The chemokine receptor CCR5 is not a necessary inflammatory mediator in kainic acid-induced hippocampal injury: evidence for a compensatory effect by increased CCR2 and CCR3

Chemokines and their receptors have been strongly implicated in the inflammatory process. However, their roles in excitotoxic brain injury are largely unknown. In this study we used C-C chemokine receptor 5 (CCR5) knockout (KO) mice to investigate the role of CCR5 in neurodegeneration induced by intranasal administration of the excitotoxin kainic acid (KA). Although KA treatment resulted in an increased CCR5 mRNA level in the hippocampi of wild-type mice, a CCR5 deficiency in KO mice did not affect either the clinical and pathological changes in vivo or the neuronal susceptibilities to KA insult in vitro. KA treatment stimulated mRNA expression of the monocyte chemoattractant protein-2 (MCP-2) in both the wild-type and KO mice. KA treatment did not affect mRNA levels for the macrophage inflammatory protein-1alpha (MIP-1alpha) or the regulated upon activation normal T cells expressed and secreted protein (RANTES) in either wild-type or CCR5 KO mice. CCR2 mRNA expression was undetectable in the hippocampi of wild-type mice regardless of KA treatment. In contrast, CCR5 KO mice showed CCR2 mRNA expression that was remarkably increased after KA treatment. KA treatment did not affect CCR3 mRNA expression in the wild-type mice, whereas KO mice showed both a higher basal level of CCR3 mRNA expression as well as a strong upregulation following KA treatment. These results indicate that CCR5 is not a necessary inflammatory mediator in KA induced neurodegeneration. The roles of CCR5 in excitotoxic injury in CCR5 deficient mice are compensated by increased CCR2 and CCR3 expression, which share the common MCP-2 ligand with CCR5.     

Endogenous reductions in N-methyl-d-aspartate receptor activity inhibit nitric oxide production in the anoxic freshwater turtle cortex

Increased nitric oxide (NO) production from hypoxic mammalian neurons increases cerebral blood flow (CBF) but also glutamatergic excitotoxicity and DNA fragmentation. Anoxia-tolerant freshwater turtles have evolved NO-independent mechanisms to increase CBF; however, the mechanism(s) of NO regulation are not understood. In turtle cortex, anoxia or NMDAR blockade depressed NO production by 27+/-3% and 41+/-5%, respectively. NMDAR antagonists also reduced the subsequent anoxic decrease in NO by 74+/-6%, suggesting the majority of the anoxic decrease is due to endogenous suppression of NMDAR activity. Prevention of NO-mediated damage during the transition to and from anoxia may be incidental to natural reductions of NMDAR activity in the anoxic turtle cortex.     

Reduction of excitotoxicity and associated leukocyte recruitment by a broad-spectrum matrix metalloproteinase inhibitor

An important step in the cascade leading to neuronal cell death is degradation of laminin and other components of the brain extracellular matrix by microglia-derived proteases. Excitotoxic cell death of murine hippocampal neurones in vivo can be prevented by inhibitors of tissue plasminogen activator (tPA) or by inhibitors of plasmin. Plasmin is a potent activator of the matrix metalloproteinases (MMPs), which are made by resident and recruited leukocytes following CNS injury. In this study, we show, using Taqman RT-PCR, that MMP mRNAs, but not other calcium-dependent proteases such as calpain mRNAs, are acutely up-regulated after an excitotoxic challenge in vivo. alpha(2)-antiplasmin or BB-3103, a broad-spectrum inhibitor of the MMPs, co-injected with kainic acid into the striatum, inhibits excitotoxic cell death in the rat striatum, and reduces both the number of recruited macrophages and the size of the lesion. We also show that leukocyte populations differentially express MMPs, which may account, in part, for the expression profile we observe in the challenged brain. Our results show that inhibition of the MMPs in the rat will prevent kainic acid-induced cell death in the brain. These studies suggest that MMP inhibitors have therapeutic potential for use in stroke, and support the increasing evidence that microglial activation may contribute to neuronal cell death.     

Age- and Sex-Dependent Laterality of Rat Hippocampal Cholinergic System in Relation to Animal Models of Neurodevelopmental and Neurodegenerative Disorders

Studies suggest age- and sex-dependent structural and functional patterns of human cerebral lateralization underlie hemisphere specialization and its alterations in schizophrenia. Recent works report sexual dimorphism of neurons in the hippocampal formation and specialization of hemispheres in rats. Our experiments indicate for the first time functional lateralization of the high-affinity choline uptake (HACU) system directly associated with a synthesis of acetylcholine in the hippocampus of Wistar rats. The markedly increased HACU activity was found in the left compared to the right hippocampus of adult male but not female animals. Lineweaver-Burk plot analysis revealed a statistically significant increase of Vmax in the left hippocampus of 14-day-old when compared to 7-day-old males. It appears that laterality of HACU occurs during late postnatal maturation, and its degree is markedly enhanced after puberty and attenuated during aging. Quinolinic acid (QUIN), an endogenous agonist of N-methyl-D-aspartate type glutamate receptors, was used in this study to evaluate the neurodevelopmental hypothesis of schizophrenia. It is known that elevated levels of QUIN accompany viral infections, increasing the risk of developing schizophrenia. Bilateral intracerebroventricular application of QUIN (250 nmoles/ventricle) to pups aged 12 days significantly impaired the cholinergic hippocampal system of adolescent male and female rats and reversed lateralization of male HACU. Morphological analysis indicated marked changes in brain lesion sizes (extensive 24 h and moderate 38 days after the operation). Asymmetry of lesions was observed in the majority of cases, but the left hemisphere was not generally more vulnerable to QUIN effects than the right side. Moreover, no lateral differences were found between lesioned hippocampi in the specific binding of [3H]hemicholinium-3 (10%-15% loss of binding sites when compared to sham-operated animals). In summary, our results indicate a symmetrical drop in the number of choline carriers of lesioned male rats but a asymmetrical decrease in the activity of remaing carriers, suggesting defects in processes of sexual brain differentiation, leading under normal conditions to the higher activity of carriers in the left hippocampus. The data demonstrate viral infection-mediated alterations in normal patterns of brain asymmetry and are discussed in relation to animal models of neurodevelopmental and neurodegenerative diseases.     

The Analysis of Neurovascular Remodeling in Entorhino-hippocampal Organotypic Slice Cultures

Ischemic brain injury is among the most common and devastating conditions compromising proper brain function and often leads to persisting functional deficits in the affected patients. Despite intensive research efforts, there is still no effective treatment option available that reduces neuronal injury and protects neurons in the ischemic areas from delayed secondary death. Research in this area typically involves the use of elaborate and problematic animal models. Entorhino-hippocampal organotypic slice cultures challenged with oxygen and glucose deprivation (OGD) are established in vitro models which mimic cerebral ischemia. The novel aspect of this study is that changes of the brain blood vessels are studied in addition to neuronal changes and the reaction of both the neuronal compartment and the vascular compartment can be compared and correlated. The methods presented in this protocol substantially broaden the potential applications of the organotypic slice culture approach. The induction of OGD or hypoxia alone can be applied by rather simple means in organotypic slice cultures and leads to reliable and reproducible damage in the neural tissue. This is in stark contrast to the complicated and problematic animal experiments inducing stroke and ischemia in vivo. By broadening the analysis to include the study of the reaction of the vasculature could provide new ways on how to preserve and restore brain functions. The slice culture approach presented here might develop into an attractive and important tool for the study of ischemic brain injury and might be useful for testing potential therapeutic measures aimed at neuroprotection.     

Stereotaxic Surgery for Excitotoxic Lesion of Specific Brain Areas in the Adult Rat

Many behavioral functions in mammals, including rodents and humans, are mediated principally by discrete brain regions. A common method for discerning the function of various brain regions for behavior or other experimental outcomes is to implement a localized ablation of function. In humans, patient populations with localized brain lesions are often studied for deficits, in hopes of revealing the underlying function of the damaged area. In rodents, one can experimentally induce lesions of specific brain regions.Lesion can be accomplished in several ways. Electrolytic lesions can cause localized damage but will damage a variety of cell types as well as traversing fibers from other brain regions that happen to be near the lesion site. Inducible genetic techniques using cell-type specific promoters may also enable site-specific targeting. These techniques are complex and not always practical depending on the target brain area. Excitotoxic lesion using stereotaxic surgery, by contrast, is one of the most reliable and practical methods of lesioning excitatory neurons without damaging local glial cells or traversing fibers.Here, we present a protocol for stereotaxic infusion of the excitotoxin, N-methyl-D-aspartate (NMDA), into the basolateral amygdala complex. Using anatomical indications, we apply stereotaxic coordinates to determine the location of our target brain region and lower an injection needle in place just above the target. We then infuse our excitotoxin into the brain, resulting in excitotoxic death of nearby neurons. While our experimental subject of choice is a rat, the same methods can be applied to other mammals, with the appropriate adjustments in equipment and coordinates.This method can be used on a variety of brain regions, including the basolateral amygdala^1-6, other amygdala nuclei^{6, 7}, hippocampus⁸, entorhinal cortex⁹ and prefrontal cortex¹⁰. It can also be used to infuse biological compounds such as viral vectors^{1, 11}. The basic stereotaxic technique could also be adapted for implantation of more permanent osmotic pumps, allowing more prolonged exposure to a compound of interest.     

小鼠脑室内注射神经干细胞裂解液促进谷氨酸盐诱导的兴奋性神经元损伤的修复

先前的研究已经证实,阿魏酸钠诱导分化的PC12细胞裂解液的无细胞滤液具有改善抑郁症样模型大鼠的行为学障碍、上调其海马和大脑皮质神经生长因子(nerve growth factor,NGF)和脑源性神经营养因子(brain-derived neurotrophic factor,BDNF)的表达、增加海马神经干细胞(neural stem cells,NSCs)/神经前体细胞(neural progenitor cells)增殖的效果。该研究的目的在于探讨神经干细胞裂解液的无细胞滤液(cell-free filtrate ofneural stem cell lysates,FNSCL)脑室内注射促进谷氨酸盐诱导的成年小鼠兴奋性神经元损伤修复的可能性。成年小鼠谷氨酸单钠(monosodiumglutamate,MSG,2.0g/(kg·d))灌胃,连续10日,造成兴奋性神经元损伤模型。自孕15 d的昆明种小鼠取胎脑,分离、培养神经干细胞,免疫细胞化学法检测巢蛋白(nestin)抗原,制备神经干细胞裂解液的无细胞滤液。MSG+NSCs组动物在MSG灌胃后接收脑室内NSCs移植,MSG+FNSCL组动物在MSG灌胃...     

Autoradiographic localization of γ-aminobutyric acidA receptors within the ventral tegmental area

Destruction of intrinsic neurons in the ventral tegmental area (VTA) with the excitotoxin, quinolinic acid produced a significant decrease (80%) in [³H]muscimol binding to GABA_A receptors within the parabrachial pigmented and paranigral nuclei of the VTA. Selective destruction of the dopaminergic neurons with 6-hydroxydopamine (6-OHDA) did not reduce [³H]muscimol binding within the VTA. However, the destruction of dopaminergic neurons did produce an increase (20%) in [³H]muscimol binding contralateral to the lesion, suggesting a reduction in the GABAergic innervation to this region. Additionally, destruction of the VTA afferents with quinolinic acid injections in the medial accumbens failed to produce alterations in [³H]muscimol binding within the VTA. These results are consistent with the predominant localization of GABA_A receptors to non-dopaminergic neurons intrinsic to the VTA.Special issue dedicated to Dr. Frederick E. Samson