In vitro model of the blood-brain barrier established by co-culture of primary cerebral microvascular endothelial and astrocyte cells

Drugs for the treatment and prevention of nervous system diseases must permeate the bloodbrain barrier to take effect.In vitro models of the blood-brain barrier are therefore important in the investigation of drug permeation mechanisms.However,to date,no unified method has been described for establishing a blood-brain barrier model.Here,we modified an in vitro model of the blood-brain barrier by seeding brain microvascular endothelial cells and astrocytes from newborn rats on a polyester Transwell cell culture membrane with 0.4-μm pores,and conducted transepithelial electrical resistance measurements,leakage tests and assays for specific bloodbrain barrier enzymes.We show that the permeability of our model is as low as that of the bloodbrain barrier in vivo.Our model will be a valuable tool in the study of the mechanisms of action of neuroprotective drugs.     

Activation of the Notch signaling pathway promotes neurovascular repair after traumatic brain injury

The Notch signaling pathway plays a key role in angiogenesis and endothelial cell formation, but it remains unclear whether it is involved in vascular repair by endothelial progenitor cells after traumatic brain injury. Therefore, in the present study, we controlled the Notch signaling pathway using overexpression and knockdown constructs. Activation of the Notch signaling pathway by Notch1 or Jagged1 overexpression enhanced the migration, invasiveness and angiogenic ability of endothelial progenitor cells. Suppression of the Notch signaling pathway with Notch1 or Jagged1 si RNAs reduced the migratory capacity, invasiveness and angiogenic ability of endothelial progenitor cells. Activation of the Notch signaling pathway in vivo in a rat model of mild traumatic brain injury promoted neurovascular repair. These findings suggest that the activation of the Notch signaling pathway promotes blood vessel formation and tissue repair after brain trauma.     

Membrane transporter proteins: a challenge for CNS drug development

Drug transporters are membrane proteins present in various tissues such as the lymphocytes, intestine, liver, kidney, testis, placenta, and central nervous system. These transporters play a significant role in drug absorption and distribution to organic systems, particularly if the organs are protected by blood-organ barriers, such as the blood-brain barrier or the maternal-fetal barrier. In contrast to neurotransmitters and receptor-coupled transporters or other modes of interneuronal transmission, drug transporters are not directly involved in specific neuronal functions, but provide global protection to the central nervous system. The lack of capillary fenestration, the low pinocytic activity and the tight junctions between brain capillary and choroid plexus endothelial cells represent further gatekeepers limiting the entrance of endogenous and exogenous compounds into the central nervous system. Drug transport is a result of the concerted action of efflux and influx pumps (transporters) located both in the basolateral and apical membranes of brain capillary and choroid plexus endothelial cells. By regulating efflux and influx of endogenous or exogenous substances, the blood-brain barrier and, to a lesser extent the blood-cerebrospinal barrier in the ventricles, represents the main interface between the central nervous system and the blood, i.e., the rest of the body. As drug distribution to organs is dependent on the affinity of a substrate for a specific transport system, membrane transporter proteins are increasingly recognized as a key determinant of drug disposition. Many drug transporters are members of the adenosine triphosphate (ATP)-binding cassette (ABC) transporter superfamily or the solute-linked carrier (SLC) class. The multidrug resistance protein MDR1 (ABCB1), also called P-glycoprotein, the multidrug resistance-associated proteins MRP1 (ABCC1) and MRP2 (ABCC2), and the breast cancer-resistance protein BCRP (ABCG2) are ATP-dependent efflux transporters expressed in the blood-brain barrier They belong to the superfamily of ABC transporters, which export drugs from the intracellular to the extracellular milieu. Members of the SLC class of solute carriers include, for example, organic ion transporting peptides, organic cation transporters, and organic ion transporters. They are ATP-independent polypeptides principally expressed at the basolateral membrane of brain capillary and choroid plexus endothelial cells that also mediate drug transport through central nervous system barriers.     

Axon regeneration impediment: the role of paired immunoglobulin-like receptor B

Regenerative capacity is weak after central nervous system injury because of the absence of an enhancing microenvironment and presence of an inhibitory microenvironment for neuronal and axonal repair. In addition to the Nogo receptor(Ng R), the paired immunoglobulin-like receptor B(Pir B) is a recently discovered coreceptor of Nogo, myelin-associated glycoprotein, and myelin oligodendrocyte glycoprotein. Concurrent blocking of Ng R and Pir B almost completely eliminates the inhibitory effect of myelin-associated inhibitory molecules on axonal regeneration. Pir B participates in a key pathological process of the nervous system, specifically axonal regeneration inhibition. Pir B is an inhibitory receptor similar to Ng R, but their effects are not identical. This study summarizes the structure, distribution, relationship with common nervous system diseases, and known mechanisms of Pir B, and concludes that Pir B is also distributed in cells of the immune and hematopoietic systems. Further investigations are needed to determine if immunomodulation and blood cell migration involve inhibition of axonal regeneration.     

PAR-1激活剂对体外培养的脑血管内皮细胞的影响

目的探讨蛋白酶激活受体-1(protease activated receptor-1,PAR-1)激活剂SFLLRN对体外培养的脑血管内皮细胞的影响,明确凝血酶增加血脑屏障通透性的可能机制。方法将脑微血管内皮细胞在体外进行培养,在细胞的培养液中加入SFLLRN或凝血酶,应用相差显微镜动态观察SFLLRN或TM对内皮细胞形态的影响,应用免疫组织细胞化学技术检测基质金属蛋白酶2(matrix metalloproteinase-2,MMP-2)表达的变化。结果 SFLLRN加入后1h内皮细胞开始收缩,细胞面积变小,细胞间隙增宽,细胞收缩程度具有时间依赖性,至12h时细胞面积仅为处理前的20%。凝血酶加入培养液后,细胞形态学的变化方式与SFLL-TRN组相似。SFLLTRN孵育后6h,内皮细胞MMP-2的表达水平开始增加,24小时达高峰,与对照组比较,存在明显统计学差异(P0.01),与TM组比较,无明显统计学差异(P0.05)。结论凝血酶通过激活PAR-1,使内皮细胞发生收缩,促进MMP-2表达,是凝血酶增加BBB通透性的可能机制。     

Acupuncture inhibits Notch1 and Hes1 protein expression in the basal ganglia of rats with cerebral hemorrhage

Notch pathway activation maintains neural stem cells in a proliferating state and increases nerve repair capacity. To date, studies have rarely focused on changes or damage to signal transduction pathways during cerebral hemorrhage. Here, we examined the effect of acupuncture in a rat model of cerebral hemorrhage. We examined four groups: in the control group, rats received no treatment. In the model group, cerebral hemorrhage models were established by infusing non-heparinized blood into the brain. In the acupuncture group, modeled rats had Baihui(DU20) and Qubin(GB7) acupoints treated once a day for 30 minutes. In the DAPT group, modeled rats had 0.15 μg/m L DAPT solution(10 m L) infused into the brain. Immunohistochemistry and western blot results showed that acupuncture effectively inhibits Notch1 and Hes1 protein expression in rat basal ganglia. These inhibitory effects were identical to DAPT, a Notch signaling pathway inhibitor. Our results suggest that acupuncture has a neuroprotective effect on cerebral hemorrhage by inhibiting Notch-Hes signaling pathway transduction in rat basal ganglia after cerebral hemorrhage.     

Restoring nervous system structure and function using tissue engineered living scaffolds

Neural tissue engineering is premised on the integration of engineered living tissue with the host nervous system to directly restore lost function or to augment regenerative capacity following nervous system injury or neurodegenerative disease. Disconnection of axon pathways – the long-distance fibers connecting specialized regions of the central nervous system or relaying peripheral signals – is a common feature of many neurological disorders and injury. However, functional axonal regeneration rarely occurs due to extreme distances to targets, absence of directed guidance, and the presence of inhibitory factors in the central nervous system, resulting in devastating effects on cognitive and sensorimotor function. To address this need, we are pursuing multiple strategies using tissue engineered “living scaffolds”, which are preformed three-dimensional constructs consisting of living neural cells in a defined, often anisotropic architecture. Living scaffolds are designed to restore function by serving as a living labeled pathway for targeted axonal regeneration – mimicking key developmental mechanisms– or by restoring lost neural circuitry via direct replacement of neurons and axonal tracts. We are currently utilizing preformed living scaffolds consisting of neuronal clusters spanned by long axonal tracts as regenerative bridges to facilitate long-distance axonal regeneration and for targeted neurosurgical reconstruction of local circuits in the brain. Although there are formidable challenges in preclinical and clinical advancement, these living tissue engineered constructs represent a promising strategy to facilitate nervous system repair and functional recovery.     

Activation of immediate-early response gene c-Fos protein in the rat paralimbic cortices after myocardial infarction

c-Fos is a good biological marker for detecting the pathogenesis of central nervous system disorders. Few studies are reported on the change in myocardial infarction-induced c-Fos expression in the paralimbic regions. Thus, in this study, we investigated the changes in c-Fos expression in the rat cingulate and piriform cortices after myocardial infarction. Neuronal degeneration in cingulate and piriform cortices after myocardial infarction was detected using cresyl violet staining, Neu N immunohistochemistry and Fluoro-Jade B histofluorescence staining. c-Fos-immunoreactive cells were observed in cingulate and piriform cortices at 3 days after myocardial infarction and peaked at 7 and 14 days after myocardial infarction. But they were hardly observed at 56 days after myocardial infarction. The chronological change of c-Fos expression determined by western blot analysis was basically the same as that of c-Fos immunoreactivity. These results indicate that myocardial infarction can cause the chronological change of immediate-early response gene c-Fos protein expression, which might be associated with the neural activity induced by myocardial infarction.     

Inflammation and cutaneous nervous system involvement in hypertrophic scarring

This study aimed to use a mouse model of hypertrophic scarring by mechanical loading on the dorsum of mice to determine whether the nervous system of the skin and inflammation participates in hypertrophic scarring. Results of hematoxylin-eosin and immunohistochemical staining demonstrated that inflammation contributed to the formation of a hypertrophic scar and increased the nerve density in scar tissue.Western blot assay verified that interleukin-13 expression was increased in scar tissue. These findings suggest that inflammation and the cutaneous nervous system play a role in hypertrophic scar formation.     

Effects of total saponins of Panax notoginseng on immature neuroblasts in the adult olfactory bulb following global cerebral ischemia/reperfusion

The main active components extracted from Panax notoginseng are total saponins. They have been shown to inhibit platelet aggregation, increase cerebral blood flow, improve neurological behavior, decrease infarct volume and promote proliferation and differentiation of neural stem cells in the hippocampus and lateral ventricles. However, there is a lack of studies on whether total saponins of Panax notoginseng have potential benefits on immature neuroblasts in the olfactory bulb following ischemia and reperfusion. This study established a rat model of global cerebral ischemia and reperfusion using four-vessel occlusion. Rats were administered total saponins of Panax notoginseng at 75 mg/kg intraperitoneally 30 minutes after ischemia then once a day, for either 7 or 14 days. Total saponins of Panax notoginseng enhanced the number of doublecortin(DCX)+ neural progenitor cells and increased co-localization of DCX with neuronal nuclei and phosphorylated c AMP response element-binding/DCX+ neural progenitor cells in the olfactory bulb at 7 and 14 days post ischemia. These findings indicate that following global brain ischemia/reperfusion, total saponins of Panax notoginseng promote differentiation of DCX+ cells expressing immature neuroblasts in the olfactory bulb and the underlying mechanism is related to the activation of the signaling pathway of cyclic adenosine monophosphate response element binding protein.     

Structural plasticity of the adult brain: how animal models help us understand brain changes in depression and systemic disorders related to depression

The brain interprets experiences and translates them into behavioral and physiological responses. Stressful events are those which are threatening or, at the very least, unexpected and surprising, and the physiological and behavioral responses are intended to promote adaptation via a process called "allostasis. " Chemical mediators of allostasis include cortisol and adrenalin from the adrenal glands, other hormones, and neurotransmitters, the parasympathetic and sympathetic nervous systems, and cytokines and chemokines from the immune system. Two brain structures, the amygdala and hippocampus, play key roles in interpreting what is stressful and determining appropriate responses. The hippocampus, a key structure for memories of events and contexts, expresses receptors that enable it to respond to glucocorticoid hormones in the blood, it undergoes atrophy in a number of psychiatric disorders; it also responds to stressors with changes in excitability, decreased dendritic branching, and reduction in number of neurons in the dentate gyrus. The amygdala, which is important for "emotional memories, " becomes hyperactive in posttraumatic stress disorder and depressive illness, in animal models of stress, there is evidence for growth and hypertrophy of nerve cells in the amygdala. Changes in the brain after acute and chronic stressors mirror the pattern seen in the metabolic, cardiovascular, and immune systems, that is, short-term adaptation (allostasis) followed by long-term damage (allostatic load), eg, atherosclerosis, fat deposition obesity, bone demineralization, and impaired immune function. Allostatic load of this kind is seen in major depressive illness and may also be expressed in other chronic anxiety and mood disorders.     

Neuroplasticity in addictive disorders

Compulsive drug-taking behavior develops in vulnerable individuals who ingest substances that activate the reward system. This intense activation produces learned associations to cues that predict drug availability. With repetition the reward system becomes reflexively activated by cues alone, leading to a drive toward drug-taking. The central nervous system changes underlying this conditioned behavior are just beginning to be understood. New treatments aimed at this neuroplasticity are being tested in animal models. The clinical significance of these brain changes is that addiction, once established, becomes a chronic illness with relapses and remissions, it therefore requires chronic treatment with medications and behavioral therapies based on an understanding of the fundamental nature of these changes in the brain.     

Ethanol extract of Oenanthe javanica increases cell proliferation and neuroblast differentiation in the adolescent rat dentate gyrus

Oenanthe javanica is an aquatic perennial herb that belongs to the Oenanthe genus in Apiaceae family, and it displays well-known medicinal properties such as protective effects against glutamate-induced neurotoxicity. However, few studies regarding effects of Oenanthe javanica on neurogenesis in the brain have been reported. In this study, we examined the effects of a normal diet and a diet containing ethanol extract of Oenanthe javanica on cell proliferation and neuroblast differentiation in the subgranular zone of the hippocampal dentate gyrus of adolescent rats using Ki-67(an endogenous marker for cell proliferation) and doublecortin(a marker for neuroblast). Our results showed that Oenanthe javanica extract significantly increased the number of Ki-67-immunoreactive cells and doublecortin-immunoreactive neuroblasts in the subgranular zone of the dentate gyrus in the adolescent rats. In addition, the immunoreactivity of brain-derived neurotrophic factor was significantly increased in the dentate gyrus of the Oenanthe javanica extract-treated group compared with the control group. However, we did not find that vascular endothelial growth factor expression was increased in the Oenanthe javanica extract-treated group compared with the control group. These results indicate that Oenanthe javanica extract improves cell proliferation and neuroblast differentiation by increasing brain-derived neurotrophic factor immunoreactivity in the rat dentate gyrus.     

Connexin: a potential novel target for protecting the central nervous system?

Connexin subunits are proteins that form gap junction channels, and play an important role in communication between adjacent cells. This review article discusses the function of connexins/hemichannels/gap junctions under physiological conditions, and summarizes the findings regarding the role of connexins/hemichannels/gap junctions in the physiological and pathological mechanisms underlying central nervous system diseases such as brain ischemia, traumatic brain and spinal cord injury, epilepsy, brain and spinal cord tumor, migraine, neuroautoimmune disease, Alzheimer’s disease, Parkinson’s disease, X-linked Charcot-Marie-Tooth disease, Pelizaeus-Merzbacher-like disease, spastic paraplegia and maxillofacial dysplasia. Connexins are considered to be a potential novel target for protecting the central nervous system.     

Chitosan-collagen porous scaffold and bone marrow mesenchymal stem cell transplantation for ischemic stroke

In this study, we successfully constructed a composite of bone marrow mesenchymal stem cells and a chitosan-collagen scaffold in vitro, transplanted either the composite or bone marrow mesenchymal stem cells alone into the ischemic area in animal models, and compared their effects. At 14 days after co-transplantation of bone marrow mesenchymal stem cells and the hitosan-collagen scaffold, neurological function recovered noticeably. Vascular endothelial growth factor expression and nestin-labeled neural precursor cells were detected in the ischemic area, surrounding tissue, hippocampal dentate gyrus and subventricular zone. Simultaneously, a high level of expression of glial fibrillary acidic protein and a low level of expression of neuron-specific enolase were visible in Brd U-labeled bone marrow mesenchymal stem cells. These findings suggest that transplantation of a composite of bone marrow mesenchymal stem cells and a chitosan-collagen scaffold has a neuroprotective effect following ischemic stroke.     

The role of the Rho/ROCK signaling pathway in inhibiting axonal regeneration in the central nervous system

The Rho/Rho-associated coiled-coil containing protein kinase (Rho/ROCK) pathway is a major signaling pathway in the central nervous system, transducing inhibitory signals to block regeneration. After central nervous system damage, the main cause of impaired regeneration is the presence of factors that strongly inhibit regeneration in the surrounding microenvironment. These factors signal through the Rho/ROCK signaling pathway to inhibit regeneration. Therefore, a thorough understanding of the Rho/ROCK signaling pathway is crucial for advancing studies on regeneration and repair of the injured central nervous system.     

Brain activation and inhibition after acupuncture at Taichong and Taixi: resting-state functional magnetic resonance imaging

Acupuncture can induce changes in the brain. However, the majority of studies to date have focused on a single acupoint at a time. In the present study, we observed activity changes in the brains of healthy volunteers before and after acupuncture at Taichong(LR3) and Taixi(KI3) using resting-state functional magnetic resonance imaging. Fifteen healthy volunteers underwent resting-state functional magnetic resonance imaging of the brain 15 minutes before acupuncture, then received acupuncture at Taichong and Taixi using the nail-pressing needle insertion method, after which the needle was retained in place for 30 minutes. Fifteen minutes after withdrawal of the needle, the volunteers underwent a further session of resting-state functional magnetic resonance imaging, which revealed that the amplitude of low-frequency fluctuation, a measure of spontaneous neuronal activity, increased mainly in the cerebral occipital lobe and middle occipital gyrus(Brodmann area 18/19), inferior occipital gyrus(Brodmann area 18) and cuneus(Brodmann area 18), but decreased mainly in the gyrus rectus of the frontal lobe(Brodmann area 11), inferior frontal gyrus(Brodmann area 44) and the center of the posterior lobe of the cerebellum. The present findings indicate that acupuncture at Taichong and Taixi specifically promote blood flow and activation in the brain areas related to vision, emotion and cognition, and inhibit brain areas related to emotion, attention, phonological and semantic processing, and memory.     

Structural and functional connectivity in traumatic brain injury

Traumatic brain injury survivors often experience cognitive deficits and neuropsychiatric symptoms.However,the neurobiological mechanisms underlying specific impairments are not fully understood.Advances in neuroimaging techniques(such as diffusion tensor imaging and functional MRI)have given us new insights on structural and functional connectivity patterns of the human brain in both health and disease.The connectome derived from connectivity maps reflects the entire constellation of distributed brain networks.Using these powerful neuroimaging approaches,changes at the microstructural level can be detected through regional and global properties of neuronal networks.Here we will review recent developments in the study of brain network abnormalities in traumatic brain injury,mainly focusing on structural and functional connectivity.Some connectomic studies have provided interesting insights into the neurological dysfunction that occurs following traumatic brain injury.These techniques could eventually be helpful in developing imaging biomarkers of cognitive and neurobehavioral sequelae,as well as predicting outcome and prognosis.     

Nogo-A expression dynamically varies after spinal cord injury

The mechanism involved in neural regeneration after spinal cord injury is unclear. The myelin-derived protein Nogo-A, which is specific to the central nervous system, has been identified to negatively affect the cytoskeleton and growth program of axotomized neurons. Studies have shown that Nogo-A exerts immediate and chronic inhibitory effects on neurite outgrowth. In vivo, inhibitors of Nogo-A have been shown to lead to a marked enhancement of regenerative axon extension. We established a spinal cord injury model in rats using a free-falling weight drop device to subsequently investigate Nogo-A expression. Nogo-A mR NA and protein expression and immunoreactivity were detected in spinal cord tissue using real-time quantitative PCR, immunohistochemistry and western blot analysis. At 24 hours after spinal cord injury, Nogo-A protein and mR NA expression was low in the injured group compared with control and sham-operated groups. The levels then continued to drop further and were at their lowest at 3 days, rapidly rose to a peak after 7 days, and then gradually declined again after 14 days. These changes were observed at both the mR NA and protein level. The transient decrease observed early after injury followed by high levels for a few days indicates Nogo-A expression is time dependent. This may contribute to the lack of regeneration in the central nervous system after spinal cord injury. The dynamic variation of Nogo-A should be taken into account in the treatment of spinal cord injury.     

Short-term use of antiepileptic drugs is neurotoxic to the immature brain

Previous studies have shown that the long-term use of antiepileptic drugs can cause nervous system damage. However, short-term antiepileptic drug treatment is frequently given to infants, especially neonates, to control seizure. Whether the short-term use of antiepileptic drugs is neurotoxic remains unclear. In the present study, immature rats, 3–21 days of age, were intraperitoneally injected with phenobarbital and/or topiramate for 3 consecutive days. Hematoxylin-eosin and immunohistochemical staining revealed that phenobarbital and topiramate, individually or in combination, were cytotoxic to hippocampal CA1 neurons and inhibited the expression of Glu R1 and NR2 B, excitatory glutamate receptor subunits. Furthermore, the combination of the two drugs caused greater damage than either drug alone. The results demonstrate that the short-term use of antiepileptic drugs damages neurons in the immature brain and that the combined use of antiepileptic drugs exacerbates damage. Our findings suggest that clinicians should consider the potential neurotoxic risk associated with the combined use of antiepileptic drugs in the treatment of seizure.