ventricle enlargement. 50 and 100 mg/kg DFX also reduced ICH-induced ventricle enlargement, caudate atrophy and ICH-induced neurological deficits in aged rats. However, while 10 mg/kg DFX reduced ventricle enlargement and forelimb placing deficits, it did not reduce caudate atrophy and corner turn deficits.
During the long-term experiments, body weight and mean arterial blood pressure were monitored. DFX treatment did not affect the body weight change among the ICH groups. Also, in sham rats, there were no differences between DFX- and vehicle-treated groups.
Conclusions: These results indicate that DFX can reduce ICH-induced brain injury in aged as well as young rats and that a dose higher than 10 mg/kg is the optimal dose of DFX in this model.
743. The protective effects of ishcemic postconditioning on potential inflammatory factors invovled in focal ischemia in rats
D. Wei, X. Gao, X. Chen and H. Zhao
Stanford University, Stanford, California, USA
Objectives: Ischemic postconditioning, which refers to a series of mechanical interruptions of reperfusion after ischemia, reduces infarction,
1
but the protective mechanisms remain largely unknown. In this study we investigated its protective effects on some potential inflammatory molecules, including galectin-9, Tim-3, COX-2, iNOS and nitrotyrosine after stroke. Tim-3, which is a member of the T cell immunoglobulin and mucin domain (Tim) family, is activated by its ligand, galectin-9. It plays a double role, either promoting or inhibiting inflammatory response, depending on the cell types it activates.
2
COX-2, iNOS and nitrotyrosine are well-known inflammatory molecules.
Methods: Focal ischemia was induced by permanent distal MCA occlusion combined with 30 mins of bilateral CCA occlusion in rats. For postconditioning,
1
after 30 secs of CCA reperfusion, the CCAs were occluded again by tightening sutures around the CCA for 10 secs, followed with another 2 cycles of 30 secs reperfusion and 10 secs occlusion (total of 3 cycles). Western blot and immunofluorescence staining were used to examine protein expression. To detect the effects of immune suppression on protein expression, minocycline hydrochloride was intraperitoneally (i.p) injected at 60 mins before and 8 h after the MCA occlusion.
Results: Ischemic postconditioning significantly reduced infarct size after focal ischemia. The results of Western blot indicate that Tim-3 expression was increased after stroke, which was inhibited by postconditioning (n = 6/group, P<0.001). The results of confocal microscopy indicate that galecin-9 was expressed on blood vessels 1 h after stroke, and on neurons from 5 h to 24 h. Such an expression was inhibited by postconditioning. Postconditioning also blocked iNOS and nitrotyrosine expression after stroke. However, postconditioning had no protective effects on COX-2 expression induced by stroke. At last, we found that minocycline injection attenuated increases in Tim-3 and iNOS, but promoted COX-2 expression.
Conclusions: the galectin-9/Tim-3 pathway is involved in brain injury after stroke. Ischemic postconditioning blocks increases in galectin-9, Tim-3, iNOS and nitrotyrosine, but promotes COX-2 expression. Minocycline, an inflammatory inhibitor, executed similar effects as postconditioning. Therefore, we conclude that ischemic postconditioning may prevent brain injury by regulating galectin-9/Tim-3 pathways, and inhibiting iNOS and nitrotyrosine activity.
488. Reduction of activation of microglia/macrophage confers neuroprotection following intracerebral hemorrhage
M. Xue and V.W. Yong
Clinical Neurosciences and Hotchkiss Brain Institute, University of Calgary, Calgary, AB, Canada
Objectives: Microglia, the resident immune cells of the CNS, become activated following CNS injuries including intracerebral hemorrhage (ICH). Correspondingly, monocytes from the circulation migrate into areas of CNS injury to become macrophages. These activated cells are often called ‘microglia/macrophage’ since it is difficult to differentiate between them in situ using immunohistochemistry. Activated microglia/microphages are capable of releasing numerous cytotoxic mediators such as matrix metalloproteinases, free radicals, chemokines and cytokines, but they can also produce neurotrophic factors such as NGF, FGF and BDNF. The degree of activation of microglia/macrophage is often correspondent with the extent of resulting pathology, and it is uncertain whether the microglia/macrophage reaction promotes or reduces the evolving pathology. We tested the hypothesis that the activation of microglia/macrophages promotes neuronal injury in the acute period after ICH and that inhibiting their activity attenuates brain injury and improves outcomes following ICH.
Methods: 10-μl of autologous blood obtained from tail was introduced into the right striatum of adult male mice (7 to 9 weeks old) to produce ICH injury. C57/B6 wildtype mice were used to evaluate the time course of brain injury from 1 to 7 day(s). We determined the area of brain damage and the extent of neuronal death and microglia/macrophage activity. We also used transgenic CD11b thymidine kinase (CD11b-TK) mice where proliferating microglia/macrophages were removed by ganciclovir treatment. These mice were killed at 3 days after ICH injury and brain sections were stained for various parameters.
Results: We found that ICH injury resulted in activation of microglia/macrophages through 1 to 7 day(s) of insult. The area of brain damage peaked at 2 to 3 days; the number of dying neurons peaked at 1 to 3 days, while the activation of microglia/macrophages peaked at 3 to 4 days. These findings led us to choose day 3 for further studies. We found that the activation of microglia/macrophages was significantly reduced in CD11b-TK transgenic mice with ganciclovir treatment after ICH compared to various injury control groups, including wildtype mice with ganciclovir treatment after ICH. Correspondingly, the area of brain damage and the extent of neuronal death were also reduced in CD11b-TK mice with diminished microglia/macrophage activity. Ongoing work with pharmacological deactivators of microglia will be presented at the meeting.
Conclusions: Activation of microglia/macrophages in the early periods after ICH promotes brain injury. These results shed light on the advent of new medications for ICH patients, including microglia deactivators.
171. Docosahexaenoic acid complexed to human albumin is neuroprotective after focal cerebral ischemia in rats: therapeutic window study
L. Belayev, L. Khoutorova, K.D. Atkins and N.G. Bazan
Neuroscience, Louisiana State University, New Orleans, Louisiana, USA
Introduction: We have previously shown that DHA complexed to human albumin (DHA-Alb) is markedly neuroprotective at relatively moderate-low doses compared with native human serum albumin (Alb) in the setting of focal cerebral ischemia in rat (Belayev et al, 2005). In this study, we defined the therapeutic window within which this therapy would confer neurobehavioral neuroprotection.
Methods: Physiologically controlled male Sprague-Dawley rats received 2 h middle cerebral artery occlusion (MCAo) by retrograde insertion of an intraluminal suture coated with poly-L-lysine (Belayev et al, 1996). The drug (DHA-Alb, 4 mg/kg or Alb, 25%, 0.5% of body weight), was administered i.v. at either 2, 3, 4, 5, 6 or 7 h after onset of stroke (n = 4 to 5 per group). Control group received saline at 1 h after onset of reperfusion. The neurological status was evaluated during occlusion (60 mins), and on day 1, 2, 3 and 7 after MCAo; a grading scale of 0 to 12 was employed, as previously described. Seven days after MCAo, brains were perfusion-fixed and infarct volume was measured.
Results: The physiological variables were entirely comparable among all groups. There were no significant differences with respect to rectal and cranial temperatures, arterial blood gases or arterial blood glucose. Alb and DHA-Alb treatment, started at 2, 3 and 4 h after onset of stroke significantly improved neurological score compared to saline, but there was no significant difference between DHA-Alb and Alb groups. Rats given DHA-Alb at 5 and 6 h after onset of stroke neurobehavioral improvement, however, exceeded that of native ALB at 72 h (5 h, DHA-Alb: 5.9±0.9 versus Alb: 7.3±0.6 and 6 h, 3.4±0.5 versus 6.2±1.1, respectively) and on day 7 (3 h, DHA-Alb: 4.2±0.9 versus 6.4±0.5 and 4 h, 2.8±0.5 versus 5.8±1.1, respectively, P<0.05). There was no significant neurological improvement when treatment was delayed until 7 h. Total (cortical and subcortical) corrected infarct volume was significantly reduced by DHA-Alb treatment compared to Alb treated rats, even when initiated as late as 5 and 6 h after onset of stroke (50.3±14 and 29.2±16 versus 118.4±23 mm3, respectively). Cortical infarct volumes were also significantly reduced by treatment with DHA-Alb in the same 5 and 6 h groups compared to Alb (22.7±9 and 15.1±4 versus 66.9±14.4 mm3, respectively). Subcortical infarct areas and volumes were not significantly different between DHA-Alb and Alb groups.
Conclusions: The DHA-Alb complex affords high-grade neurobehavioral neuroprotection in focal cerebral ischemia, equaling or exceeding that afforded by native ALB, at considerably moderate doses. Even DHA-Alb therapy was initiated as late as 5 and 6 h after onset of MCAo, it improved the behavioral score and reduced infarct volume. This is clearly clinically advantageous in that moderate DHA-Alb doses would be expected to reduce the likelihood of acute intravascular volume overload and congestive heart failure sometimes induced when patients with compromised cardiovascular function are treated with high-moderate doses human albumin therapy.
