Roger Strong

Reperfusion injury : Demonstration of brain damage produced by reperfusion after transient focal ischemia in rats

During reperfusion after ischemia, deleterious biochemical processes can be triggered that may antagonize the beneficial effects of reperfusion. Research into the understanding and treatment of reperfusion injury (RI) is an important objective in the new era of reperfusion therapy for stroke. To investigate RI, permanent and reversible unilateral middle cerebral artery/common carotid artery (MCA/CCA) occlusion (monitored by laser Doppler) of variable duration in Long-Evans (LE) and spontaneously hypertensive (SH) rats and unilateral MCA and bilateral CCA occlusion in selected LE rats was induced. In LE rats, infarct volume after 24 hours of permanent unilateral MCA/CCA occlusion was 31.1 ± 34.6 mm3 and was only 28% of the infarct volume after 120 to 300 minutes of reversible occlusion plus 24 hours of reperfusion, indicating that 72% of the damage of ischemia/reperfusion is produced by RI. When reversible ischemia was prolonged to 480 and 1080 minutes, infarct volume was 39.6 mm3 and 16.6 mm3, respectively, being indistinguishable from the damage produced by permanent ischemia and significantly smaller than damage after 120 to 300 minutes of ischemia. Reperfusion injury was not seen in SH rats or with bilateral CCA occlusion in LE rats, in which perfusion is reduced more profoundly. Reperfusion injury was ameliorated by the protein synthesis inhibitor cycloheximide or spin-trap agent N-tert-butyl-alpha-phenylnitrone pretreatment.

15d-Prostaglandin J2 Activates Peroxisome Proliferator-Activated Receptor-γ, Promotes Expression of Catalase, and Reduces Inflammation, Behavioral Dysfunction, and Neuronal Loss after Intracerebral Hemorrhage in Rats:

Peroxisome proliferator-activated receptor-γ (PPARγ) is a transcription factor that regulates the expression of various gene products that are essential in lipid and glucose metabolism, as well as that of the peroxisome-enriched antioxidant enzyme, catalase. Activation of PPARγ is linked to anti-inflammatory activities and is beneficial for cardiovascular diseases. However, little is known about its role in intracerebral hemorrhage (ICH). 15-Deoxy-Δ12,14-prostaglandin J2 (15d-PGJ2) acts as a physiologic agonist for PPARγ. In this study, we found that injection of 15d-PGJ2 into the locus of striatal hematoma increased PPARγ-deoxyribonucleic acid (DNA) binding activity and the expression of catalase messenger ribonucleic acid (mRNA) and protein in the perihemorrhagic area. Additionally, 15d-PGJ2 significantly reduced nuclear factor-κB (NF-κB) activation and prevented neutrophil infiltration measured by myeloperoxidase (MPO) immunoassay, and also reduced cell apoptosis measured by terminal deoxynucleotide transferase dUTP nick-end labeling (TUNEL). In addition, 15d-PGJ2 reduced behavioral dysfunction produced by the ICH. Altogether, our findings indicate that injection of 15d-PGJ2 at the onset of ICH is associated with activation of PPARγ and elevation of catalase expression, suppression of NF-κB activity, and restricted neutrophil infiltration. All these events predicted reduced behavioral deficit and neuronal damage.

Hematoma resolution as a target for intracerebral hemorrhage treatment: Role for peroxisome proliferator‐activated receptor γ in microglia/macrophages

Phagocytosis is necessary to eliminate the hematoma after intracerebral hemorrhage (ICH); however, release of proinflammatory mediators and free radicals during phagocyte activation is toxic to neighboring cells, leading to secondary brain injury. Promotion of phagocytosis in a timely and efficient manner may limit the toxic effects of persistent blood products on surrounding tissue and may be important for recovery after ICH.

Nuclear Factor-κB and Cell Death After Experimental Intracerebral Hemorrhage in Rats

Background and Purpose—Nuclear factor-κB (NF-κB) is a ubiquitous transcription factor that, when activated, translocates to the nucleus, binds to DNA, and promotes transcription of many target genes. Its activation has been demonstrated in chronic inflammatory conditions, cerebral ischemia, and apoptotic cell death. The present study evaluated the presence and activation of NF-κB in relation to cell death surrounding intracerebral hemorrhage (ICH). Methods—Striatal ICH was induced in rats by the double blood injection method. Animals were killed 2, 8, and 24 hours and 4 days after ICH. To examine changes in NF-κB protein, Western blot was performed on brain extract. We determined NF-κB activity using electrophoretic mobility shift assay (EMSA) and immunohistochemistry, using an antibody that only recognizes active NF-κB. DNA fragmentation was detected with terminal deoxynucleotidyl transferase–mediated uridine 5′-triphosphate-biotin nick end-labeling (TUNEL) staining. Results—Western blot analysis of the ...

Early Exclusive Use of the Affected Forelimb After Moderate Transient Focal Ischemia in Rats: Functional and Anatomic Outcome

BACKGROUND AND PURPOSE Previous work by researchers in our laboratory has shown that in the rat, the exclusive use of the affected forelimb during an early critical period exaggerates lesion volume and retards functional recovery after electrolytic lesions of the forelimb sensorimotor cortex. In the present study, we examined the effects of exclusive use of the affected forelimb after middle cerebral artery occlusion (MCAO). METHODS Ischemia of moderate severity was produced in male Long-Evans rats through 45 minutes of occlusion of the left middle cerebral and both common carotid arteries. Exclusive use of either the affected or unaffected forelimb was forced through immobilization of either the ipsilateral (MCAO+ipsi) or contralateral (MCAO+contra) forelimb, respectively, for 10 days in a plaster cast, or the animal was left uncasted (MCAO+nocast). Sham surgeries were performed, and animals were also casted for 10 days or left uncasted. Sensorimotor testing was performed during days 17 to 38. At the end of sensorimotor testing, cognitive performance was tested with use of the Morris water maze. In a separate experiment, temperatures and corticosterone levels were measured during the 10-day period after 45-minute ischemia and casting. RESULTS The MCAO+ipsi group performed worse on sensorimotor tasks than the MCAO+contra, MCAO+nocast, and sham groups. Infarct volume was significantly larger in the MCAO+ipsi group than in the sham and MCAO+contra groups but not in the MCAO+nocast group. No group differences were found with the Morris water maze, and no group differences were found in either temperature or plasma corticosterone level. CONCLUSIONS The exclusive use of the affected forelimb immediately after focal ischemia has detrimental effects on sensorimotor function that cannot be attributed to hyperthermia or stress.

Autologous bone marrow mononuclear cells enhance recovery after acute ischemic stroke in young and middle-aged rats

We investigated intra-arterially administered autologous bone marrow mononuclear cells (MNCs) in rats with acute ischemic stroke. Long Evans rats (2 to 3 months or 12 months old) underwent tandem reversible common carotid artery (CCA)/middle cerebral artery (MCA) occlusion (CCAo/MCAo) for 3 h and then 24 h later underwent tibial bone marrow harvest. Ten million or 4 million cells were re-injected by an intra-carotid infusion. Control animals underwent marrow needle insertion and then saline injection into the carotid artery. Animals were assessed on a battery of neurological tests. MNCs in the ischemic brain were tracked using Q-dot nanocrystal labeling. Infarct volume and cytokines in the ischemia-affected brain were analyzed. Cell-treated animals in the younger and older groups showed improvement from 7 to 30 days after stroke compared with vehicle-treated animals. MNCs significantly reduced infarct volume compared with saline. There was a significant reduction in tumor necrosis factor-α, interleukin-1α (IL-1α), IL-β, IL-6, and a significant increase in IL-10 in injured brains harvested from the cell-treated groups compared with saline controls. Labeled MNCs were found in the peri-infarcted area at 1 h and exponentially decreased over the ensuing week after injection. Autologous bone marrow MNCs can be safely harvested from rodents after stroke, migrate to the peri-infarct area, enhance recovery, and modulate the post-ischemic inflammatory response.

Transcription Factor Nrf2 Protects the Brain From Damage Produced by Intracerebral Hemorrhage

Background and Purpose— Intracerebral hemorrhage (ICH) remains a major medical problem for which there is no effective treatment. Oxidative and cytotoxic damage plays an important role in ICH pathogenesis and may represent a target for treatment of ICH. Recent studies have suggested that nuclear factor–erythroid 2–related factor 2 (Nrf2), a pleiotropic transcription factor, may play a key role in protecting cells from cytotoxic/oxidative damage. This study evaluated the role of Nrf2 in protecting the brain from ICH-mediated damage. Methods— Sprague-Dawley rats and Nrf2-deficient or control mice received intracerebral injection of autologous blood to mimic ICH. Sulforaphane was used to activate Nrf2. Oxidative stress, the presence of myeloperoxidase-positive cells (neutrophils) in ICH-affected brains, and behavioral dysfunction were assessed to determine the extent of ICH-mediated damage. Results— Sulforaphane activated Nrf2 in ICH-affected brain tissue and reduced neutrophil count, oxidative damage, and behavioral deficits caused by ICH. Nrf2-deficient mice demonstrated more severe neurologic deficits after ICH and did not benefit from the protective effect of sulforaphane. Conclusions— Nrf2 may represent a strategic target for ICH therapies.

Cell death in experimental intracerebral hemorrhage: The “black hole” model of hemorrhagic damage

Intracerebral hemorrhage (ICH) has a poor prognosis that may be the consequence of the hematomas effect on adjacent and remote brain regions. Little is known about the mechanism, location, and severity of such effects. In this study, rats subjected to intracerebral blood injection were examined at 100 days. Stereology (neuronal count and density) and volume measures in the perihematoma rim, the adjacent and overlying brain, and the substantia nigra pars reticulata (SNr) were compared with contralateral brain regions at 100 days and the perihemorrhage region at 24 hours and 7 days. In addition, cytochrome c release was investigated at 24 hours, 3 days, and 7 days. At 100 days, post‐ICH rats showed no difference in neuronal density in the perihemorrhagic scar region or regions of the striatum immediately surrounding and distal to the perihemorrhage scar. The cell density index in the ipsilateral field was 16.2 ± 3.8 versus the contralateral control field of 15.6 ± 3.2 (not significant). Volume measurements of the ipsilateral striatum revealed a 20% decrease that was compensated by an increase in ipsilateral ventricular size. The area of the initial ICH as measured by magnetic resonance imaging correlated with the degree of atrophy. In the region immediately surrounding the hematoma, cytochrome c immunoreactivity increased at 24 hours and 3 days, and returned toward baseline by day 7. At 24 hours, stereology in the peri‐ICH region showed decreased density in the region where cytochrome c immunoreactivity was the highest. Neuronal density of the ipsilateral SNr was significantly less than the contralateral side (9.6 ± 1.9 vs 11.6 ± 2.3). Histologic damage from ICH occurred mainly in the immediate perihemorrhage region. Except for SNr, we found no evidence of neuronal loss in distal regions. We have termed this continued destruction of neurons, which occurs over at least 3 days as the neurons come into proximity to the hematoma, the “black hole” model of hemorrhagic damage.

Neuronal PPARγ Deficiency Increases Susceptibility to Brain Damage after Cerebral Ischemia

Peroxisome proliferator-activated receptor gamma (PPARγ) plays a role in regulating a myriad of biological processes in virtually all brain cell types, including neurons. We and others have reported recently that drugs which activate PPARγ are effective in reducing damage to brain in distinct models of brain disease, including ischemia. However, the cell type responsible for PPARγ-mediated protection has not been established. In response to ischemia, PPARγ gene is robustly upregulated in neurons, suggesting that neuronal PPARγ may be a primary target for PPARγ-agonist-mediated neuroprotection. To understand the contribution of neuronal PPARγ to ischemic injury, we generated conditional neuron-specific PPARγ knock-out mice (N-PPARγ-KO). These mice are viable and appeared to be normal with respect to their gross behavior and brain anatomy. However, neuronal PPARγ deficiency caused these mice to experience significantly more brain damage and oxidative stress in response to middle cerebral artery occlusion. The primary cortical neurons harvested from N-PPARγ-KO mice, but not astroglia, exposed to ischemia in vitro demonstrated more damage and a reduced expression of numerous key gene products that could explain increased vulnerability, including SOD1 (superoxide dismutase 1), catalase, glutathione S-transferase, uncoupling protein-1, or transcription factor liver X receptor-α. Also, PPARγ agonist-based neuroprotective effect was lost in neurons from N-PPARγ neurons. Therefore, we conclude that PPARγ in neurons play an essential protective function and that PPARγ agonists may have utility in neuronal self-defense, in addition to their well established anti-inflammatory effect.

Ethanol Plus Caffeine (Caffeinol) for Treatment of Ischemic Stroke. Preclinical Experience

Background and Purpose— Ethanol and caffeine are 2 common psychoactive dietary components. We have recently shown that low-dose ethanol plus caffeine results in a 70% to 80% reduction of infarct volume after reversible common carotid/middle cerebral artery (CCA/MCA) occlusion in rats. The combination (caffeinol) was effective after either oral pretreatment or intravenous administration starting up to 2 hours after stroke onset. Ethanol alone aggravated ischemic damage, while caffeine alone was without effect. Daily caffeinol for 2 weeks before ischemia eliminated the neuroprotection seen with acute treatment (tolerance). The purpose of our present study was to further characterize the properties of caffeinol as a possible treatment for ischemic stroke. Methods— The transient CCA/MCA occlusion model was used in all experiments. Five sets of experiments were conducted (1) to test the effectiveness of various doses of ethanol (0.2 to 0.65 g/kg) and caffeine (3 to 10 mg/kg) in the caffeinol mixture; (2) to test whether the neuroprotective dose of caffeinol can improve behavioral dysfunction; (3) to test whether chronic ethanol or caffeine before ischemia will affect efficacy of caffeinol treatment; (4) to test whether the protective effect of caffeinol can be improved by pairing it with 35°C hypothermia; and (5) to test whether caffeinol affects frequency of hemorrhage after administration of recombinant tissue plasminogen activator (rtPA) in ischemic animals. Results— Doses as low as 0.2 g/kg of ethanol and 6 mg/kg of caffeine in the caffeinol were effective in reducing cortical infarct volume and behavioral dysfunction after transient CCA/MCA occlusion. Daily exposure to ethanol but not caffeine before CCA/MCA occlusion eliminated the therapeutic efficacy of acute caffeinol treatment, similar to the tolerance observed after chronic exposure to caffeinol. The therapeutic effect of caffeinol could be further improved by pairing it with mild intraischemic hypothermia, and caffeinol did not increase hemorrhagic infarction when given in combination with rtPA. Conclusions— Low doses of caffeinol, equivalent to no more than 2 to 3 cups of strong coffee and 1 cocktail, are consistently and highly neuroprotective, are well tolerated, can be added to other therapies to increase the effect of each, and do not interfere with or complicate rtPA therapy. Caffeinol is an appropriate candidate for clinical trial in stroke patients, although it may be less effective in patients with regular alcohol intake.