Increased oxidative stress during hyperglycemic cerebral ischemia

In this review, we summarize the role of hyperglycemia during cerebral ischemia. Hyperglycemia occurring during experimental and clinical stroke has been associated with increased cerebral damage. Increased oxidative stress resulting from hyperglycemia is believed to contribute to the exacerbated damage. More specifically, superoxide, nitric oxide and peroxynitrite are believed to play an important role in cerebral damage. This also involves increased recruitment of various blood cells to the ischemic zone that contribute to inflammation. We present data from our group and others that demonstrate that free radical production is increased during hyperglycemic stroke in rodents. Recent data suggest that inflammation is an important component of ischemic damage under both normo- and hyperglycemic conditions. We summarize numerous studies that indicate that a variety of antioxidant (inhibition of free radical production, scavenging of free radicals and increasing free radical degradation) and anti-inflammatory strategies decrease cerebral infarction. Finally, we compare the success of some of these strategies in clinical trials compared to the animal models.     

Synaptosomal glutamate uptake in a model of experimental cerebral ischemia

In the present investigation we studied the synaptosomal uptake of glutamate in brain omogenate of Mongolian gerbils submitted to bilateral common carotid occlusion, with and without subsequent return of blood flow. The results show that glutamate uptake after ischemia is reduced by about 35% The damage appears to be persistent, since return of blood flow restores uptake only slightly. The membrane alterations occurring in ischemia could explain the persistence of glutamate transporter impairment. Besides the blockade of NMDA receptors, the stimulation and/or the protection of the uptake systems for glutamate could be of help in preventing neuronal ischemic damage.     

Augmentation of nitrite therapy in cerebral ischemia by NMDA receptor inhibition

Our recent work suggested that early infusion of nitrite might represent a novel therapeutic approach for acute ischemic stroke. In this study, we sought to examine the therapeutic time window of nitrite in an experimental stroke model, and to develop combined strategies for augmenting its protective effects. Nitrite was infused at various times after ischemia to rats subjected to transient or permanent focal ischemia. Nitrite was infused with memantine to prevent the potential toxicity. Infarct volumes, functional outcomes, microhypoxic areas, and oxidative stress were measured. Nitrite reduced the infarction volume and enhanced functional recovery when administered within 3 and 1.5 h in the transient and permanent model, respectively. Combined therapy with nitrite and memantine prolonged the time window up to 4.5 h. The potential oxidative toxicities of nitrite were significantly inhibited by memantine. The combination therapy of nitrite and memantine may be a feasible therapeutic approach for acute ischemic stroke.     

Intracellular regions of potassium channels: Kv2.1 and heag

Intracellular regions of voltage-gated potassium channels often comprise the largest part of the channel protein, and yet the functional role of these regions is not fully understood. For the Kv2.1 channel, although there are differences in activation kinetics between rat and human channels, there are, for instance, no differences in movement of the S4 region between the two channels, and indeed our mutagenesis studies have identified interacting residues in both the N- and C -terminal intracellular regions that are responsible for these functional effects. Furthermore, using FRET with fluorescent-tagged Kv2.1 channels, we have shown movement of the C-termini relative to the N-termini during activation. Such interactions and movements of the intracellular regions of the channel appear to form part of the channel gating machinery. Heag1 and heag2 channels also display differing activation properties, despite their considerable homology. By a chimeric approach, we have shown that these differences in activation kinetics are determined by multiple interacting regions in the N-terminus and membrane-spanning regions. Furthermore, alanine mutations of many residues in the C-terminal cyclic nucleotide binding domain affect activation kinetics. The data again suggest interacting regions between N- and C- termini that participate in the conformational changes during channel activation. Using a mass-spectrometry approach, we have identified α-tubulin and a heat shock protein as binding to the C-terminus of the heag2 channel, and α-tubulin itself has functional effects on channel activation kinetics. Clearly, the intracellular regions of these ion channels (and most likely many other ion channels too) are important regions in determining channel function. EBSA Satellite Meeting: Ion channels, Leeds, July 2007.     

Effect of repeated allogeneic bone marrow mononuclear cell transplantation on brain injury following transient focal cerebral ischemia in rats

Aims

Transplantation of bone marrow mononuclear cells (BMMCs) exerts neuroprotection against cerebral ischemia. We examined the therapeutic timepoint of allogeneic BMMC transplantation in a rat model of focal cerebral ischemia, and determined the effects of repeated transplantation outside the therapeutic window.

Main methods

Male Sprague–Dawley rats were subjected to 90 minute focal cerebral ischemia, followed by intravenous administration of 1 × 10⁷ allogeneic BMMCs or vehicle at 0, 3 or 6 h after reperfusion or 2 × 10⁷ BMMCs 6 h after reperfusion. Other rats administered 1 × 10⁷ BMMCs at 6 h after reperfusion received additional BMMC transplantation or vehicle 9 h after reperfusion. Infarct volumes, neurological deficit scores and immunohistochemistry were evaluated 24 or 72 h after reperfusion.

Key findings

Infarct volumes at 24 h were significantly decreased in transplantation rats at 0 and 3 h, but not at 6 h, after reperfusion, compared to vehicle-treatment. Even high dose BMMC transplantation at 6 h after reperfusion was ineffective. Repeated BMMC transplantation at 6 and 9 h after reperfusion reduced infarct volumes and significantly improved neurological deficit scores at 24 and 72 h. Immunohistochemistry showed repeated BMMC transplantation reduced ionized calcium-binding adapter molecule 1, 4-hydroxy-2-nonenal and 8-hydroxydeoxyguanosine expression at 24 and 72 h after reperfusion.

Significance

Intravenous allogeneic BMMCs were neuroprotective following transient focal cerebral ischemia, and the therapeutic time window of BMMC transplantation was > 3 h and < 6 h after reperfusion in this model. Repeated transplantation at 6 and 9 h after reperfusion suppressed inflammation and oxidative stress in ischemic brains, resulting in improved neuroprotection.     

The roles of intracellular regions in the activation of voltage-dependent potassium channels

The involvement of the transmembrane regions S2, S3 and S4 in the activation of potassium channels by depolarization has been well clarified. However, a role of the intracellular regions in channel function is emerging. Here we review recent evidence for the roles of intracellular regions in the functioning of members of two families of channels. The Kv2.1 potassium channel, a member of the voltage activated Kv family, has long intracellular regions. By mutagenesis studies and expression in oocytes, we identify residues in both the N- and C-terminal regions that contribute to determining activation kinetics of this channel. It seems that the C-terminus wraps around the N-terminus and interacts with it functionally. The voltage-activated ether-a-go-go (eag) channels also have long intracellular regions. Despite considerable homology, eag1 and eag2 channels display different activation kinetics. By making chimeras between these channels and again expressing in oocytes, we show that residues in both the N-terminal region and the membrane-spanning region are involved in determining these differences in activation kinetics. The intracellular N- and C-terminal regions are likely to continue to prove fertile regions in future investigations into the functioning of ion channels.Presented at the Biophysical Society Meeting on Ion channels—from structure to disease held in May 2003, Rennes, France     

Evidence for two-pore domain potassium channels in rat cerebral arteries

Little is known about the presence and function of two-pore domain K(+) (K(2P)) channels in vascular smooth muscle cells (VSMCs). Five members of the K(2P) channel family are known to be directly activated by arachidonic acid (AA). The purpose of this study was to determine 1) whether AA-sensitive K(2P) channels are expressed in cerebral VSMCs and 2) whether AA dilates the rat middle cerebral artery (MCA) by increasing K+ currents in VSMCs via an atypical K+ channel. RT-PCR revealed message for the following AA-sensitive K(2P) channels in rat MCA: tandem of P domains in weak inward rectifier K+ (TWIK-2), TWIK-related K+ (TREK-1 and TREK-2), TWIK-related AA-stimulated K+ (TRAAK), and TWIK-related halothane-inhibited K+ (THIK-1) channels. However, in isolated VSMCs, only message for TWIK-2 was found. Western blotting showed that TWIK-2 is present in MCA, and immunohistochemistry further demonstrated its presence in VSMCs. AA (10-100 microM) dilated MCAs through an endothelium-independent mechanism. AA-induced dilation was not affected by inhibition of cyclooxygenase, epoxygenase, or lipoxygenase or inhibition of classical K+ channels with 10 mM TEA, 3 mM 4-aminopyridine, 10 microM glibenclamide, or 100 microM Ba2+. AA-induced dilations were blocked by 50 mM K+, indicating involvement of a K+ channel. AA (10 microM) increased whole cell K+ currents in dispersed cerebral VSMCs. AA-induced currents were not affected by inhibitors of the AA metabolic pathways or blockade of classical K+ channels. We conclude that AA dilates the rat MCA and increases K+ currents in VSMCs via an atypical K+ channel that is likely a member of the K(2P) channel family.     

脑出血与脑缺血动物模型脾淋巴细胞的蛋白质组学研究

目的：通过比较正常与脑出血及脑缺血模型大鼠脾淋巴细胞蛋白质表达的差异,初步探讨细胞免疫功能与脑血管病之间的关系。方法：将SD大鼠随机分为正常组、脑出血模型组（采用VII型胶原酶诱导脑出血）和局灶性脑缺血模型组（采用线栓法造成大脑中动脉阻塞）,分离大鼠脾淋巴细胞,提取总蛋白质后进行双向凝胶电泳,考马斯亮蓝染色,PDQUEST软件分析,对差异蛋白质点采用基质辅助激光解析电离质谱（MALDI-TOF-MS）技术进行鉴定并分析。结果：胶质细胞成熟因子 等9个蛋白在脑出血和脑缺血模型组表达上调,膜联蛋白III在脑出血和脑缺血模型组表达下调。结论：建立了分辨率高重复性较好的脑出血及局灶性脑缺血脾淋巴细胞总蛋白的双向凝胶电泳图谱,并鉴定一些与脑血管病脑损伤相关的差异表达蛋白质,为深入研究脑血管病细胞免疫功能改变与脑血管病之间的关系奠定了基础。     

Roles of surface residues of intracellular domains of heag potassium channels

Ether-a-go-go potassium channels have large intracellular regions containing ‘Per-Ant-Sim’ (PAS) and cyclic nucleotide binding (cNBD) domains at the N- and C-termini, respectively. In heag1 and heag2 channels, recent studies have suggested that the N- and C-terminal domains interact, and affect activation properties. Here, we have studied the effect of mutations of residues on the surfaces of PAS and cNBD domains. For this, we introduced alanine and lysine mutations in heag1 channels, and recorded currents by two-electrode voltage clamp. In both the PAS domain and the cNBD domain, contiguous areas of conserved residues on the surfaces of these domains were found which affected the activation kinetics of the channel. Next, we investigated possible effects of mutations on domain interactions of PAS and cNBD proteins in heag2 by co-expressing these domain proteins followed by analysis with native gels and western blotting. We found oligomeric association between these domains. Mutations F30A and A609K (on the surfaces of the PAS and cNBD domains, respectively) affected oligomeric compositions of these domains when proteins for PAS and cNBD domains were expressed together. Taken together, the data suggest that the PAS and cNBD domains form interacting oligomers that have roles in channel function.     

N-terminal PDZ-binding domain in Kv1 potassium channels

We have investigated the interactions of prototypical PDZ domains with both the C- and N-termini of Kv1.5 and other Kv channels. A combination of in vitro binding and yeast two-hybrid assays unexpectedly showed that PDZ domains derived from PSD95 bind both the C- and N-termini of the channels with comparable avidity. From doubly transfected HEK293 cells, Kv1.5 was found to co-immunoprecipitate with the PDZ protein, irrespective of the presence of the canonical C-terminal PDZ-binding motif in Kv1.5. Imaging analysis of the same HEK cell lines demonstrated that co-localization of Kv1.5 with PSD95 at the cell surface is similarly independent of the canonical PDZ-binding motif. Deletion analysis localized the N-terminal PDZ-binding site in Kv1.5 to the T1 region of the channel. Co-expression of PSD95 with Kv1.5 N- and C-terminal deletions in HEK cells had contrasting effects on the magnitudes of the potassium currents across the membranes of these cells. These findings may have important implications for the regulation of channel expression and function by PDZ proteins like PSD95.     

Intra-arterial transplantation of bone marrow mononuclear cells immediately after reperfusion decreases brain injury after focal ischemia in rats

AIMS: Transplantation of bone marrow cells has been reported to exert neuroprotection against cerebral ischemia. However, the effect of bone marrow mononuclear cells (BMMCs) administered immediately after reperfusion has rarely been investigated. The present study was designed to examine whether brain injury in response to transient focal ischemia can be ameliorated by BMMC administration immediately after reperfusion in rats, and to determine whether there are differences in the route of administration. MAIN METHODS: Autologous BMMCs were obtained from each rat. Rats were then subjected to transient focal ischemia followed by BMMC administration via the ipsilateral carotid artery (IA group) or the femoral vein (IV group) immediately after reperfusion. Control rats underwent the same procedure but received vehicle injection. Infarct volume was compared among the groups 24 h and 7 days after reperfusion. BMMCs were fluorescently labeled with PKH26 prior to administration to track transplanted cells. KEY FINDINGS: Total infarct volume decreased in the IA group, but not in the IV group, when compared to the vehicle group. In the ipsilateral hemisphere, PKH26 positive cell count was greater in the IA group than in the IV group. Motor function, assessed with a rotarod test, improved in the IA group compared to the vehicle group. SIGNIFICANCE: These results show significant neuroprotection after transient focal ischemia by 1x10(7) autologous BMMCs administered intra-arterially, but not intravenously, immediately after reperfusion in rats. The larger number of transplanted BMMCs in the brain during the early stage of reperfusion may be responsible for the protective effect.     

Mechanosensitive potassium channels in locust muscle membrane

Patch clamp recordings have been made from adult locust (Schistocerca gregaria) muscle membrane to study the mechanosensitivity of potassium channels (BK and IK) in cell-attached patches by transiently applying measured pressures to the contents of the patch pipettes. The aim of the investigations was to demonstrate a novel gating behaviour by pressure of the BK channel in contrast to the familiar behaviour of the IK channel. The open probability (p ₀) of the IK channel increased rapidly in response to a pressure step and monotonically during a pressure ramp. This gating was readily repeatable and rapidly reversible. The relationship between ln[p ₀/(1–p ₀)] and transmembrane pressure was linear. In comparison, p ₀ for the BK channel was also increased by pressure, but its gating was delayed, cumulative, and hysteretic. Received: 12 July 1998 / Revised version: 7 October 1998 / Accepted: 7 October 1998     

Sulfur dioxide derivative modulation of potassium channels in rat ventricular myocytes

The effects of sulfur dioxide (SO2) derivatives (bisulfite and sulfite, 1:3 M/M) on voltage-dependent potassium current in isolated adult rat ventricular myocyte were investigated using the whole cell patch-clamp technique. SO2 derivatives (10 microM) increased transient outward potassium current (I(to)) and inward rectifier potassium current (I(K1)), but did not affect the steady-state outward potassium current (I(ss)). SO2 derivatives significantly shifted the steady-state activation curve of I(to) toward the more negative potential at the V(h) point, but shifted the inactivation curve to more positive potential. SO2 derivatives markedly shifted the curve of time-dependent recovery of I(to) from the steady-state inactivation to the left, and accelerated the recovery of I(to) from inactivation. In addition, SO2 derivatives also significantly change the inactivation time constants of I(to) with increasing fast time constant and decreasing slow time constant. These results indicated a possible correlation between the change of properties of potassium channel and SO2 inhalation toxicity, which might cause cardiac myocyte injury through increasing extracellular potassium via voltage-gated potassium channels.     

Effects of pretreatment with PACAP on the infarct size and functional outcome in rat permanent focal cerebral ischemia

PACAP exerts neuroprotective effects under various neurotoxic conditions in vitro. In vivo, it reduces brain damage after global and transient focal ischemia. The present study investigated whether PACAP has neuroprotective effects when applied before the onset of permanent ischemia. Rats were given bolus injections of PACAP38 intracerebroventricularly, and then underwent permanent middle cerebral artery occlusion. The results show that 2 μg of PACAP significantly reduced the infarct size measured 12 and 24 h after the onset of ischemia. No further reduction was obtained by a 7-day pretreatment. PACAP also ameliorated certain sensorimotor deficits. Our present study provides further evidence for the neuroprotective effects of PACAP, and implies that it might be a promising preventive therapeutic agent in ameliorating ischemic brain damage.     

双侧迷走神经和颈总动脉联合切断法建立急性脑缺血大鼠模型

目的建立一种操作简单的急性脑缺血动物模型。方法取雄性Wistar大鼠40只,体重200～230g,手术前禁食12 h,自由饮水,随机分为对照组A、B、C组及模型D组,共4组,每组10只。即A组:假手术组,仅切开颈部两侧皮肤,分离双侧颈总动脉和迷走神经,不切断,然后缝合;B组:仅切断双侧颈部迷走神经;C组:结扎并切断双侧颈总动脉(CCA);D组:联合组,即结扎并切断双侧颈总动脉,同时切断双侧颈部迷走神经。观察各组大鼠手术后的脑缺血症状,记录各组大鼠在8h内的死亡情况,超过8h死亡的动物按8h计,计算死亡率和死亡时间。结果 A组大鼠没有脑缺血症状,无死亡;B组大鼠无脑缺血症状,呼吸变慢变深,心率血压上升,但无死亡;C组大鼠部分出现脑缺血症状,眼睑下垂,活动能力低下,自发运动减少,有些大鼠术后自发运动增加,在8 h内无死亡;D组大鼠大多数出现较为明显的脑缺血症状,在8 h内全部死亡。结论采取同时结扎并切断大鼠双侧颈总动脉和双侧颈部迷走神经的方法,可以建立急性脑缺血大鼠动物模型,此方法具有手术简单,成功率高,术后动物缓慢死亡的特点。     

Three classes of potassium channels in large monopolar cells of the blowfly Calliphora vicina

Summary We have used single electrode voltage clamp in the intact animal and whole-cell recording from dissociated cell bodies to investigate the properties of potassium conductances in large monopolar cells (LMCs) of the first optic ganglion of the blowfly Calliphora vicina. Two classes of voltage gated potassium conductances were found: a delayed rectifier current (Kd) with slow inactivation (_inac = 1–3 sec), and an A current (Ka) showing both faster inactivation (_inac = 21 ms) and also more rapid activation. The reversal potential of both currents is ca. -90 mV with 2 mM [K_o] and 140 mM [K_i], and follows the Nernst slope with increasing [K_o]. The voltage operating range of Ka is unusually negative, with the mid point of the steady-state inactivation curve (V₅₀) at- 101 mV. V₅₀ for Kd is - 84 mV. Although no inward currents were detected, for technical reasons their presence cannot be excluded.In inside-out patches from LMC soma membranes the single channels underlying the currents both have a conductance of ca. 20 pS in symmetrical 140 mM K solutions and channel densities may be as high as 10/m². Less frequently, inside-out patches contained a large conductance (110 pS) calcium-activated potassium channel which existed almost exclusively in a rapidly flickering mode. Open probability increased with depolarization and Ca concentrations greater than 40 nM.In whole-cell recordings, dissociated LMC cell bodies fall into two classes with respect to their voltage sensitive currents: 37 % of cells only showed Kd; the remainder (63%) were dominated by Ka with a variable (0–30%) contribution from Kd. In the intact animal, intracellular recordings from LMCs, combined with dye-marking, indicate that cells expressing only Kd are type L3 cells, whilst L1 and L2 express predominantly Ka. Since L1 and L2 have resting potentials of ca. - 40 mV and maximum hyperpolarizations reaching -90 mV only transiently, inactivation of Ka is unlikely to be removed under most physiological conditions. In contrast, L3 cells have a more negative resting potential (–60 mV) and Kd should play a significant role in signal-shaping, in particular contributing to the falling phase of a prominent spike-like transient in response to dimming.Abbreviations Ka A current - Kd delayed rectifier - LMC large monopolar cell - L1-L3 classes thereof - TTX tetrodotoxin     

Differential activation of protein kinase C isoforms following chemical ischemia in rat cerebral cortex slices

The aim of the current study was to characterize the effects of chemical ischemia and reperfusion at the transductional level in the brain. Protein kinase C isoforms (, β₁, β₂, γ, δ and ) total levels and their distribution in the particulate and cytosolic compartments were investigated in superfused rat cerebral cortex slices: (i) under control conditions; (ii) immediately after a 5-min treatment with 10 mM NaN₃, combined with 2 mM 2-deoxyglucose (chemical ischemia); (iii) 1 h after chemical ischemia (reperfusion). In control samples, all the PKC isoforms were detected; immediately after chemical ischemia, PKC β₁, δ and isoforms total levels (cytosol + particulate) were increased by 2.9, 2.7 and 9.9 times, respectively, while isoform was slightly reduced and γ isoform was no longer detectable. After reperfusion, the changes displayed by , β₁, γ, δ and were maintained and even potentiated, moreover, an increase in β₂ (by 41 ± 12%) total levels became significant. Chemical ischemia-induced a significant translocation to the particulate compartment of PKC isoform, which following reperfusion was found only in the cytosol. PKC β₁ and δ isoforms particulate levels were significantly higher both in ischemic and in reperfused samples than in the controls. Conversely, following reperfusion, PKC β₂ and isoforms displayed a reduction in their particulate to total level ratios. The intracellular calcium chelator, 1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid, 1 mM, but not the N-methyl-d-asparate receptor antagonist, MK-801, 1 μM, prevented the translocation of β₁ isoform observed during ischemia. Both drugs were effective in counteracting reperfusion-induced changes in β₂ and isoforms, suggesting the involvement of glutamate-induced calcium overload. These findings demonstrate that: (i) PKC isoforms participate differently in neurotoxicity/neuroprotection events; (ii) the changes observed following chemical ischemia are pharmacologically modulable; (iii) the protocol of in vitro chemical ischemia is suitable for drug screening.