To identify suitable cell lines for a mimetic system of in vivo blood-brain barrier (BBB) for drug permeability assessment, we characterized two immortalized cell lines, ECV304 and bEnd3 in the respect of the tightness, tight junction proteins, P-glycoprotein (P-gp) function and discriminative brain penetration. The ECV304 monoculture achieved higher transendothelial electrical resistance (TEER) and lower permeability to Lucifer yellow than bEnd3. However, co-culture with rat glioma C6 cells impaired the integrity of ECV304 and bEnd3 cell layers perhaps due to the heterogeneity among C6 cells in inducing BBB characteristics. The immunostaining of ZO-1 delivered distinct bands along cell borders on both cell lines while those of occludin and claudin-5 were diffused and weak. P-gp functionality was only proved in bEnd3 by Rhodamine 123 (R123) uptake assay. A permeability test of reference compounds displayed a similar rank order (digoxin < R123 < quinidine, verapamil < propranolol) in ECV304 and bEnd3 cells. In comparison with bEnd3, ECV304 developed tighter barrier for the passage of reference compounds and higher discrimination between transcellular and paracellular transport. However, the monoculture models of ECV304 and bEnd3 fail to achieve the sufficient tightness of in vitro BBB permeability models with high TEER and evident immunostaining of tight junction proteins. Further strategies to enhance the paracellular tightness of both cell lines to mimic in vivo BBB tight barrier deserve to be conducted.
Apocynin (4-hydroxy-3-methoxyacetophenone) is a natural polyphenolic compound with multiple biological activities. In the present study, a series of apocynin derivatives were designed and synthesized. The in silico ADMET prediction, blood-brain barrier (BBB) penetration assay, anti-NADPH oxidase activity, reactive oxygen species (ROS) levels, and anti-glioma effects of these apocynin derivatives were evaluated. The anti-glioma mechanisms of candidate compounds were studied by flow cytometer and Western blot. The results showed that D31 exhibited higher BBB penetration, increased ROS generations and significant anti-glioma effects both in vitro and in vivo. Further studies showed that D31 inhibited the activations of NF-κB pathway. Overall, our data demonstrated that D31 inhibited growth and induced apoptosis of glioma, which might be caused by ROS-related NF-κB activation. The current study suggested that D31 could be further explored for its potential use in anti-glioma therapy.
In the process of screening cell-type-specific genes, we identified juxtanodin (JN), an oligodendroglial protein featuring a putative C-terminal actin-binding domain. At the cellular level, JN in the rat CNS colocalized with 2′,3′-cyclic nucleotide-3′-phosphodiesterase (CNPase), a cytoskeleton-related oligodendroglial protein. In the myelin sheath, JN was found mainly in the abaxon and the lateral few terminal loops. Its apposition to the myelinated axon, through the latter, defined an axonal subregion, herewith termed juxtanode, at the Ranvier node-paranode junction. During forebrain ontogenesis, JN expression paralleled that of MBPs but lagged behind CNPase. Juxtanodin transfection promoted arborization of cultured OLN-93 cells and augmented endogenous CNPase expression and transport to the process arbors of cultured primary oligodendrocyte precursors. These results reveal JN as a cytoskeleton-related oligodendroglial protein that delineates the juxtanode and might serve oligodendrocyte motility, differentiation, or myelin-axon signaling. Functionally, JN may be involved in CNS myelination and/or specialization of the node of Ranvier.
Abstract NADPH-diaphorase (N-d) activity is commonly used to identify NOS-ergic neurons. In our previous study, N-d positive neuritic dystrophy and spheroid termed aging-related N-d Body is discovered in the lumbosacral spinal cord in the normal aging rats. Histological studies also reveal that N-d positive neurodegenerative changes occur in the gracile nucleus. We re-examined N-d activity in gracile nucleus in aged rat. We found N-d positive neuritic dystrophy and spheroid also occurred in the cuneatus nucleus and spinal trigeminal nucleus. Besides regular coronal section, longitudinal oriented dystrophic neurites were detected in the sagittal and horizontal section in gracile nucleus and dorsal column. We fziurther examined the medullary oblongata with regular classical histology including Golgi staining, immunocytochemistry of NOS and phosphorylated tau protein, neuronal tracing method with wheat germ agglutinin conjugated alexa-fluor-488 through sciatic nerve, and spinal cord transection at thoracic level. Most of N-d positive neuritic dystrophy and spheroid did not showed colocalization with NOS or phosphorylated tau protein. Neuronal tracing and spinal cord transection revealed that N-d dystrophic neurites in gracile nucleus originated from terminal of sensory projection from spinal cord and peripheral somatic input. The results suggested that aging-related N-d dystrophy in the gracile nucleus was unique morphological feature. In conclusion, it was postulated that the N-d dystrophy as a morphological marker of aging degenerative damage in normal aged organisms.
The endogenous neurotransmitter, noradrenaline, exerts anti-inflammatory and neuroprotective effects in vivo and in vitro. Reduced noradrenaline levels results in increased inflammation and neuronal damage. The primary source of noradrenaline in the central nervous system is tyrosine hydroxylase (TH)‑positive neurons, located in the locus coeruleus (LC). TH is the rate‑limiting enzyme for noradrenaline synthesis; therefore, regulation of TH protein expression and intrinsic enzyme activity represents the central means for controlling the synthesis of noradrenaline. Catalpol is an iridoid glycoside purified from Rehmannia glutinosa Libosch, which exerts a neuroprotective effect in multiple sclerosis (MS). The present study used an experimental mouse model of autoimmune encephalomyelitis to verify the neuroprotective effects of catalpol. Significant improvements in the clinical scores were observed in catalpol‑treated mice. Furthermore, catalpol increased TH expression and increased noradrenaline levels in the spinal cord. In primary cultures, catalpol exerted a neuroprotective effect in rat LC neurons by increasing the noradrenaline output. These results suggested that drugs targeting LC survival and function, including catalpol, may be able to benefit patients with MS.
Abstract The aging-related changes of NADPH-diaphorase (NADPH-d) in the spinal cord were studied in aged dogs. At all levels of the spinal cord examined, NADPH-d activities were present in neurons and fibers in the superficial dorsal horn, dorsal commissure and in neurons around the central canal. In addition, the sympathetic autonomic nucleus in the thoracic and rostral lumbar segments exhibited prominent NADPH-d cellular staining whereas the sacral parasympathetic nucleus (SPN) in the sacral segments was not well stained. Interestingly, we found abundant NADPH-d positive enlarged-diameter fibers termed megaloneurite, which characteristically occurred in the aged sacral segments, distributed in the dorsal gray commissure (DGC), lateral collateral pathway (LCP) the lateral fasciculi and the central canal compared with the cervical, thoracic and lumbar segments. The dense, abnormal NADPH-d megaloneurites occurred in extending from dorsal entry zone through lamina I along with the lateral boundary of the dorsal horn to the region of the SPN. These fibers were prominent in the S1-S3 segments but not in adjacent segments L5-L7 and Cx1 or in thoracolumbar segments and cervical segments. Double staining with GFAP, NeuN, CGRP, MAP2 and Iba1, NADPH-d megaloneurite colocalized with vasoactive intestinal peptide. Presumably, the megaloneurites may represent, in part, visceral afferent projections to the SPN and/or DGC. The NADPH-d megaloneurites in the aged sacral spinal cord indicated some anomalous changes in the neurites, which might account for a disturbance in the aging pathway of the autonomic and sensory nerve in the pelvic visceral organs.
Ischemia-reperfusion (I/R) injury contributes to the morbidity and mortality of ischemic strokes. As an in vitro model, oxygen-glucose deprivation and reperfusion (OGD/R) exposure induces neuronal injury. Low-dose ethanol preconditioning (EtOH-PC) was reported to alleviate neuronal apoptosis during OGD/R. However, whether the mitochondrial BKCa (mitoBKCa) channel is involved in the neuroprotective effect of EtOH-PC during OGD/R is not clearly defined. This study attempts to explore the mediation of the mitoBKCa channel in the neuroprotective effect of EtOH-PC on OGD/R-induced neuronal apoptosis and the underlying mechanisms. OGD/R model was established using primary cortical neurons that were preincubated with ethanol. Subsequently, the cell viability was measured by CCK-8 assay, and the apoptotic cells were determined by TUNEL assay. Annexin V/7-AAD staining and mitochondrial membrane potential using JC-10 were detected by flow cytometry. Western blot analysis was performed to check the apoptosis-related proteins. In the mixed primary culture, 95% neurofilament-positive cells were cortical neurons. Low-dose EtOH-PC (10 mmol/L) for 24 h significantly attenuated the OGD2h/R24h-induced neuronal apoptosis through activating the BKCa channel. Further investigations suggested that ethanol pretreatment increased the mitochondrial membrane potential (MMP) and downregulated the production of cleaved caspase 3 in OGD/R-injured neurons by activating the mitoBKCa channel. Low-dose ethanol pretreatment significantly attenuated the OGD/R-induced neuronal apoptosis mediated by the mitoBKCa channel which modulated the mitochondrial function by impeding the uncontrolled opening of mitochondrial permeability transition pore (MPTP).
To study the clinical characteristics of cognitive impairment in patients with hemorrhagic stroke, Birmingham cognitive screen test, mini-mental state examination, Montreal cognitive assessment was used to evaluate the cognitive function of acute hemorrhagic stroke and the incidence and clinical characteristics of cognitive impairment was evaluated. Seventy acute hemorrhagic stroke patients were enrolled in the study and the abnormal rates of Birmingham cognitive screen test, mini-mental state examination and Montreal cognitive assessment were 81.42, 33.34 and 41.5 %, respectively. Birmingham cognitive screen test showed that patients with acute hypertensive intracerebral hemorrhage have cognitive impairment in many aspects, especially in language function, memory, attention, executive function, number and application ability and the damage rate was more than 30 %. Different degrees of cognitive impairment involving multiple cognitive domains could occur in the acute stage of hemorrhagic stroke, among which language ability, memory, execution and application ability were the most common. Compared to minimental state examination and Montreal cognitive assessment, Birmingham cognitive screen test has a higher detection rate of cognitive impairment after hypertensive hemorrhage and a more comprehensive evaluation of cognitive function.
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