Jaroslaw Aronowski

The hematopoietic factor G-CSF is a neuronal ligand that counteracts programmed cell death and drives neurogenesis

G-CSF is a potent hematopoietic factor that enhances survival and drives differentiation of myeloid lineage cells, resulting in the generation of neutrophilic granulocytes. Here, we show that G-CSF passes the intact blood-brain barrier and reduces infarct volume in 2 different rat models of acute stroke. G-CSF displays strong anti-apoptotic activity in mature neurons and activates multiple cell survival pathways. Both G-CSF and its receptor are widely expressed by neurons in the CNS, and their expression is induced by ischemia, which suggests an autocrine protective signaling mechanism. Surprisingly, the G-CSF receptor was also expressed by adult neural stem cells, and G-CSF induced neuronal differentiation in vitro. G-CSF markedly improved long-term behavioral outcome after cortical ischemia, while stimulating neural progenitor response in vivo, providing a link to functional recovery. Thus, G-CSF is an endogenous ligand in the CNS that has a dual activity beneficial both in counteracting acute neuronal degeneration and contributing to long-term plasticity after cerebral ischemia. We therefore propose G-CSF as a potential new drug for stroke and neurodegenerative diseases.

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.

New horizons for primary intracerebral hemorrhage treatment: experience from preclinical studies

Abstract Intracerebral hemorrhage (ICH) remains a major medical problem, for which there is no effective treatment. However, extensive experimental and clinical research carried out in recent years has brought to light new exciting ideas for novel potential treatments. First, it was well documented that the management of hypertension helps to prevent new and recurrent ICH. Also, development of new guidelines for management of hypertension after the onset of the ICH may help in more effective ICH treatment. Existing contemporary data collected from preclinical studies indicates that ICH–induced inflammation represents a key factor leading to secondary brain damage, suggesting that some anti–inflammatory approaches can be used to treat hemorrhagic stroke. In this article, beyond discussing implications related to hypertension, we will summarize important (but not all) new discoveries connecting the role of inflammation to ICH pathology. Selected aspects of inflammatory response including the role of cytokines, transcription factor nuclear factor–kB, microglia activation, astrogliosis, and complement activation will be introduced. We will also discuss the role for reactive oxygen species and metalloproteinases in ICH pathogenesis and introduce basic knowledge on the nature of ICH–induced cell death including apoptosis. Potential targets for intervention and translation will be discussed.

Sulforaphane reduces infarct volume following focal cerebral ischemia in rodents

Stroke is the third leading cause of death and disability in the United States. As several biochemical mechanisms have been proposed to contribute to stroke pathophysiology, treatments acting on multiple targets may be desirable. Sulforaphane (SUL), a naturally occurring isothiocyanate present in cruciferous vegetables, has been shown to induce the expression of multiple NF-E2-related factor-2 (Nrf2) responsive genes. In the present study, we demonstrate that systemically administered SUL can enter the brain as determined by increased mRNA and protein levels of the Nrf2-responsive gene heme oxygenase 1 (HO-1). Delayed administration (15 min) of a single dose of SUL significantly decreased cerebral infarct volume following focal ischemia, suggesting a potential therapeutic value for this compound.

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 PURPOSEnPrevious 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).nnnMETHODSnIschemia 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.nnnRESULTSnThe 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.nnnCONCLUSIONSnThe exclusive use of the affected forelimb immediately after focal ischemia has detrimental effects on sensorimotor function that cannot be attributed to hyperthermia or stress.

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.

Altered PPARγ expression and activation after transient focal ischemia in rats

Stroke is a devastating disease with limited treatment options. Recently, we found that the peroxisome proliferator‐activated receptor‐γ (PPARγ) agonists troglitazone and pioglitazone reduce injury and inflammation in a rat model of transient cerebral ischemia. The mechanism of this protection is unclear, as these agents can act through PPAR‐γ activation or through PPAR‐γ‐independent mechanisms. Therefore, we examined PPAR‐γ expression, DNA binding and transcriptional activity following stroke. In addition, we used a PPAR‐γ antagonist, T0070907, to determine the role of PPAR‐γ during ischemia. Using immunohistochemical techniques and real‐time PCR, we found low levels of PPAR‐γ mRNA and PPAR‐γ immunoreactivity in nonischemic brain; however, PPAR‐γ expression dramatically increased in ischemic neurons, peaking 24u2003h following middle cerebral artery occlusion. Interestingly, we found that in both vehicle‐ and agonist‐treated brains, DNA binding was reduced in the ischemic hemisphere relative to the contralateral hemisphere. Expression of a PPAR‐γ target gene, lipoprotein lipase, was also reduced in ischemic relative to nonischemic brain. Both DNA binding and lipoprotein lipase expression were increased by the addition of the PPAR‐γ agonist rosiglitazone. Finally, we found that rosiglitazone‐mediated protection after stroke was reversed by the PPAR‐γ antagonist T0070907. Interestingly, infarction size was also increased by T0070907 in the absence of PPAR‐γ agonist, suggesting that endogenous PPAR‐γ ligands may mitigate the effects of cerebral ischemia.