Novel Neuroprotective Agents to Treat Neonatal Hypoxic-Ischemic Encephalopathy: Inter-Alpha Inhibitor Proteins

Perinatal hypoxia-ischemia (HI) is a major cause of brain injury and mortality in neonates. Hypoxic-ischemic encephalopathy (HIE) predisposes infants to long-term cognitive deficits that influence their quality of life and place a large burden on society. The only approved treatment to protect the brain after HI is therapeutic hypothermia, which has limited effectiveness, a narrow therapeutic time window, and is not considered safe for treatment of premature infants. Alternative or adjunctive therapies are needed to improve outcomes of full-term and premature infants after exposure to HI. Inter-alpha inhibitor proteins (IAIPs) are immunomodulatory molecules that are proposed to limit the progression of neonatal inflammatory conditions, such as sepsis. Inflammation exacerbates neonatal HIE and suggests that IAIPs could attenuate HI-related brain injury and improve cognitive outcomes associated with HIE. Recent studies have shown that intraperitoneal treatment with IAIPs can decrease neuronal and non-neuronal cell death, attenuate glial responses and leukocyte invasion, and provide long-term behavioral benefits in neonatal rat models of HI-related brain injury. The present review summarizes these findings and outlines the remaining experimental analyses necessary to determine the clinical applicability of this promising neuroprotective treatment for neonatal HI-related brain injury.     

Time Course of Changes in the Neurovascular Unit after Hypoxic-Ischemic Injury in Neonatal Rats

Exposure to hypoxic-ischemic (HI) insults in newborns can predispose them to severe neurological sequela. The mechanisms underlying HI-related brain injury have not been completely elucidated. The neurovascular unit (NVU) is a composite of structures that protect the brain from the influx of detrimental molecules. Changes in the NVU after HI are important because they could reveal endogenous neuroprotective pathways in the cerebral microvasculature. Furthermore, the time course of changes in the NVU after exposure to HI in the newborn remains to be determined. In this study, we examined the effects of severe HI on the time course of changes in the NVU in neonatal rats. Brains were collected from rats exposed to right carotid artery ligation and 2 h of hypoxia on postnatal day 7 with recovery for 6 or 48 h after exposure to sham treatment (Sham) or HI. The right HI and left hypoxic alone sides of the brains were examined by quantitative immunohistochemistry for vascular density (laminin), pericyte vascular coverage (PDGFRβ), astrocyte vascular coverage (GFAP), and claudin-5 expression in the microvasculature of the cerebral cortex, white matter, and hippocampus. HI-related brain injury in neonatal rats was associated with increases in vascular density in the cortex and hippocampus 48 h after HI as well as neurovascular remodeling, including loss of pericyte coverage in the cortex and increases in claudin-5 in the hippocampus 6 h after HI. Astrocyte coverage was not affected by HI injury. The time course of the responses in the different components of the NVU varied after exposure to HI. There were also differential regional responses in the elements of the NVU in response to HI and hypoxia alone.     

Neuroelectric Mechanisms of Delayed Cerebral Ischemia after Aneurysmal Subarachnoid Hemorrhage

Delayed cerebral ischemia (DCI) remains a challenging but very important condition, because DCI is preventable and treatable for improving functional outcomes after aneurysmal subarachnoid hemorrhage (SAH). The pathologies underlying DCI are multifactorial. Classical approaches to DCI focus exclusively on preventing and treating the reduction of blood flow supply. However, recently, glutamate-mediated neuroelectric disruptions, such as excitotoxicity, cortical spreading depolarization and seizures, and epileptiform discharges, have been reported to occur in high frequencies in association with DCI development after SAH. Each of the neuroelectric disruptions can trigger the other, which augments metabolic demand. If increased metabolic demand exceeds the impaired blood supply, the mismatch leads to relative ischemia, resulting in DCI. The neuroelectric disruption also induces inverted vasoconstrictive neurovascular coupling in compromised brain tissues after SAH, causing DCI. Although glutamates and the receptors may play central roles in the development of excitotoxicity, cortical spreading ischemia and epileptic activity-related events, more studies are needed to clarify the pathophysiology and to develop novel therapeutic strategies for preventing or treating neuroelectric disruption-related DCI after SAH. This article reviews the recent advancement in research on neuroelectric disruption after SAH.     

Effects of Three Different Doses of Inter-Alpha Inhibitor Proteins on Severe Hypoxia–Ischemia-Related Brain Injury in Neonatal Rats

Hypoxia–ischemia (HI)-related brain injury is an important cause of morbidity and long-standing disability in newborns. We have previously shown that human plasma-derived inter-alpha inhibitor proteins (hIAIPs) attenuate HI-related brain injury in neonatal rats. The optimal dose of hIAIPs for their neuroprotective effects and improvement in behavioral outcomes remains to be determined. We examined the efficacy of 30, 60, or 90 mg/kg of hIAIPs administered to neonatal rats after exposure to HI for 2 h. Postnatal day 7 (P7) Wistar rats were exposed to either sham-surgery or unilateral HI (right carotid artery ligation, 2 h of 8% O₂) brain injury. A placebo, 30, 60, or 90 mg/kg of hIAIPs were injected intraperitoneally at 0, 24 and 48 h after HI (n = 9–10/sex). We carried out the following behavioral analyses: P8 (righting reflex), P9 (negative geotaxis) and P10 (open-field task). Rats were humanely killed on P10 and their brains were stained with cresyl violet. Male extension/contraction responses and female righting reflex times were higher in the HI placebo groups than the sham groups. Female open-field exploration was lower in the HI placebo group than the sham group. hIAIPs attenuated these behavioral deficits. However, the magnitude of the responses did not vary by hIAIP dose. hIAIPs reduced male brain infarct volumes in a manner that correlated with improved behavioral outcomes. Increasing the hIAIP dose from 30 to 90 mg/kg did not further accentuate the hIAIP-related decreases in infarct volumes. We conclude that larger doses of hIAIPs did not provide additional benefits over the 30 mg/kg dose for behavior tasks or reductions in infarct volumes in neonatal rats after exposure to severe HI.     

Long-Term Changes in Axon Calibers after Injury: Observations on the Mouse Corticospinal Tract

White matter pathology is common across a wide spectrum of neurological diseases. Characterizing this pathology is important for both a mechanistic understanding of neurological diseases as well as for the development of neuroimaging biomarkers. Although axonal calibers can vary by orders of magnitude, they are tightly regulated and related to neuronal function, and changes in axon calibers have been reported in several diseases and their models. In this study, we utilize the impact acceleration model of traumatic brain injury (IA-TBI) to assess early and late changes in the axon diameter distribution (ADD) of the mouse corticospinal tract using Airyscan and electron microscopy. We find that axon calibers follow a lognormal distribution whose parameters significantly change after injury. While IA-TBI leads to 30% loss of corticospinal axons by day 7 with a bias for larger axons, at 21 days after injury we find a significant redistribution of axon frequencies that is driven by a reduction in large-caliber axons in the absence of detectable degeneration. We postulate that changes in ADD features may reflect a functional adaptation of injured neural systems. Moreover, we find that ADD features offer an accurate way to discriminate between injured and non-injured mice. Exploring injury-related ADD signatures by histology or new emerging neuroimaging modalities may offer a more nuanced and comprehensive way to characterize white matter pathology and may also have the potential to generate novel biomarkers of injury.     

The Effect of Treadmill Training Pre-Exercise on Glutamate Receptor Expression in Rats after Cerebral Ischemia

Physical exercise has been demonstrated to be neuroprotective in both clinical and laboratory settings. However, the exact mechanism underlying this effect is unclear. Our study aimed to investigate whether pre-ischemic treadmill training could serve as a form of ischemic preconditioning in a rat model undergoing middle cerebral artery occlusion (MCAO). Thirty-six rats were divided into three groups: a sham control group, a non-exercise with operation group and an exercise with operation group. After treadmill training, ischemia was induced by occluding the MCA for 2 h, followed by reperfusion. Half of the rats in each group were sacrificed for mRNA detection of mGluR5 and NR2B 80 min after occlusion. The remaining animals were evaluated for neurological deficits by behavioral scoring and then decapitated to assess the infarct volume. The mRNA expression of mGluR5 and NR2B was detected by real-time PCR. The results suggest that pre-ischemic treadmill training may induce brain ischemic tolerance by reducing the mRNA levels of mGluR5 and NR2B, and thus, the results indicate that physical exercise might be an effective method to establish ischemic preconditioning.     

The Quinazoline Otaplimastat (SP-8203) Reduces the Hemorrhagic Transformation and Mortality Aggravated after Delayed rtPA-Induced Thrombolysis in Cerebral Ischemia

Acute ischemic stroke is the leading cause of morbidity and mortality worldwide. Recombinant tissue plasminogen activator (rtPA) is the only agent clinically approved by FDA for patients with acute ischemic stroke. However, delayed treatment of rtPA (e.g., more than 3 h after stroke onset) exacerbates ischemic brain damage by causing intracerebral hemorrhage and increasing neurotoxicity. In the present study, we investigated whether the neuroprotant otaplimastat reduced delayed rtPA treatment-evoked neurotoxicity in male Sprague Dawley rats subjected to embolic middle cerebral artery occlusion (eMCAO). Otaplimastat reduced cerebral infarct size and edema and improved neurobehavioral deficits. In particular, otaplimastat markedly reduced intracerebral hemorrhagic transformation and mortality triggered by delayed rtPA treatment, consequently extending the therapeutic time window of rtPA. We further found that ischemia-evoked extracellular matrix metalloproteases (MMPs) expression was closely correlated with cerebral hemorrhagic transformation and brain damage. In ischemic conditions, delayed rtPA treatment further increased brain injury via synergistic expression of MMPs in vascular endothelial cells. In oxygen-glucose-deprived endothelial cells, otaplimastat suppressed the activity rather than protein expression of MMPs by restoring the level of tissue inhibitor of metalloproteinase (TIMP) suppressed in ischemia, and consequently reduced vascular permeation. This paper shows that otaplimastat under clinical trials is a new drug which can inhibit stroke on its own and extend the therapeutic time window of rtPA, especially when administered in combination with rtPA.     

Neuroprotective Properties of Picroside II in a Rat Model of Focal Cerebral Ischemia

The aim of this study was to explore the effect of picroside II on neuronal apoptosis and the expression of caspase-3 and poly ADP-ribose polymerase (PARP) following middle cerebral artery occlusion/reperfusion in male Wistar rats. Picroside II (10 mg/kg) was administered intravenously into the tail vein of the animals. The neurological function deficits were evaluated with the Bederson's test and the cerebral infarction volume was visualized with tetrazolium chloride (TTC) staining. The apoptotic cells were counted by in situ terminal deoxynucleotidyl transferase-mediated biotinylated deoxyuridine triphosphate nick end labeling (TUNEL) assay. The immunohistochemistry stain and enzyme linked immunosorbent assay (ELISA) was used to determine the expressions of caspase-3 and PARP in brain tissue. The results indicated that rats in the control group showed neurological function deficit and cerebral infarction in ischemic hemisphere after two hours ischemia followed by 22 hours reperfusion. Caspase-3 and PARP expressions were also profound in the cortex, the striatum and the hippocampus, along with increased apoptotic cells in this group. Bederson's score, infarction volume, and expressions of caspase-3 and PARP, as well as apoptosis in the treatment group were, however, significantly decreased compared to those in the control group indicating that intravenous treatment with picroside II might be beneficial to inhibit neuronal apoptosis and, thus, to improve the neurological function of rats upon cerebral ischemia reperfusion injury.     

Relationship between Neuronal Damage/Death and Astrogliosis in the Cerebral Motor Cortex of Gerbil Models of Mild and Severe Ischemia and Reperfusion Injury

Neuronal loss (death) occurs selectively in vulnerable brain regions after ischemic insults. Astrogliosis is accompanied by neuronal death. It can change the molecular expression and morphology of astrocytes following ischemic insults. However, little is known about cerebral ischemia and reperfusion injury that can variously lead to damage of astrocytes according to the degree of ischemic injury, which is related to neuronal damage/death. Thus, the purpose of this study was to examine the relationship between damage to cortical neurons and astrocytes using gerbil models of mild and severe transient forebrain ischemia induced by blocking the blood supply to the forebrain for five or 15 min. Significant ischemia tFI-induced neuronal death occurred in the deep layers (layers V and VI) of the motor cortex: neuronal death occurred earlier and more severely in gerbils with severe ischemia than in gerbils with mild ischemia. Distinct astrogliosis was detected in layers V and VI. It gradually increased with time after both ischemiae. The astrogliosis was significantly higher in severe ischemia than in mild ischemia. The ischemia-induced increase of glial fibrillary acidic protein (GFAP; a maker of astrocyte) expression in severe ischemia was significantly higher than that in mild ischemia. However, GFAP-immunoreactive astrocytes were apparently damaged two days after both ischemiae. At five days after ischemiae, astrocyte endfeet around capillary endothelial cells were severely ruptured. They were more severely ruptured by severe ischemia than by mild ischemia. However, the number of astrocytes stained with S100 was significantly higher in severe ischemia than in mild ischemia. These results indicate that the degree of astrogliosis, including the disruption (loss) of astrocyte endfeet following ischemia and reperfusion in the forebrain, might depend on the severity of ischemia and that the degree of ischemia-induced neuronal damage may be associated with the degree of astrogliosis.     

Lack of Glutamate Receptor Subunit Expression Changes in Hippocampal Dentate Gyrus after Experimental Traumatic Brain Injury in a Rodent Model of Depression

Traumatic brain injury (TBI) affects over 69 million people annually worldwide, and those with pre-existing depression have worse recovery. The molecular mechanisms that may contribute to poor recovery after TBI with co-morbid depression have not been established. TBI and depression have many commonalities including volume changes, myelin disruption, changes in proliferation, and changes in glutamatergic signaling. We used a well-established animal model of depression, the Wistar Kyoto (WKY) rat, to elucidate changes after TBI that may influence the recovery trajectory. We compared the histological and molecular outcomes in the hippocampal dentate gyrus after experimental TBI using the lateral fluid percussion injury (LFPI) in the WKY and the parent Wistar (WIS) strain. We showed that WKY had exaggerated myelin loss after LFPI and baseline deficits in proliferation. In addition, we showed that while after LFPI WIS rats exhibited glutamate receptor subunit changes, namely increased GluN2B, the WKY rats failed to show such injury-related changes. These differential responses to LFPI helped to elucidate the molecular characteristics that influence poor recovery after TBI in those with pre-existing depression and may lead to targets for future therapeutic interventions.     

Neuroprotective Effects of Ischemic Preconditioning and Postconditioning on Global Brain Ischemia in Rats through the Same Effect on Inhibition of Apoptosis

Transient forebrain or global ischemia induces neuronal death in vulnerable CA1 pyramidal cells with many features. A brief period of ischemia, i.e., ischemic preconditioning, or a modified reperfusion such as ischemic postconditioning, can afford robust protection of CA1 neurons against ischemic challenge. Therefore, we investigated the effect of ischemic preconditioning and postconditioning on neural cell apoptosis in rats. The result showed that both ischemic preconditioning and postconditioning may attenuate the neural cell death and DNA fragment in the hippocampal CA1 region. Further western blot study suggested that ischemic preconditioning and postconditioning down-regulates the protein of cleaved caspase-3, caspase-6, caspase-9 and Bax, but up-regulates the protein Bcl-2. These findings suggest that ischemic preconditioning and postconditioning have a neuroprotective role on global brain ischemia in rats through the same effect on inhibition of apoptosis.     

白藜芦醇对大鼠脑缺血再灌注氧化应激损伤的影响

目的探讨白藜芦醇(Resveratrol,Res)对大鼠脑缺血再灌注氧化应激损伤的影响。方法将SD大鼠随机分为假手术组(S组)、缺血再灌注组(I/R组,线栓法复制大鼠右侧大脑中动脉栓塞模型)、Res低剂量组(15 mg/kg,I/R+R1组)和Res高剂量组(30 mg/kg,I/R+R2组),于缺血2 h再灌注24 h进行神经功能缺损评分;化学比色法测定大鼠血清和脑组织中丙二醛(Malondialdehyde,MDA)含量及超氧化物歧化酶(Superoxide dismutase,SOD)活性;TTC法测定脑梗死体积;干湿重法测定脑含水量,HE染色观察脑组织的病理改变。结果与I/R组相比,Res能改善大鼠脑缺血再灌注损伤后的神经功能缺失(P<0.01),降低血清及脑组织中MDA的含量(P<0.01),提高SOD活性(P<0.01),缩小脑梗死体积(P<0.01),降低损伤侧脑含水量(P<0.01),改善脑组织的病理变化,且呈剂量依赖性。结论 Res对大鼠局灶性脑缺血再灌注氧化应激损伤具有良好的保护作用,其机制可能与清除自由基,减轻氧化性损伤有关。     

蛋白质沉淀结晶研究进展

论述了国内外生物大分子 (蛋白质、酶等 )沉淀结晶的研究现状和进展 ,着重从结晶热力学、粒子聚集、结晶的成核与晶体生长 ,以及场的作用等方面阐述了蛋白质沉淀结晶过程的特定现象与可能的结晶机理 ,对蛋白质的沉淀结晶过程作了全面的描述 ,并提出未来研究方向 ,为蛋白质结构分析、新药设计、生化研究以及工业化生产提供一定的基础     

The Effect of TGF-β1 Reduced Functionality on the Expression of Selected Synaptic Proteins and Electrophysiological Parameters: Implications of Changes Observed in Acute Hepatic Encephalopathy

Decreased platelet count represents a feature of acute liver failure (ALF) pathogenesis. Platelets are the reservoir of transforming growth factor 1 (TGF-β1), a multipotent cytokine involved in the maintenance of, i.a., central nervous system homeostasis. Here, we analyzed the effect of a decrease in TGF-β1 active form on synaptic proteins levels, and brain electrophysiology, in mice after intraperitoneal (ip) administration of TGF-β1 antibody (anti-TGF-β1; 1 mg/mL). Next, we correlated it with a thrombocytopenia-induced TGF-β1 decrease, documented in an azoxymethane-induced (AOM; 100 mM ip) model of ALF, and clarified the impact of TGF-β1 decrease on blood–brain barrier functionality. The increase of both synaptophysin and synaptotagmin in the cytosolic fraction, and its reduction in a membrane fraction, were confirmed in the AOM mice brains. Both proteins’ decrease in analyzed fractions occurred in anti-TGF-β1 mice. In turn, an increase in postsynaptic (NR1 subunit of N-methyl-D-aspartate receptor, postsynaptic density protein 95, gephyrin) proteins in the AOM brain cortex, but a selective compensatory increase of NR1 subunit in anti-TGF-β mice, was observed. The alterations of synaptic proteins levels were not translated on electrophysiological parameters in the anti-TGF-β1 model. The results suggest the impairment of synaptic vesicles docking to the postsynaptic membrane in the AOM model. Nevertheless, changes in synaptic protein level in the anti-TGF-β1 mice do not affect neurotransmission and may not contribute to neurologic deficits in AOM mice.     

HS-01缓蚀剂的评价筛选

针对常减压蒸馏装置三顶冷凝系统的腐蚀特点，利用先进的腐蚀速率测定仪，在实验室条件下，评价筛选出缓蚀效果较好的HS-01缓蚀剂，在常减压装置应用后，获得了理想的工业应用效果。     

图像处理技术在阻垢剂性能评定方法的应用

针对目前阻垢剂性能评定方法的不足,鉴于图像处理技术在检测领域中越来越多的应用,首次提出基于图像处理技术的阻垢剂性能快速评定方法.本方法以CaCl2为母液,Na2CO3为滴定液,通过连续自动滴加至过饱和,直至晶核形成,最终形成沉淀.利用摄像机对这一过程进行连续采样,对图像进行处理得出溶液状态变化与图像特征值变化的对应关系,并找出图像特征曲线的突变点(拐点),以此评定阻垢剂的性能.试验得到了与标准方法一致的结论,且具有重复性好、评定快捷、操作简便等特点.     

双咪唑啉缓蚀剂抑制油田CO_2腐蚀的性能研究

以葵二酸、三乙烯四胺和氯化苄为原料,合成了双咪唑啉缓蚀剂。通过电化学阻抗法,研究了双咪唑啉缓蚀剂在饱和CO_2油田采出水介质中对J55油管钢的缓蚀性能,并对两口CO_2腐蚀严重的油井进行了现场加注试验。结果表明,双咪唑啉缓蚀剂在油田水介质中抑制CO_2腐蚀性能优异:加入缓蚀剂后可使电荷传递电阻大幅增加,在缓蚀剂浓度为150ml/L时,缓蚀率可达96.3%;两口试验井加注缓蚀剂后,腐蚀速率大幅下降,缓蚀率可达85%以上,现场使用效果优异。     

The Role of CCL2/CCR2 Axis in Cerebral Ischemia-Reperfusion Injury and Treatment: From Animal Experiments to Clinical Trials

C-C motif chemokine ligand 2 (CCL2) is a member of the monocyte chemokine protein family, which binds to its receptor CCR2 to induce monocyte infiltration and mediate inflammation. The CCL2/CCR2 signaling pathway participates in the transduction of neuroinflammatory information between all types of cells in the central nervous system. Animal studies and clinical trials have shown that CCL2/CCR2 mediate the pathological process of ischemic stroke, and a higher CCL2 level in serum is associated with a higher risk of any form of stroke. In the acute phase of cerebral ischemia-reperfusion, the expression of CCL2/CCR2 is increased in the ischemic penumbra, which promotes neuroinflammation and enhances brain injury. In the later phase, it participates in the migration of neuroblasts to the ischemic area and promotes the recovery of neurological function. CCL2/CCR2 gene knockout or activity inhibition can reduce the nerve inflammation and brain injury induced by cerebral ischemia-reperfusion, suggesting that the development of drugs regulating the activity of the CCL2/CCR2 signaling pathway could be used to prevent and treat the cell injury in the acute phase and promote the recovery of neurological function in the chronic phase in ischemic stroke patients.