Multiple sites of vasopressin synthesis in the injured brain

Previous studies have indicated that the primary targets for vasopressin actions on the injured brain are the cerebrovascular endothelium and astrocytes, and that vasopressin amplifies the posttraumatic production of proinflammatory mediators. Here, the controlled cortical impact model of traumatic brain injury in rats was used to identify the sources of vasopressin in the injured brain. Injury increased vasopressin synthesis in the hypothalamus and cerebral cortex adjacent to the posttraumatic lesion. In the cortex, vasopressin was predominantly produced by activated microglia/macrophages, and, to a lesser extent, by the cerebrovascular endothelium. These data further support the pathophysiological role of vasopressin in brain injury.     

Intravenous transplantation of bone marrow mesenchymal stem cells promotes neural regeneration after traumatic brain injury

To investigate the supplement of lost nerve cells in rats with traumatic brain injury by intravenous administration of allogenic bone marrow mesenchymal stem cells, this study established a Wistar rat model of traumatic brain injury by weight drop impact acceleration method and administered 3 × 106 rat bone marrow mesenchymal stem cells via the lateral tail vein. At 14 days after cell transplantation, bone marrow mesenchymal stem cells differentiated into neurons and astrocytes in injured rat cerebral cortex and rat neurological function was improved significantly. These findings suggest that intravenously administered bone marrow mesenchymal stem cells can promote nerve cell regeneration in injured cerebral cortex, which supplement the lost nerve cells.     

Gonadal hormones down-regulate reactive gliosis and astrocyte proliferation after a penetrating brain injury

Astrocytes are a target for gonadal steroids in the normal brain. The putative modulation by gonadal hormones of the astrocytic reaction to brain injury was assessed in this study. Male and female adult Wistar albino rats were gonadectomized and, one month later, their brains were lesioned by a longitudinal incision crossing the parietal cerebral cortex, the CA1 field of the dorsal hippocampus and the dentate gyrus. Males were injected either with testosterone (20 μg/rat) or vehicle immediately after surgery. Females were injected either with 17β estradiol (250 μg/rat), progesterone (500 μg/rat) or vehicle. Hormonal injections were repeated 24 and 48 h after brain injury. All animals received injections of 5′-bromodeoxyuridine (BrdU) to label proliferating cells. Histological sections from the brain of animals killed 72 h after surgery were used for the double immunohistochemical localization of BrdU and glial fibrillary acidic protein (GFAP). The number of GFAP-immunoreactive astrocytes and the number of double labelled astrocytes (GFAP+BrdU) were recorded as a function of the distance to the lesion site in the parietal cerebral cortex, the CA1 field of the hippocampus and the dentate gyrus. Testosterone, estradiol and progesterone treatments resulted in a significant decrease in the number of GFAP-immunolabelled reactive astrocytes in the vicinity of the wound. The number of double labelled cells and the labelling index (proportion of GFAP-immunoreactive astrocytes labelled with BrdU) varied according to the cerebral area, the distance to the wound and the sex of the animals, and were significantly decreased by gonadal steroids in all the areas examined. These results ndicate that gonadal hormones may decrease gliosis and astrocyte proliferation after a penetrating brain injury.     

Astrocyte-Dependent Vulnerability to Excitotoxicity in Spermine Oxidase-Overexpressing Mouse

Transgenic mice overexpressing spermine oxidase (SMO) in the cerebral cortex (Dach-SMO mice) showed increased vulnerability to excitotoxic brain injury and kainate-induced epileptic seizures. To investigate the mechanisms by which SMO overexpression leads to increased susceptibility to kainate excitotoxicity and seizure, in the cerebral cortex of Dach-SMO and control mice we assessed markers for astrocyte proliferation and neuron loss, and the ability of kainate to evoke glutamate release from nerve terminals and astrocyte processes. Moreover, we assessed a possible role of astrocytes in an in vitro model of epileptic-like activity in combined cortico-hippocampal slices recorded with a multi-electrode array device. In parallel, as the brain is a major metabolizer of oxygen and yet has relatively feeble protective antioxidant mechanisms, we analyzed the oxidative status of the cerebral cortex of both SMO-overexpressing and control mice by evaluating enzymatic and non-enzymatic scavengers such as metallothioneins. The main findings in the cerebral cortex of Dach-SMO mice as compared to controls are the following: astrocyte activation and neuron loss; increased oxidative stress and activation of defense mechanisms involving both neurons and astrocytes; increased susceptibility to kainate-evoked cortical epileptogenic activity, dependent on astrocyte function; appearance of a glutamate-releasing response to kainate from astrocyte processes due to activation of Ca²⁺-permeable AMPA receptors in Dach-SMO mice. We conclude that reactive astrocytosis and activation of glutamate release from astrocyte processes might contribute, together with increased reactive oxygen species production, to the vulnerability to kainate excitotoxicity in Dach-SMO mice. This mouse model with a deregulated polyamine metabolism would shed light on roles for astrocytes in increasing vulnerability to excitotoxic neuron injury.     

Osteopontin is indispensable for activation of astrocytes in injured mouse brain and primary culture

ABSTRACT

Objectives: Osteopontin (OPN) is an inflammatory cytokine inducer involved in cell proliferation and migration in inflammatory diseases or tumors. To investigate the function of OPN in astrocyte activation during brain injury, we compared OPN-deficient (OPN/KO) with wild-type (WT) mouse brains after stab wound injury and primary culture of astrocytes.

Methods: Primary cultures of astrocytes were prepared from either WT or OPN/KO postnatal mouse brains. Activation efficiency of astrocytes in primary culture was accessed using Western blotting by examining the protein levels of glial fibrillary acidic protein (GFAP) and tenascin-C (TN-C), which are markers for reactive astrocytes, following lipopolysaccharide (LPS) stimulation. Furthermore, the stab wound injury on the cerebral cortex as a brain traumatic injury model was used, and activation of astrocytes and microglial cells was investigated using immunofluorescent analysis on fixed brain sections.

Results: Primary cultures of astrocytes prepared from WT or OPN/KO postnatal mouse brains showed that only 25% of normal shaped astrocytes in a flask were produced in OPN/KO mice. The expression levels of both GFAP and TN-C were downregulated in the primary culture of astrocytes from OPN/KO mice compared with that from WT mice. By the immunofluorescent analysis on the injured brain sections, glial activation was attenuated in OPN/KO mice compared with WT mice.

Discussion: Our data suggest that OPN is essential for proper astrocytic generation in vitro culture prepared from mouse cerebral cortex. OPN is indispensable for astrocyte activation in the mouse brain injury model and in LPS stimulated primary culture.

Abbreviations: AQP4: aquaporin 4; BBB: blood brain barrier; BrdU: bromo-deoxy uridine; CNS: central nervous system; GFAP: glial fibllirary acidic protein; IgG: immunoglobulin G; LPS: lipopolysaccharide; OPN: osteopontin; OPN/KO: osteopontin-deficient; TN-C: tenascin-C     

Erratum to: Differential alterations in the distribution of voltage-gated calcium channels in aged rat cerebellum: [Brain Research 903 (2001) 247–252]

In the present study, we have investigated the spatial and temporal distribution of voltage-gated calcium channels in the gerbil model of global cerebral ischemia using immunohistochemistry. Distinct localizations of P-type (α_1A), N-type (α_1B), and L-type (α_1C and α_1D) Ca²⁺ channels were observed in the hippocampus at days 1–5 after ischemic injury. However, increased expression of N-type Ca²⁺ channels was detectable in brain regions vulnerable to ischemia only at days 2 and 3 after ischemic injury. The pyramidal cell bodies of CA1-3 areas and the granule cell bodies of the dentate gyrus were intensely stained at days 2 and 3 following ischemic injury. Transient changes in N-type Ca²⁺ channel expression were also observed in the affected cerebral cortex and striatum at days 2 and 3 after ischemic injury. Although the present study has not addressed the multiple mechanisms contributing to the intracellular free Ca²⁺ concentration ([Ca²⁺]_i) increase in the ischemic brain, the first demonstration of the transient increase in N-type Ca²⁺ channels may prove useful for future investigations.     

Protein kinase C as an early and sensitive marker of ischemia-induced progressive neuronal damage in gerbil hippocampus

In the model of transient brain ischemia of 6-min duration in gerbils we have estimated:

1. The concentration of brain gangliosides: A significant decrease to about 70% of control was observed selectively in the hippocampus at 3 and 7 d after ischemia.
2. The activity of Na⁺,K⁺-ATPase: The enzyme activity was not affected in either hippocampus nor in cerebral cortex.
3. The malonylaldehyde (MDA) concentration: The levels of MDA had increased at 30 min after ischemia up to 123 and 129% of control in hippocampus and cerebral cortex, respectively.
4. Immunoreactivity of protein kinase C detected by Western blotting: In hippocampus the early translocation toward membranes was followed by a decrease in total enzyme content at 6, 24, 72, and 96 h of postischemic recovery. Also, a sharp increase of 50 kDa isoform (PKM) was noticed immediately and at the early recovery times.

The behavior of these biochemical markers of ischemic brain injury in the hippocampus after the short (6 min) insult was contrasted with their reaction in the cerebral cortex as well as after prolongation of the ischemia to 15 min. These results taken together indicate that an early increase in PKC translocation followed by a decrease is the most symptomatic for selective, delayed, postischemic hippocampal injury, resulting from short duration (6 min) ischemia of the gerbil brain.     

Enhanced electrical responsiveness in the cerebral cortex with oral melatonin administration after a small hemorrhage near the internal capsule in rats

Intracerebral hemorrhage (ICH) can cause direct brain injury at the insult site and indirect damage in remote brain areas. Although a protective effect of melatonin (ML) has been reported for ICH, its detailed mechanisms and effects on remote brain injury remain unclear. To clarify the mechanism of indirect neuroprotection after ICH, we first investigated whether ML improved motor function after ICH and then examined the underlying mechanisms. The ICH model rat was made by collagenase injection into the left globus pallidus, adjacent to the internal capsule. ML oral administration (15 mg/kg) for 7 days after ICH resulted in significant recovery of motor function. Retrograde labeling of the corticospinal tract by Fluoro‐Gold revealed a significant increase in numbers of positive neurons in the cerebral cortex. Immunohistological analysis showed that ML treatment induced no difference in OX41‐positive activated microglia/macrophage at day 1 (D1) but a significant reduction in 8‐hydroxydeoxyguanosin‐positive cells at D7. Neutral red assay revealed that ML significantly prevented H₂O₂‐induced cell death in cultured oligodendrocytes and astrocytes but not in neurons. Electrophysiological response in the cerebral cortex area where the number of Fluoro‐Gold‐positive cells was increased was significantly improved in ML‐treated rats. These data suggest that ML improves motor abilities after ICH by protecting oligodendrocytes and astrocytes in the vicinity of the lesion in the corticospinal tract from oxidative stress and causes enhanced electrical responsiveness in the cerebral cortex remote to the ICH pathology. © 2014 Wiley Periodicals, Inc.     

Cortical neurogenesis in adult rats after ischemic brain injury: most new neurons fail to mature

The present study examines the hypothesis that endogenous neural progenitor cells isolated from the neocortex of ischemic brain can differentiate into neurons or glial cells and contribute to neural regeneration. We performed middle cerebral artery occlusion to establish a model of cerebral ischemia/reperfusion injury in adult rats. Immunohistochemical staining of the cortex 1, 3, 7, 14 or 28 days after injury revealed that neural progenitor cells double-positive for nestin and sox-2 appeared in the injured cortex 1 and 3 days post-injury, and were also positive for glial fibrillary acidic protein. New neurons were labeled using bromodeoxyuridine and different stages of maturity were identified using doublecortin, microtubule-associated protein 2 and neuronal nuclei antigen immunohistochemistry. Immature new neurons coexpressing doublecortin and bromodeoxyuridine were observed in the cortex at 3 and 7 days post-injury, and semi-mature and mature new neurons double-positive for microtubule-associated protein 2 and bromodeoxyuridine were found at 14 days post-injury. A few mature new neurons coexpressing neuronal nuclei antigen and bromodeoxyuridine were observed in the injured cortex 28 days post-injury. Glial fibrillary acidic protein/bromodeoxyuridine double-positive astrocytes were also found in the injured cortex. Our findings suggest that neural progenitor cells are present in the damaged cortex of adult rats with cerebral ischemic brain injury, and that they differentiate into astrocytes and immature neurons, but most neurons fail to reach the mature stage.     

Expression of connexin29 and 32 in the penumbra region after traumatic brain injury of mice

Connexins (Cx) are transmembrane proteins forming vertebrate gap junction channels for direct cell-cell communication. We found that the expressions of two Cx family members, Cx29 and Cx32, were progressively increased in the sharp border of injury penumbra regions after cryotraumatic brain injury. Although these two Cxs are expressed exclusively in the oligodendrocytes in the normal cerebral cortex, their expressions were increased in the astrocytes and microglia localized in the injury border. Highly selective induction of Cxs in the injury border suggests that altered Cxs may contribute to the propagations of injury-related and/or regeneration signals after acute brain injury.     

BQ788, an endothelin ET(B) receptor antagonist, attenuates stab wound injury-induced reactive astrocytes in rat brain.

Endothelins (ETs) are suggested to be involved in pathological or pathophysiological responses on brain injuries. In the present study, an involvement of ETs on activation of astrocytes in vivo was examined by using selective endothelin receptor antagonists. A stab wound injury on rat cerebral cortex increased immunoreactive ET-1 at the injured site. GFAP-positive [GFAP(+)] and vimentin-positive [Vim(+)] cells appeared at the injured site in 1 day to 2 weeks after the injury. A continuous infusion of BQ788, a selective ETB receptor antagonist, into cerebral ventricle (23 nmole/day) attenuated increase in the numbers of GFAP(+) and Vim(+) cells after the injury. FR139317, a selective ETA antagonist (23 nmole/day), slightly decreased the number of Vim(+) cells but not that of GFAP(+) cells. Increase in the number of microglia/macrophages by a stab wound injury, which was determined by Griffonia simplicifolia isolectin B4 staining, was not affected by BQ788 and FR139317. These results suggest that activation of glial ETB receptors is one of the signal cascades leading to reactive astrocytes on brain injuries.     

Immunohistochemical studies on the proliferation of reactive astrocytes and the expression of cytoskeletal proteins following brain injury in rats

The appearance of reactive astrocytes following brain injury was investigated in 4-week-old rats with special reference to their proliferation and chronological changes in the cytoskeletal proteins. Two days after the injury, glial fibrillary acidic protein (GFAP)-positive cells had increased in number around the lesion and spread to the entire ipsilateral cortex by 3 days after the injury. To investigate the distribution of mitotic cells and its chronological change, immunohistochemical staining with monoclonal antibody to bromodeoxyuridine (BrdU) was performed. BrdU-positive cells began to appear around the lesion and spread to the entire ipsilateral cortex by 3 days and their distribution was the same as that of GFAP-positive cells. To investigate the association of GFAP-positive cells with cell division, double labeling experiments using [3H]thymidine autoradiography and immunohistochemical staining with antiserum to GFAP were performed. Cells doubly labeled with GFAP and [3H]thymidine were localized in the area adjacent to the lesion, in the molecular layer of the cortex and in the white matter. By contrast, none of the cells were doubly labeled in the IInd to VIth layers of the cortex. Furthermore, only astrocytes in the former areas expressed vimentin transiently from 2 to 10 days after the injury. In the rats administered vincristine, cells arrested during mitosis were found in the regions which express vimentin. From these results, it was suggested that astrocytes in the molecular layer of the cortex and the white matter adjacent to the lesion proliferated in response to the injury and expressed vimentin transiently, then acquired GFAP, and that astrocytes in the IInd to VIth layers of the cortex became reactive astrocytes without mitosis.     

Neuronal apolipoprotein E is not synthesized in neuron after focal ischemia in rat brain

Abstract

Apolipoprotein E (ApoE) is a major apolipoprotein in the central nervous system (CNS) that plays an important role in Alzheimer's disease. It may also be involved in other CNS disorders including ischemic injury. We investigated the changes of ApoE protein and mRNA expression in the brain with middle cerebral artery occlusion (MCAO) to clarify its origin after focal ischemia in rats. Increased ApoE immunoreactivity was recognized in astrocytes 3-14 days after MCAO in the affected side of cortex, and in neurons 4-14 days after MCAO in the same area. ApoE immunoreactivity was also detected in macrophages in the ischemic core 3-14 days after MCAO. In contrast, ApoE mRNA was expressed in astrocytes and macrophages, but not in neurons. These results suggested that neuronal ApoE was not synthesized in neurons, but derived from astrocytes.     

N‐myc downstream‐regulated gene 2 protects blood–brain barrier integrity following cerebral ischemia

Disruption of the blood‐brain barrier (BBB) following cerebral ischemia is closely related to the infiltration of peripheral cells into the brain, progression of lesion formation, and clinical exacerbation. However, the mechanism that regulates BBB integrity, especially after permanent ischemia, remains unclear. Here, we present evidence that astrocytic N‐myc downstream‐regulated gene 2 (NDRG2), a differentiation‐ and stress‐associated molecule, may function as a modulator of BBB permeability following ischemic stroke, using a mouse model of permanent cerebral ischemia. Immunohistological analysis showed that the expression of NDRG2 increases dominantly in astrocytes following permanent middle cerebral artery occlusion (MCAO). Genetic deletion of Ndrg2 exhibited enhanced levels of infarct volume and accumulation of immune cells into the ipsilateral brain hemisphere following ischemia. Extravasation of serum proteins including fibrinogen and immunoglobulin, after MCAO, was enhanced at the ischemic core and perivascular region of the peri‐infarct area in the ipsilateral cortex of Ndrg2‐deficient mice. Furthermore, the expression of matrix metalloproteinases (MMPs) after MCAO markedly increased in Ndrg2^?/? mice. In culture, expression and secretion of MMP‐3 was increased in Ndrg2^?/? astrocytes, and this increase was reversed by adenovirus‐mediated re‐expression of NDRG2. These findings suggest that NDRG2, expressed in astrocytes, may play a critical role in the regulation of BBB permeability and immune cell infiltration through the modulation of MMP expression following cerebral ischemia.     

Astrocytic transforming growth factor‐beta signaling reduces subacute neuroinflammation after stroke in mice

Astrocytes limit inflammation after CNS injury, at least partially by physically containing it within an astrocytic scar at the injury border. We report here that astrocytic transforming growth factor‐beta (TGFβ) signaling is a second, distinct mechanism that astrocytes utilize to limit neuroinflammation. TGFβs are anti‐inflammatory and neuroprotective cytokines that are upregulated subacutely after stroke, during a clinically accessible time window. We have previously demonstrated that TGFβs signal to astrocytes, neurons and microglia in the stroke border days after stroke. To investigate whether TGFβ affects astrocyte immunoregulatory functions, we engineered “Ast‐Tbr2DN” mice where TGFβ signaling is inhibited specifically in astrocytes. Despite having a similar infarct size to wildtype controls, Ast‐Tbr2DN mice exhibited significantly more neuroinflammation during the subacute period after distal middle cerebral occlusion (dMCAO) stroke. The peri‐infarct cortex of Ast‐Tbr2DN mice contained over 60% more activated CD11b⁺ monocytic cells and twice as much immunostaining for the activated microglia and macrophage marker CD68 than controls. Astrocytic scarring was not altered in Ast‐Tbr2DN mice. However, Ast‐Tbr2DN mice were unable to upregulate TGF‐β1 and its activator thrombospondin‐1 2 days after dMCAO. As a result, the normal upregulation of peri‐infarct TGFβ signaling was blunted in Ast‐Tbr2DN mice. In this setting of lower TGFβ signaling and excessive neuroinflammation, we observed worse motor outcomes and late infarct expansion after photothrombotic motor cortex stroke. Taken together, these data demonstrate that TGFβ signaling is a molecular mechanism by which astrocytes limit neuroinflammation, activate TGFβ in the peri‐infarct cortex and preserve brain function during the subacute period after stroke. GLIA 2014;62:1227–1240     

Expression of insulin-like growth factor I by astrocytes in response to injury

Astrocytes are known to express several growth factors in response to injury and neurological disease. Insulin-like growth factor I (IGF-I) induces astrocytes to divide in vitro and is expressed by developing, but not adult astrocytes both in vivo and in vitro. We tested whether IGF-I is re-expressed by reactive astrocytes in response to injury. We found that astrocytes surrounding the lesioned parenchyma after introduction of a cannula through the cerebral cortex, hippocampus and midbrain contain high levels of immunoreactive IGF-I, as determined by immunocytochemistry using a highly sensitive and specific anti-IGF-I monoclonal antibody. Interestingly, the contralateral hippocampus also contained IGF-I positive astrocytes although in substantial lower numbers. Intact animals showed no detectable IGF-I immunoreactivity in astrocytes. IGF-I was detected at the first time point tested after the lesion was made, 1 week, and for at least 1 month thereafter. Reactive astrocytes expressing high levels of glial fibrillary acidic protein were found in a much wider distribution all along the lesioned area and beyond. We conclude that mechanical injury of the brain induces a specific pattern of expression of IGF-I by a subpopulation of astrocytes. These findings suggest that IGF-I is participating in the response of astrocytes to injury.     

Striatal astrocytes transdifferentiate into functional mature neurons following ischemic brain injury

To determine whether reactive astrocytes stimulated by brain injury can transdifferentiate into functional new neurons, we labeled these cells by injecting a glial fibrillary acidic protein (GFAP) targeted enhanced green fluorescence protein plasmid (pGfa2‐eGFP plasmid) into the striatum of adult rats immediately following a transient middle cerebral artery occlusion (MCAO) and performed immunolabeling with specific neuronal markers to trace the neural fates of eGFP‐expressing (GFP⁺) reactive astrocytes. The results showed that a portion of striatal GFP⁺ astrocytes could transdifferentiate into immature neurons at 1 week after MCAO and mature neurons at 2 weeks as determined by double staining GFP‐expressing cells with βIII‐tubulin (GFP⁺‐Tuj‐1⁺) and microtubule associated protein‐2 (GFP⁺‐MAP‐2⁺), respectively. GFP⁺ neurons further expressed choline acetyltransferase, glutamic acid decarboxylase, dopamine receptor D2‐like family proteins, and the N‐methyl‐d ‐aspartate receptor subunit R2, indicating that astrocyte‐derived neurons could develop into cholinergic or GABAergic neurons and express dopamine and glutamate receptors on their membranes. Electron microscopy analysis indicated that GFP⁺ neurons could form synapses with other neurons at 13 weeks after MCAO. Electrophysiological recordings revealed that action potentials and active postsynaptic currents could be recorded in the neuron‐like GFP⁺ cells but not in the astrocyte‐like GFP⁺ cells, demonstrating that new GFP⁺ neurons possessed the capacity to fire action potentials and receive synaptic inputs. These results demonstrated that striatal astrocyte‐derived new neurons participate in the rebuilding of functional neural networks, a fundamental basis for brain repair after injury. These results may lead to new therapeutic strategies for enhancing brain repair after ischemic stroke. GLIA 2015;63:1660–1670     

Region-Specific and State-Dependent Astrocyte Ca2+ Dynamics during the Sleep-Wake Cycle in Mice

Neural activity is diverse, and varies depending on brain regions and sleep/wakefulness states. However, whether astrocyte activity differs between sleep/wakefulness states, and whether there are differences in astrocyte activity among brain regions remain poorly understood. Therefore, in this study, we recorded astrocyte intracellular calcium (Ca²⁺) concentrations of mice during sleep/wakefulness states in the cortex, hippocampus, hypothalamus, cerebellum, and pons using fiber photometry. For this purpose, male transgenic mice expressing the genetically encoded ratiometric Ca²⁺ sensor YCnano50 specifically in their astrocytes were used. We demonstrated that Ca²⁺ levels in astrocytes substantially decrease during rapid eye movement (REM) sleep, and increase after the onset of wakefulness. In contrast, differences in Ca²⁺ levels during non-REM (NREM) sleep were observed among the different brain regions, and no significant decrease was observed in the hypothalamus and pons. Further analyses focusing on the transition between sleep/wakefulness states and correlation analysis with the duration of REM sleep showed that Ca²⁺ dynamics differs among brain regions, suggesting the existence of several clusters, i.e., the first comprising the cortex and hippocampus, the second comprising the hypothalamus and pons, and the third comprising the cerebellum. Our study thus demonstrated that astrocyte Ca²⁺ levels change substantially according to sleep/wakefulness states. These changes were consistent in general unlike neural activity. However, we also clarified that Ca²⁺ dynamics varies depending on the brain region, implying that astrocytes may play various physiological roles in sleep.SIGNIFICANCE STATEMENT Sleep is an instinctive behavior of many organisms. In the previous five decades, the mechanism of the neural circuits controlling sleep/wakefulness states and the neural activities associated with sleep/wakefulness states in various brain regions have been elucidated. However, whether astrocytes, which are a type of glial cell, change their activity during different sleep/wakefulness states was poorly understood. Here, we demonstrated that dynamic changes in astrocyte Ca²⁺ concentrations occur in the cortex, hippocampus, hypothalamus, cerebellum, and pons of mice during natural sleep. Further analyses demonstrated that Ca²⁺ dynamics slightly differ among different brain regions, implying that the physiological roles of astrocytes in sleep/wakefulness might vary depending on the brain region.     

Intracerebral transportation and cellular localisation of insulin-like growth factor-1 following central administration to rats with hypoxic–ischemic brain injury

Insulin-like growth factor-1 (IGF-1) has been shown to be neuroprotective when administered centrally following hypoxic–ischemic (HI) brain injury. However, the cerebral distribution and site of action of IGF-1 after intracerebroventricular (i.c.v.) administration are not known. A unilateral HI brain injury was induced in adult rats by a modified Levine method. Either ³H-IGF-1 alone, or in combination with unlabelled IGF-1, was administered into the lateral ventricle 2 h after injury. The activity of ³H-IGF-1 signal in the potentially injured cortex was compared between two treatment groups using image analysis. The regional distribution and cellular localisation of ³H-IGF-1 were examined autoradiographically in potentially injured hemispheres at 0.5 and 6 h after administration. Tritiated IGF-1 was detected predominantly in the pia mater, perivascular spaces and subcortical white matter tracts 0.5 h after administration and decreased by 6 h (p<0.05). The signals associated with the perivascular spaces and pia mater were not blocked by unlabelled IGF-1, suggesting non-saturable binding in these brain areas. IGF-1 signal was co-localised with IGF binding protein (IGFBP)-2 immunostaining in the white matter tracts where the signal was blocked by unlabelled IGF-1, suggesting competitive association. IGF-1 signal associated with neurons and glia was maximal in the cerebral cortex and less in the CA1–2 subregion of the hippocampus which were blocked by unlabelled IGF-1 (p<0.05). The signals from cortical neurons did not decrease 6 h after administration, suggesting specific and persistent binding to these cells. Our results indicate that centrally administered IGF-1 can be translocated to neurons and glia via the perivascular circulation and the ependymal cell–white matter tract pathways.     

An inhibitory effect of interferon gamma on the injury-induced astrocyte proliferation in the postmitotic rat brain

Influence of interferon gamma (IFNγ) on astrocytes proliferating in response to unilateral injury of the cerebral hemisphere was investigated in 30-day-old rats. The brain injury was followed by an immediate injection of IFNγ into the lesion cavity. On 1st or 2nd day following injury the animals were injected with [³H]thymidine and killed 4 h after the injection. The proliferating astrocytes were visualised by combination of immunocytochemical staining for glial fibrillary acidic protein (GFAP) and autoradiography. Thereafter, numbers of GFAP-immunopositive and autoradiographically-labeled astrocytes located within the region of injury were counted. On the day 1 after injury no effect of IFNγ administration was seen. However, on day 2 the average 43% reduction of the number of proliferating astrocytes was recorded. The reduction showed no dose-dependent changes. This in vivo evidence of IFNγ-induced suppression of astrocyte proliferation was considered in relation to other determinants of astrocyte reactivity existing in the injured brain.