Claire L. Gibson

G-CSF reduces infarct volume and improves functional outcome after transient focal cerebral ischemia in mice

Growth factors possess neuroprotective and neurotrophic properties in vitro, but few have been extensively studied in vivo after stroke. In the present study, we investigated the potential functional benefits of granulocyte colony-stimulating factor (G-CSF) administration after focal cerebral ischemia. Male mice underwent 60-minute middle cerebral artery occlusion (MCAO) and received G-CSF (50 μg/kg, subcutaneously) or vehicle (saline) at the onset of reperfusion. Granulocyte colony-stimulating factor-treated mice killed at 48 hours after MCAO revealed a >45% reduction (P<0.05) in lesion volume. In terms of body weight recovery, and in tests of motor (grid test and rotarod) and cognitive ability (water maze), MCAO significantly worsened the outcome in vehicle-treated mice as compared with shams (P<0.05). However, G-CSF treatment was beneficial as, compared with vehicle, this significantly improved weight recovery and motor ability. This effect was most apparent on the water maze where G-CSF-treated mice were indistinguishable from shams in terms of acquiring the task. These results indicate long-term beneficial effects of a single dose of G-CSF administered on reperfusion, and illustrate the need to further investigate the mechanisms of G-CSF action.

Progesterone suppresses the inflammatory response and nitric oxide synthase-2 expression following cerebral ischemia.

Gender differences in outcome following cerebral ischemia have frequently been observed and attributed to the actions of steroid hormones. Progesterone has been shown to possess neuroprotective properties following transient ischemia, with respect to decreasing lesion volume and improving functional recovery. The present study was designed to determine the mechanisms of progesterone neuroprotection, and whether these relate to the inflammatory response. Male mice underwent either 60 min or permanent middle cerebral artery occlusion (MCAO) and received progesterone (8 mg/kg ip) or vehicle 1 h, 6 h and 24 h post-MCAO. Forty-eight hours following transient MCAO, structural magnetic resonance imaging revealed a significant decrease in the amount of edematous tissue present in progesterone-treated mice as compared with vehicle. Using real-time PCR we found that progesterone treatment significantly suppressed the injury-induced upregulation of interleukin (IL)-1beta, transforming growth factor (TGF)beta2, and nitric oxide synthase (NOS)-2 mRNAs in the ipsilateral hemisphere while having no effect on tumor necrosis factor (TNF)-alpha mRNA expression. Progesterone treatment following permanent MCAO also resulted in a significant decrease in lesion volume. This was not apparent in mice lacking a functional NOS-2 gene. Thus, progesterone is neuroprotective in both permanent and transient ischemia, and this effect is related to the suppression of specific aspects of the inflammatory response.

Estrogens and experimental ischemic stroke: a systematic review

Estrogens are believed to provide females with endogenous protection against cerebrovascular events although clinical trials studying long-term hormone replacement have yielded disappointing results. In contrast, estrogens might be neuroprotective after experimental ischemia. We performed a systematic review of controlled experimental studies that administered estrogens before, or after, cerebral ischemia and measured lesion volume. Relevant studies were found from searching PubMed, Embase and Web of Science. From 161 identified publications, 27 studies using 1304 experimental subjects were analyzed using the Cochrane Review Manager software. Estrogens reduced lesion volume in a dose-dependent manner, after either transient (P < 0.001) or permanent (P < 0.001) ischemia and whether administered before or up to 4 h after ischemia onset; no studies assessed efficacy for later time periods. The effect size for estrogens decreased with increasing quality scores for studies of transient ischemia. Estrogens reduced lesion volume when administered to ovariectomized females and young adult males, but had no effect in intact females. Limited data were present for aged animals and the full dose-response relationship was not available in all experimental groups. On the basis of these data, estrogens are a candidate treatment for ischemic stroke, although further preclinical studies are also warranted.

G-CSF suppresses edema formation and reduces interleukin-1β expression after cerebral ischemia in mice

Granulocyte-colony stimulating factor (G-CSF) is reported to be neuroprotective after transient cerebral ischemia with respect to decreasing lesion volume and enhancing functional recovery. We investigated whether G-CSF is neuroprotective after permanent ischemia and the possible mechanisms underlying this neuroprotection. Mice underwent permanent or 60-minute middle cerebral artery occlusion (MCAO) and received G-CSF (50 μg/kg) or vehicle at the onset or 1 hour post-MCAO. Forty-eight hours after transient MCAO, structural magnetic resonance imaging revealed a significant reduction (50%) in the amount of edematous tissue present in G-CSF-treated mice (p < 0.05). G-CSF treatment also prevented a significant increase in ipsilateral brain water content that was present in vehicle-treated mice after transient (p < 0.05) and permanent (p < 0.001) MCAO. Forty-eight hours after permanent MCAO, G-CSF decreased (50%) the cortical lesion volume (p < 0.05). Using real-time polymerase chain reaction, we found that G-CSF treatment significantly suppressed (p < 0.05) the injury-induced upregulation of IL-1β mRNA while having no effect on TNFα and NOS-2 mRNA expression. This suggests that part of the neuroprotection may be attributed to the ability of G-CSF to reduce the inflammatory response.

Glial nitric oxide and ischemia

One of the responses to cerebral ischemia is an increase in the production of nitric oxide, catalyzed by enzymes expressed in both resident and infiltrating cells. The nitric oxide that is generated does contribute to the ensuing pathology, but it can also be beneficial. The effects of nitric oxide depend on the cell site of production, the amount generated, and the chemical nature of the products of further oxidation. Understanding how nitric oxide production from microglia and astrocytes contributes to ischemic pathology is important for the development and application of future therapeutics based on inhibiting or amplifying its production in the injured brain.

Cerebral Ischemic Stroke: is Gender Important?

Cerebral stroke continues to be a major cause of death and the leading cause of long-term disability in developed countries. Evidence reviewed here suggests that gender influences various aspects of the clinical spectrum of ischemic stroke, in terms of influencing how a patients present with ischemic stroke through to how they respond to treatment. In addition, this review focuses on discussing the various pathologic mechanisms of ischemic stroke that may differ according to gender and compares how intrinsic and hormonal mechanisms may account for such gender differences. All clinical trials to date investigating putative neuroprotective treatments for ischemic stroke have failed, and it may be that our understanding of the injury cascade initiated after ischemic injury is incomplete. Revealing aspects of the pathophysiological consequences of ischemic stroke that are gender specific may enable gender relevant and effective neuroprotective strategies to be identified. Thus, it is possible to conclude that gender does, in fact, have an important role in ischemic stroke and must be factored into experimental and clinical investigations of ischemic stroke.

Progesterone is neuroprotective following cerebral ischaemia in reproductively ageing female mice.

Gender differences in both vulnerability to stroke and outcome following cerebral ischaemia have frequently been observed and attributed to the action of steroid hormones. Progesterone is a candidate neuroprotective factor for stroke; however, studies are lacking which: (i) study those groups representing high risk i.e. postmenopausal females; (ii) administer progesterone solely post-ischaemia; and (iii) combine histopathological and functional assessments. Postmenopausal females, along with males, represent the group at highest risk of cerebral stroke and can be modelled using aged or ovariectomized animals. In the current study, we aimed to determine the neuroprotective effects of progesterone administration following cerebral ischaemia in aged and ovariectomized mice. Following transient middle cerebral artery occlusion, progesterone was administered at 1, 6 and 24 h post-ischaemia to aged and ovariectomized female mice. At 48 h post-ischaemia, progesterone significantly reduced the lesion volume (P < 0.05) but had no effect on neurological outcome in aged female mice. Whereas in ovariectomized mice, at 48 h post-ischaemia, progesterone treatment had no effect on the amount of lesion volume present but did significantly improve neurological outcome. In a further study of ovariectomized mice, allowed to survive for 7 days post-ischaemia, progesterone treatment significantly improved motor outcome as assessed using both the rotarod and grid test. In fact, by 7 days post-ischaemia, progesterone-treated ovariectomized mice did not differ significantly in performance compared with shams, whereas vehicle-treated ovariectomized mice displayed a significant functional impairment following ischaemia. The current study has demonstrated that progesterone has different neuroprotective effects whether it is administered to aged or ovariectomized female mice and emphasizes the need to combine histopathological and functional outcomes within the same study. In addition, as progesterone-only treatment may not improve all outcomes in all groups, therapies that combine progesterone with other neuroprotective candidates should be investigated to maximize benefit following stroke.

Granulocyte-colony stimulating factor in experimental stroke and its effects on infarct size and functional outcome: A systematic review.

BACKGROUND Granulocyte-colony stimulating factor (G-CSF) shows promise as a treatment for stroke. This systematic review assesses G-CSF in experimental ischaemic stroke. METHODS Relevant studies were identified with searches of Medline, Embase and PubMed. Data were extracted on stroke lesion size, neurological outcome and quality, and analysed using Cochrane Review Manager using random effects models; results are expressed as standardised mean difference (SMD) and odds ratio (OR). RESULTS Data were included from 19 publications incorporating 666 animals. G-CSF reduced lesion size significantly in transient (SMD -1.63, p<0.00001) but not permanent (SMD -1.56, p=0.11) focal models of ischaemia. Lesion size was reduced at all doses and with treatment commenced within 4 h of transient ischaemia. Neurological deficit (SMD -1.37, p=0.0004) and limb placement (SMD -1.88, p=0.003) improved with G-CSF; however, locomotor activity (> or =4 weeks post-ischaemia) was not (SMD 0.76, p=0.35). Death (OR 0.27, p<0.0001) was reduced with G-CSF. Median study quality was 4 (range 0-7/8); Eggers test suggested significant publication bias (p<0.001). CONCLUSIONS G-CSF significantly reduced lesion size in transient but not permanent models of ischaemic stroke. Motor impairment and death were also reduced. Further studies assessing dose response, administration time, length of ischaemia and long-term functional recovery are needed.

Nitric oxide, ischaemia and brain inflammation

Cerebral ischaemia results in the activation of three isoforms of NOS (nitric oxide synthase) that contribute to the development of and recovery from stroke pathology. This review discusses, in particular, the role of the transcriptionally activated NOS-2 (inducible NOS) isoform and summarizes the outcomes of experimental stroke studies with regard to the therapeutic utility of nitric oxide donors and NOS inhibitors.

Modulatory effects of progesterone on inducible nitric oxide synthase expression in vivo and in vitro

Nitric oxide (NO) is produced in the CNS following injury‐induced expression of inducible nitric oxide synthase (iNOS), yet its role as protective or damaging is unclear. Previous studies investigating the therapeutic potential of female sex steroids in stroke and trauma suggest that NO from this source is harmful, since oestradiol and progesterone decreased the level of iNOS expression in vitro and improved neurological outcome. We investigated the effects of progesterone on stroke‐induced expression of iNOS in mice, as well as cytokine‐induced expression of iNOS and its transcriptional activators in cells relevant to injury. We observed a significant reduction in stroke‐induced iNOS transcript in progesterone‐treated mice and in cultured macrophages. In contrast, progesterone significantly amplifed cytokine‐induced iNOS mRNA in cultured primary astrocytes, although the expression of protein was decreased. We sequenced upstream of the 1.5 kb reported iNOS promoter region and identified a potential progesterone response element (PRE). Astrocytes transiently transfected with iNOS promoter/CAT reporter gene constructs containing the PRE displayed a significant increase in induction of CAT expression after progesterone treatment, and this was diminished in cells transfected with a construct containing a disrupted PRE. These observations suggest the involvement of iNOS in the neuroprotective effects of progesterone.