Taizen Nakase

Increased apoptosis and inflammation after focal brain ischemia in mice lacking connexin43 in astrocytes

Astrocytes secrete cytokines and neurotrophic factors to neurons, consistent with a neurosupportive role for astrocytes. However, in ischemic or metabolic insults, the function of astrocytic gap junctions composed mainly from connexin43 (Cx43) remains controversial. We have previously shown that heterozygous Cx43 null mice subjected to middle cerebral artery occlusion exhibited significantly enhanced stroke volume and apoptosis compared to wild-type mice. In this study, we used mice in which the human GFAP promoter-driven cre transgene deletes the floxed Cx43 gene in astrocytes, excluding the effects from reduced Cx43 expression in many other cell types as well as astrocytes. We induced focal brain ischemia in mice lacking Cx43 in astrocytes [Cre(+)] and control littermates [Cre(-)]. Cre(+) mice showed a significantly increased stroke volume and enhanced apoptosis, detected by terminal dUTP nick-end labeling and caspase-3 immunostaining, compared to Cre(-) mice. Inflammatory response assessed by the microglial marker CD11b was amplified in the penumbra of Cre(+) mice compared to that of Cre(-) mice. Our results suggest that astrocytic gap junctions could be important for the regulation of neuronal apoptosis and the inflammatory response after stroke. These findings support the view that astrocytes play a critical role in neuroprotection during ischemic insults.

Astrocytic Gap Junctions Composed of Connexin 43 Reduce Apoptotic Neuronal Damage in Cerebral Ischemia

Background and Purpose— Astrocytes may play a vital role in neuroprotection by providing energy substrates to neurons and regulating the concentration of K+ and neurotransmitters through gap junctions. Connexin 43 (Cx43) is one of the major gap junction proteins in astrocytes. We have shown that, after focal stroke, heterozygote Cx43 null (Cx43+/−) mice exhibited larger infarction volumes than wild-type (Cx43+/+) mice. We explored the underlying mechanism by which gap junctional intercellular communication influences astrocytic activation and neuroprotection in ischemia. Methods— Both Cx43+/− and Cx43+/+ mice underwent right side permanent middle cerebral artery occlusion (MCAO). Mice were prepared by transcardial perfusion, and at 24 hours and 4 days after surgery, brains were prepared for immunohistochemistry or Western blot analysis. Results— Four days after MCAO, Cx43+/− mice showed severe apoptosis in the penumbral lesion compared with Cx43+/+ mice. The level of caspase-3 was significantly higher in the stroke lesion of Cx43+/− mice than in Cx43+/+ mice. Four days after MCAO, Cx43+/− mice showed a significantly larger infarct volume but a smaller area of astrogliosis than did Cx43+/+ mice. The penumbra of Cx43+/− mice showed an increased level of Cx30 compared with Cx43+/+ mice. Conclusions— Gap junctions may play an important role in astrocytic activation. Reactive astrocytes may reduce neuronal apoptosis under ischemia by regulating extracellular conditions through their gap junction.

Enhanced Connexin 43 immunoreactivity in penumbral areas in the human brain following ischemia

Astrocytes support neurons not only physically but also chemically by secreting neurotrophic factors and energy substrates. Moreover, astrocytes establish a glial network and communicate through gap junctions in the brain. Connexin 43 (Cx43) is one of major component proteins in astrocytic gap junctions. Heterozygote Cx43 KO mice and astrocyte specific Cx43 KO mice exhibited amplified brain damage after ischemic insults, suggesting a neuroprotective role for astrocytic gap junctions. However, some reports mentioned unfavorable effects of gap junctions in neuronal support. Therefore, the role of astrocytic gap junctions under ischemic condition remains controversial. Since these studies have been performed using animal models, we investigated the Cx43 expression in human brain after stroke. Brain slice sections were prepared from pathological samples in our hospital. Embolic stroke brains sectioned because of the stroke were considered as acute ischemic models. Multiple infarction brains sectioned because of pneumonia or cancer were considered as chronic models. We observed the levels of Cx43 in both lesioned and intact areas, and compared them with acute and chronic models. As the results, astrocytes were strongly activated in penumbral lesions both of acute and chronic ischemic models. The Cx43 immunoreactivity was significantly amplified in the penumbra of chronic model compared to that of the acute model. Neurons were well preserved in chronic model compared to acute model. These findings suggested that the brain may generate neuronal protection by increasing the levels of Cx43 and amplifying the astrocytic gap junctional intercellular communication under hypoxic condition.

Neuroprotective Role of Astrocytic Gap Junctions in Ischemic Stroke

The role of astrocytic gap junctions in ischemia remains controversial. Several studies support that astrocytic gap junctions play a role in the spread of hypoxic injury, while other reports have demonstrated that blocking astrocytic gap junctions increases neuronal death. Using a stroke model on animals in which the astrocytic gap junction protein connexin43 (Cx43) was compromised, we explored the neuroprotective role of astrocytic gap junctions. A focal brain stroke was performed on heterozygous Cx43 null [Cx43(+/−)] mice, wild type [Cx43(+/+)] mice, astrocyte-directed Cx43 deficient [Cx43fl/ fl/hGFAP-cre] mice (here designated as Cre(+) mice), and their corresponding controls [Cx43fl/fl] (here designated as Cre(−) mice). Four days following stroke, ischemic lesions were measured for size and analyzed immunohistochemically. Stroke volume was significantly larger in Cx43(+/−) and Cre(+) mice compared to Cx43(+/+) and Cre(−) mice, respectively. Apoptosis as detected by TUNEL labeling and caspase-3 immunostaining was amplified in Cx43(+/−) and Cre(+) mice compared to their control groups. Furthermore, increased inflammation as characterized by the immunohistochemical staining of the microglial marker CD11b was observed in the Cre(+) mice penumbra. Astrocytic gap junctions may reduce apoptosis and inflammation in the penumbra following ischemic insult, suggesting that coupled astrocytes fulfill a neuroprotective role under ischemic stroke conditions.

The impact of inflammation on the pathogenesis and prognosis of ischemic stroke

The inflammation plays a critical role in the stroke onset and even in the worsening of the lesions. Therefore, the investigation of inflammatory response in the acute stage may contribute to improve the treatment of ischemic stroke. High-sensitive CRP (hsCRP), IL-6 and TNFalpha were measured as inflammatory markers on admission and in the 28th day after the onset. Oxidized LDL was measured simultaneously, since it can be a marker of reactive oxygen species which reflect the activity of inflammation. Ischemic stroke patients within 24 h after the onset (n=105) were included in this study. All patients were classified into cardioembolism, large-artery atherosclerosis, lacunar infarction, branch atheromatous disease and arterial dissection groups based on the findings of MRI and MRA and clinical records. Oxidized LDL was significantly increased in the acute phase of all cases. The amplified level of IL-6 was related to the worse outcome. The increase of TNFalpha in lacunar infarction was statistically correlated to the neurological severity on admission. In conclusion, IL-6 may predict not only the severity of the stroke lesions but also the outcome of patients. TNFalpha may suggest the small arterial lesions.

Free radical scavenger, edaravone, reduces the lesion size of lacunar infarction in human brain ischemic stroke

BackgroundAlthough free radicals have been reported to play a role in the expansion of ischemic brain lesions, the effect of free radical scavengers is still under debate. In this study, the temporal profile of ischemic stroke lesion sizes was assessed for more than one year to evaluate the effect of edaravone which might reduce ischemic damage.MethodsWe sequentially enrolled acute ischemic stroke patients, who admitted between April 2003 and March 2004, into the edaravone(-) group (n = 83) and, who admitted between April 2004 and March 2005, into the edaravone(+) group (n = 93). Because, edaravone has been used as the standard treatment after April 2004 in our hospital. To assess the temporal profile of the stroke lesion size, the ratio of the area [T2-weighted magnetic resonance images (T2WI)/iffusion-weighted magnetic resonance images (DWI)] were calculated. Observations on T2WI were continued beyond one year, and observational times were classified into subacute (1-2 months after the onset), early chronic (3-6 month), late chronic (7-12 months) and old (≥13 months) stages. Neurological deficits were assessed by the National Institutes of Health Stroke Scale upon admission and at discharge and by the modified Rankin Scale at 1 year following stroke onset.ResultsStroke lesion size was significantly attenuated in the edaravone(+) group compared with the edaravone(-) group in the period of early and late chronic observational stages. However, this reduction in lesion size was significant within a year and only for the small-vessel occlusion stroke patients treated with edaravone. Moreover, patients with small-vessel occlusion strokes that were treated with edaravone showed significant neurological improvement during their hospital stay, although there were no significant differences in outcome one year after the stroke.ConclusionEdaravone treatment reduced the volume of the infarct and improved neurological deficits during the subacute period, especially in the small-vessel occlusion strokes.

Musical anhedonia: Selective loss of emotional experience in listening to music

Recent case studies have suggested that emotion perception and emotional experience of music have independent cognitive processing. We report a patient who showed selective impairment of emotional experience only in listening to music, that is musical anhednia. A 71-year-old right-handed man developed an infarction in the right parietal lobe. He found himself unable to experience emotion in listening to music, even to which he had listened pleasantly before the illness. In neuropsychological assessments, his intellectual, memory, and constructional abilities were normal. Speech audiometry and recognition of environmental sounds were within normal limits. Neuromusicological assessments revealed no abnormality in the perception of elementary components of music, expression and emotion perception of music. Brain MRI identified the infarct lesion in the right inferior parietal lobule. These findings suggest that emotional experience of music could be selectively impaired without any disturbance of other musical, neuropsychological abilities. The right parietal lobe might participate in emotional experience in listening to music.

Amplified expression of uncoupling proteins in human brain ischemic lesions

Uncoupling proteins (UCPs) are reported to regulate mitochondrial respiration and energy metabolism during hibernation. Recently, it has been reported that UCP2 and UCP5 might reduce free radical stress in the ischemic condition in in vitro models, suggesting both as potential neuroprotective agents. We therefore investigated the levels of UCP2 and UCP5 expression in the lesion of human brain infarction. Brain slice sections were prepared from pathological samples collected at our hospital. Embolic stroke brains sectioned because of the stroke (n = 5) and multiple brain infarction with several stroke episodes (n = 4) were selected for this study. We observed the amount of UCP2 and UCP5 expression in both lesioned and intact areas, and compared them between embolic stroke and multiple infarcton cases. The results showed that the expression of UCP2 and UCP5 was significantly elevated in the ischemic lesions compared to the intact area. UCP5 expression in the lesions was higher in multiple infarction cases than in embolic stroke cases. In conclusion, brains may respond to neuroprotection through the increased expression of UCP2 and UCP5 under ischemic conditions. Moreover, UCP5 may respond to repetitive ischemic stresses or have a long‐term effect.

Clinical Evaluation of Lacunar Infarction and Branch Atheromatous Disease

Patients with branch atheromatous disease (BAD) are more likely to experience neurologic deficits compared with those with lacunar infarction (LI), although both disorders are forms of intracranial deep brain infarction. We clinically evaluated patients with BAD (n = 42) and LI (n = 57) to investigate why patients with BAD tend to experience progressing stroke. Patients presenting to our hospital with acute ischemic stroke between April 2008 and March 2009 were screened. LI was defined as an intracerebral lesion <15 mm in diameter and fewer than 3 slices or a lesion within the pontine parenchyma. BAD was defined as an intracerebral lesion of ≥ 15 mm in diameter and more than 3 slices or a lesion extending to the surface of the pontine base observed on diffusion-weighted magnetic resonance imaging. Progressing stroke was defined as a >2-point increase in the National Institutes of Health Stroke Scale within 48 hours of stroke onset. Progressing stroke was significantly more prevalent in the BAD group compared with the LI group (38.1% vs 12.3%). Diabetes mellitus with a high low-density lipoprotein level was significantly prevalent in patients with progressing BAD. When BAD in the cerebrum and BAD in the pons were analyzed separately, a low-density lipoprotein level >140 mg/dL was the most prevalent risk factor for progressing BAD in the cerebrum, and patient age was the strongest risk factor for progressing BAD in the pons. Vascular lesions asvsessed by magnetic resonance angiography were significantly abundant in both progressing LI and BAD. Our findings suggest that BAD may have a poorer prognosis than LI. Poorly controlled diabetes and hyperlipidemia could lead to atherosclerosis of the branch artery, resulting in worsening of BAD.

Ischemia Alters the Expression of Connexins in the Aged Human Brain

Although the function of astrocytic gap junctions under ischemia is still under debate, increased expression of connexin 43 (Cx43) has been observed in ischemic brain lesions, suggesting that astrocytic gap junctions could provide neuronal protection against ischemic insult. Moreover, different connexin subtypes may play different roles in pathological conditions. We used immunohistochemical analysis to investigate alterations in the expression of connexin subtypes in human stroke brains. Seven samples, sectioned after brain embolic stroke, were used for the analysis. Data, evaluated semiquantitatively by computer-assisted densitometry, was compared between the intact hemisphere and ischemic lesions. The results showed that the coexpression of Cx32 and Cx45 with neuronal markers was significantly increased in ischemic lesions. Cx43 expression was significantly increased in the colocalization with astrocytes and relatively increased in the colocalization with neuronal marker in ischemic lesions. Therefore, Cx32, Cx43, and Cx45 may respond differently to ischemic insult in terms of neuroprotection.