Characteristics of _L -citrulline transport through blood-brain barrier in the brain capillary endothelial cell line (TR-BBB cells)

Lee, Kyeong-Eun,Kang, Young-Sook S. Karger Medical and Scientific Publishers 2017 JOURNAL OF BIOMEDICAL SCIENCE -BASEL- Vol.24 No.1

<P><B>Background</B></P><P><SUB>L</SUB>-Citrulline is a neutral amino acid and a major precursor of <SUB>L</SUB>-arginine in the nitric oxide (NO) cycle. Recently it has been reported that <SUB>L</SUB>-citrulline prevents neuronal cell death and protects cerebrovascular injury, therefore, <SUB>L</SUB>-citrulline may have a neuroprotective effect to improve cerebrovascular dysfunction. Therefore, we aimed to clarify the brain transport mechanism of <SUB>L</SUB>-citrulline through blood-brain barrier (BBB) using the conditionally immortalized rat brain capillary endothelial cell line (TR-BBB cells), as an in vitro model of the BBB.</P><P><B>Methods</B></P><P>The uptake study of [<SUP>14</SUP>C] L-citrulline, quantitative real-time polymerase chain reaction (PCR) analysis, and rLAT1, system b<SUP>0,+</SUP>, and CAT1 small interfering RNA study were performed in TR-BBB cells.</P><P><B>Results</B></P><P>The uptake of [<SUP>14</SUP>C] <SUB>L</SUB>-citrulline was a time-dependent, but ion-independent manner in TR-BBB cells. The transport process involved two saturable components with a Michaelis–Menten constant of 30.9 ± 1.0 μM (Km<SUB>1</SUB>) and 1.69 ± 0.43 mM (Km<SUB>2</SUB>). The uptake of [<SUP>14</SUP>C] <SUB>L</SUB>-citrulline in TR-BBB cells was significantly inhibited by neutral and cationic amino acids, but not by anionic amino acids. In addition, [<SUP>14</SUP>C]<SUB>L</SUB>-citrulline uptake in the cells was markedly inhibited by 2-aminobicyclo-(2,2,1)-heptane-2-carboxylic acid (BCH), which is the inhibitor of the large neutral amino acid transporter 1 (LAT1), B<SUP>0</SUP>, B<SUP>0,+</SUP> and harmaline, the inhibitor of system b<SUP>0,+</SUP>. Gabapentin and <SUB>L</SUB>-dopa as the substrates of LAT1 competitively inhibited the uptake of [<SUP>14</SUP>C] <SUB>L</SUB>-citrulline. IC<SUB>50</SUB> values for <SUB>L</SUB>-dopa, gabapentin, <SUB>L</SUB>-phenylalanine and <SUB>L</SUB>-arginine were 501 μM, 223 μM, 68.9 μM and 33.4 mM, respectively. The expression of mRNA for LAT1 was predominantly increased 187-fold in comparison with that of system b<SUP>0,+</SUP> in TR-BBB cells. In the studies of LAT1, system b<SUP>0,+</SUP> and CAT1 knockdown via siRNA transfection into TR-BBB cells, the transcript level of LAT1 and [<SUP>14</SUP>C] <SUB>L</SUB>-citrulline uptake by LAT1 siRNA were significantly reduced compared with those by control siRNA in TR-BBB cells.</P><P><B>Conclusions</B></P><P>Our results suggest that transport of <SUB>L</SUB>-citrulline is mainly mediated by LAT1 in TR-BBB cells. Delivery strategy for LAT1-mediated transport and supply of L-citrulline to the brain may serve as therapeutic approaches to improve its neuroprotective effect in patients with cerebrovascular disease.</P>

Pattern recognition analysis of dynamic susceptibility contrast (DSC)‐MRI curves automatically segments tissue areas with intact blood–brain barrier in a rat stroke model: A feasibility and comparison study

Jin, Seokha,Han, SoHyun,Stoyanova, Radka,Ackerstaff, Ellen,Cho, HyungJoon RADIOLOGICAL SOCIETY OF NORTH AMERICA INC 2020 JOURNAL OF MAGNETIC RESONANCE IMAGING Vol.51 No.5

<P><B>Background</B></P><P>The manual segmentation of intact blood–brain barrier (BBB) regions in the stroke brain is cumbersome, due to the coexistence of infarction, large blood vessels, ventricles, and intact BBB regions, specifically in areas with weak signal enhancement following contrast agent injection.</P><P><B>Hypothesis</B></P><P>That from dynamic susceptibility contrast (DSC)‐MRI alone, without user intervention, regions of weak BBB damage can be segmented based on the leakage‐related parameter <I>K</I><SUB><I>2</I></SUB> and the extent of intact BBB regions, needed to estimate <I>K</I><SUB><I>2</I></SUB> values, determined.</P><P><B>Study Type</B></P><P>Feasibility.</P><P><B>Animal Model</B></P><P>Ten female Sprague–Dawley rats (SD, 200–250g) underwent 1‐hour middle carotid artery occlusion (MCAO) and 1‐day reperfusion. Two SD rats underwent 1‐hour MCAO with 3‐day and 5‐day reperfusion.</P><P><B>Field Strength/Sequence</B></P><P>7T; ADC and T<SUB>1</SUB> maps using diffusion‐weighted echo planar imaging (EPI) and relaxation enhancement (RARE) with variable repetition time (TR), respectively. dynamic contrast‐enhanced (DCE)‐MRI using FLASH. DSC‐MRI using gradient‐echo EPI.</P><P><B>Assessment</B></P><P>Constrained nonnegative matrix factorization (cNMF) was applied to the dynamic ΔR2*‐curves of DSC‐MRI (<4 min) in a BBB‐disrupted rat model. Areas of voxels with intact BBB, classified by automated cNMF analyses, were then used in estimating <I>K</I><SUB><I>1</I></SUB> and <I>K</I><SUB><I>2</I></SUB> values, and compared with corresponding values from manually‐derived areas.</P><P><B>Statistical Tests</B></P><P>Mean ± standard deviation of ΔT<SUB>1</SUB>‐differences between ischemic and healthy areas were displayed with unpaired Student's <I>t</I>‐tests. Scatterplots were displayed with slopes and intercepts and Pearson's <I>r</I> values were evaluated between <I>K</I><SUB><I>2</I></SUB> maps obtained with automatic (cNMF)‐ and manually‐derived regions of interest (ROIs) of the intact BBB region.</P><P><B>Results</B></P><P>Mildly BBB‐damaged areas (indistinguishable from DCE‐MRI (10 min) parameters) were automatically segmented. Areas of voxels with intact BBB, classified by automated cNMF, matched closely the corresponding, manually‐derived areas when respective areas were used in estimating <I>K</I><SUB><I>2</I></SUB> maps (Pearson's <I>r</I> = 0.97, 12 slices).</P><P><B>Data Conclusion</B></P><P>Automatic segmentation of short DSC‐MRI data alone successfully identified areas with intact and compromised BBB in the stroke brain and compared favorably with manual segmentation.</P><P><B>Level of Evidence:</B> 3</P><P><B>Technical Efficacy:</B> Stage 1</P><P>J. Magn. Reson. Imaging 2020;51:1369–1381.</P>

치밀이음부 구조단백질인 Occludin에 대한 활성산소종의 영향

이희상,김대진,손동섭,정봉수,최형택,심규민,이금정,조혜진,김석중,이종찬,정윤희,김성수,이원복,Lee, Hee-Sang,Kim, Dae-Jin,Sohn, Dong-Suep,Jeong, Bong-Su,Choi, Hyung-Taek,Sim, Kyu-Min,Lee, Keum-Jeong,Cho, Hye-Jin,Kim, Suk-Joong,Lee, Jong-Chan,J 한국현미경학회 2004 Applied microscopy Vol.34 No.4

Cerebral microvessel endothelial cells that form blood-brain barrier (BBB) have tight junction for maintaining brain homeostasis. Occludin, one of tight junction protein, is crucial for BBB function. $H_2O_2$ induced occludin changes and effects in bovine brain BBB endothelial cells were examined in this study. The decrease of transendothelial electrical resistance (TEER) by $H_2O_2$ was due to disruption of occludin localization. Cytotoxicity test revealed that $H_2O_2$ did not cause cell death below 1 mM $H_2O_2$ within 4 hr. $H_2O_2$ caused intermittent disruption and loss of occludin at tight junctions and occludin disappeared with dose dependent manner from tight junction in confocal laser microscopy. But Western blot revealed that the total amounts of occludin increased by $H_2O_2$ administration. Transmission electron microscopy revealed that the ultrastructure of tight junction was not changed by $H_2O_2$. These data suggest that functional disruption of BBB by $H_2O_2$ was due to the localized loss of occludin in tight junction, but the expression of occludin increased in order to compensate the disrupted function in BBB. 뇌에서 혈액뇌장벽을 형성하는 내피세포는 치밀이음부를 통해 뇌의 항상성을 유지하고 있다. 치밀이음부의 단백질 중의 하나인 occludin은 뇌혈관장벽(BBB)의 기능을 유지하는 중요한 단백질로 인식되고 있다. 본 실험에서는 소의 뇌에서 배양된 BBB 내피세포에서 활성산소종의 하나인 $H_2O_2$에 의해 일어나는 occludin 단백질의 변화를 관찰하였다. $H_2O_2$에 의해 TEER가 감소하는 것은 occludin의 재분포에 의한 것이었다. 세포독성은 4시간내에서는 1mM $H_2O_2$ 이하에서는 나타나지 않았다. Confocal laser microscope으로 관찰한 결과, $H_2O_2$에 의해 occludin은 치밀이음부에서 중간중간이 사라져 감소해 있었고, 이러한 양상은 $H_2O_2$의 용량과 노출시간에 비례하였다. 그러나 Western blot 결과, occludin의 총량은 증가하였다. 투과전자현미경 관찰을 통해 $H_2O_2$가 세포사이의 결합의 구조에 뚜렷한 변화를 미치지 않는 것을 알 수 있었다. 이를 통해 $H_2O_2$에 의한 BBB 기능소실은 occludin이 치밀이음부에서 부분적으로 사라지는 것에 의하지만, 세포는 기능손상을 복구하기위한 방편으로 이 단백질의 생산을 더욱 증가시키는 것으로 생각된다.

Isothiazolinone (IT) biocides increase mitochondrial damage-mediated dysfunction and enhance hypoxic- or glycative-stress in blood-brain barrier

Donghyun Kim(김동현),Ok-Nam Bae(배옥남) 환경독성보건학회 2021 한국독성학회 심포지움 및 학술발표회 Vol.2021 No.5

The role of blood-brain barrier (BBB) dysfunction in various neurological/vascular diseases is well established. BBB is a gatekeeper, which strictly regulates the molecular transportation between systemic circulation and the central nervous system (CNS). Since environmental substances can enter the blood circulation and be exposed to vascular endothelium, it is important to evaluate these substances altering the function of BBB. Isothiazolinone (IT) biocides are potent anti-bacterial substances commonly used as preservatives and disinfectants. In this study, we used three representing IT biocide, benzisothiazolinone (BIT), 4,5-dichloro-2-n-octyl-isothiazolin-3-one (DCOIT), and 2-n-octyl-4-isothiazolin-3-one (OIT) to investigate its effect on the function of in vitro BBB models. We compared the IC50 (50% inhibitory concentration) of three IT biocides on mitochondrial metabolic capacity in bEND.3 cells. And the effects of these substances on oxidative stress and mitochondrial bioenergetics were examined as the molecular mechanism of BBB dysfunction. Of note, augmentation effects of IT biocide on metabolic capacity in brain ECs under hypoxic- or glycative-stress, simulating the ischemic- or diabetic-pathological condition, respectively, were observed. Here, we suggested that the IT biocides induced the BBB dysfunction through the increased oxidative stress and damage on mitochondrial bioenergetics or dynamics and that these substances might increase the hypoxic or glycative stress in the brain endothelium.

Involvement of a Novel Organic Cation Transporter in Paeonol Transport Across the Blood-Brain Barrier

Asmita Gyawali,Sokhoeurn Krol,강영숙 한국응용약물학회 2019 Biomolecules & Therapeutics(구 응용약물학회지) Vol.27 No.3

Paeonol has neuroprotective function, which could be useful for improving central nervous system disorder. The purpose of this study was to characterize the functional mechanism involved in brain transport of paeonol through blood-brain barrier (BBB). Brain transport of paeonol was characterized by internal carotid artery perfusion (ICAP), carotid artery single injection technique (brain uptake index, BUI) and intravenous (IV) injection technique in vivo. The transport mechanism of paeonol was examined using conditionally immortalized rat brain capillary endothelial cell line (TR-BBB) as an in vitro model of BBB. Brain volume of distribution (VD) of [3H]paeonol in rat brain was about 6-fold higher than that of [14C]sucrose, the vascular space marker of BBB. The uptake of [3H]paeonol was concentration-dependent. Brain volume of distribution of paeonol and BUI as in vivo and inhibition of analog as in vitro studies presented significant reduction effect in the presence of unlabeled lipophilic compounds such as paeonol, imperatorin, diphenhydramine, pyrilamine, tramadol and ALC during the uptake of [3H]paeonol. In addition, the uptake significantly decreased and increased at the acidic and alkaline pH in both extracellular and intracellular study, respectively. In the presence of metabolic inhibitor, the uptake reduced significantly but not affected by sodium free or membrane potential disruption. Similarly, paeonol uptake was not affected on OCTN2 or rPMAT siRNA transfection BBB cells. Interestingly. Paeonol is actively transported from the blood to brain across the BBB by a carrier mediated transporter system.

Development and application of a bispecific antibody penetrating the blood-brain-barrier (BBB)

Weon-Kyoo YOU 한국생물공학회 2021 한국생물공학회 학술대회 Vol.2021 No.10

One of major challenges to develop effective therapeutics for neurodegenerative diseases is due to lack of the ability to penetrate Blood-Brain-Barrier (BBB) rather than lack of potency or efficacy of therapeutics. In order to overcome this hurdle, ABL Bio has developed a bispecific platform (Grabody-BTM) that can improve BBB-penetrating activity through the receptor-mediated transcytosis (RMT) pathway in the brain endothelial cells. The most advanced development program using this platform is ABL301, a bispecific antibody binding to aggregated alpha-synuclein as well as RMT target. ABL301 is being developed for the treatment of Parkinson’s disease (PD) via specific binding against alpha-synuclein, a main pathological aggregate in the brain of PD patients. ABL301’s alpha-synuclein binding moiety is highly specific for aggregated form with no cross-reactivity against other synuclein homologs. In addition, ABL301 has shown a better in vivo efficacy in preclinical animal models of PD via improved BBB-penetrating ability compared to its parental alpha-synuclein-binding monoclonal antibody. ABL301 could be developed as a potent first-in-class therapeutic antibody for the treatment of synucleinopathy with improved BBB penetration. ABL Bio’s bispecific platform (Grabody-BTM) can be also used and applied for the development of other biologics to treat different neurodegenerative diseases.

쥐의 뇌 미세혈관 내피세포를 이용한 뇌혈관장벽 내피세포의 배양과 특성

이희상,김석중,김대진,정윤희,김성수,이원복,김경용,Lee, Hee-Sang,Kim, Seok-Jung,Kim, Dae-Jin,Chung, Yoon-Hee,Kim, Sung-Su,Lee, Won-Bok,Kim, Kyung-Yong 한국현미경학회 2005 Applied microscopy Vol.35 No.4

쥐의 뇌로부터 미세혈관에서 분리하고 배양한 내피세포의 특성을 현미경관찰, 면역염색과 전기저항을 측정해 관찰하였다. 미세혈관 내피세포는 배양 후 $5{\sim}6$일 경에 단층을 형성하였으며 특징적으로 소용돌이치는 모양을 나타냈다. 내피세포 단층의 전기저항은 배양 후 5일 경까지 따라 증가하였고 이후로는 감소하였다. 면역형광염색에서 anti-GFAP, anti-GalC, anti-neurofilament 160/200 kD antibody에 대한 면역반응을 거의 찾아볼 수 없었어 별아교세포, 희소돌기아교세포 및 신경세포에 의한 우려할 만한 오염은 배제할 수 있었다. vWF 항원에 대한 면역반응은 내피세포의 세포질에 Weibel-Palade 과립이 전반적으로 퍼져 있었다. 치밀이음부를 구성하는 occludin, ZO-1, ZO-2에 대한 면역반응은 내피세포의 접촉부위에서 매우 특징적으로 나타나고 있었다. 요약하면 쥐의 뇌 미세혈관 내피세포를 분리, 배양하여 형태학적, 조직면역 화학적 방법과 전기저항을 측정하여 내피세포의 특성을 확인하였다. 이 생체외 혈액뇌장벽 모델은 앞으로 진행될 액뇌장벽의 특징을 규명하고자 하는 시험관내 실험에 유용하게 이용될 수 있을 것이다. The characteristics of primary cultured rat brain microvessel endothelial cells (RBMECs) were studied using microscopy, immunohistochemistry and measuring of transendothelial electrical resistance (TER). The RBMECs formed a monolayer by $5{\sim}6$ days after plating and showed characteristics of whirling appearance. The TER increased until day 5 and decreased then. There was few immunoreaction with anti-GFAP, anti-GalC, anti-neurofilament 160/200 kD antibodies. So the contamination of astrocyte, oligodendrocyte, and neuron. could be ruled out.. Immunoreaction to vWF antigen was widespread througout the cytoplasm as Weibel-Palade granule. Immunoreaction to tight junction proteins, i.e. occludin, ZO-1, and ZO-2 was seen at cell contact. In summary, RBMECs isolated and cultured showed morphological, immunohistochemical and electrical characteristics of blood-brain barrier (BBB). The in vitro BBB model can be used in studying characteristics of in vivo BBB.

Bi-Functional Aspects of Peptide Decorated PLGA Nanocarriers for Enhanced Translocation Across the Blood-Brain Barrier through Macropinocytosis

Ane Nishitha Vijayan,Janani Indrakumar,Sankaranarayanan Gomathinayagam,Kodiveri Muthukaliannan Gothandam,Purna Sai Korrapati 한국고분자학회 2022 Macromolecular Research Vol.30 No.8

The blood-brain barrier (BBB) curtails the permeability of neuroprotective drugs to the brain thus restricting the effective delivery of therapeutics for neurodegenerative disorders. Recently, greater emphasis has been given for polymeric nanoparticles as a potential delivery system to transport drugs across the blood-brain barrier. This study focuses on the cellular route, localization and enhancement of uptake of drug loaded polymeric nanoparticles for delivery across the blood-brain barrier. We have optimized and synthesized polylactic-co-glycolic acid (PLGA) nanoparticles as a carrier for the delivery of drugs across the barrier. Cell penetrating peptide trans-activating transcriptor (TAT) was conjugated with the polymer through covalent bonding for increasing the efficiency of drug delivery across the BBB. Rhodamine-B was used as a model drug to study the release of drugs from the synthesized nanoparticle and finally the in vivo uptake in a mice model was checked. The size of the synthesized nanoparticles was in the nanometer range and the release profile revealed a rapid release appropriate for brain delivery. The cellular uptake experiments revealed that the peptide conjugated nanoparticle was readily taken up by the cells through macropinocytosis. Finally, to overcome the challenges for drugs to cross the BBB in an in vivo system, we have tracked the bioavailability of the nanoparticles in a mice model. Here we report an enhanced uptake of the peptide functionalized drug delivery carrier to successfully deliver and track therapeutic molecules across the blood-brain barrier in vivo.

Effect of Endurance Exercise and Methamphetamine Administration on the Expression of Blood-Brain Barrier Related Factors and BDNF in Hippocampus of Male Sprague-Dawley Rat

정수련 한국운동생리학회 2022 운동과학 Vol.31 No.3

PURPOSE: This study aimed to investigate the effect of short-term endurance exercise and methamphetamine administration on bloodbrain barrier (BBB)-related marker and brain-derived neurotrophic factor (BDNF) in the hippocampus of male Sprague-Dawley (SD) rats. METHODS: Forty male SD-rats were randomly assigned to 4 groups and treated for 2 weeks. For the methamphetamine group, 1 mg/ kg (+)-S-Methamphetamine hydrochloride (METH) was intraperitoneally injected daily, and an endurance exercise was performed at 21 m/min for 60 minutes. All treatments were performed daily, and METH or saline was administered after exercise. After 2 weeks of treatment, the head was decapitated under anesthesia, and the hippocampus was separated for western blotting. RESULTS: Two weeks of endurance exercise significantly increased the expression of occludin and claudin-5 in the hippocampus of the rats. Similarly, phosphorylation of CREB/BDNF and AKT/GSK3 signaling pathways were significantly increased. This phenomenon was also observed in the exercise and METH co-treatment groups. However, the single treatment of 1 mg/kg METH did not affect tight junction protein and BDNF expression. CONCLUSIONS: Endurance exercise increased the expression of BBB-related proteins and neurogenesis-related molecules, which were not inhibited by METH administration.

흰쥐 궁둥신경 손상 후 전침에 의한 척수 내 NeuN 발현에 미치는 영향

조미숙,Cho, Mi-Suk 대한물리치료학회 2011 대한물리치료학회지 Vol.23 No.2

Purpose: The purpose of this study was to investigate the effect of electroacupuncture on NeuN expression in ventral horn motor neurons of spinal cord, changes in pain thresholdchanges in motor function in rats with partially dissected sciatic nerves. Method: A total of 120 male Sprague-Dawley rats were randomly divided into two groups, a control group and a group administered electroacupuncture at ST36, LI11 and SP9 with 120 Hz and 0.5 mA. Animals were sacrificed on days 1, 3, 7, 14 and 28 after nerve injury (the sciatic nerve was partially dissected). The pain threshold was recorded by an Analgesia? meter and a BBB? score was calculated for motor function. After preparing lumbar spinal cord slide sections, they were immunostained with NeuN antisera (1:2,500). Results: The numbers of NeuN immunoreactive neuronsin the electroacupuncture group was increased compared to the control group. The numbers of NeuN immunoreactive neurons on days 14 and 28 day were different (p<0.05), as were the numbers on days 3 and 7 (p<0.01). The pain threshold BBB score for the electroacupuncture group was higher than for controls. Conclusion: The increase in pain threshold, BBB-score and number of NeuN immunoreactive neurons inventral horn motor neurons of spinal cord in rats withnerve dissection showed that electroacupuncture can attenuate pain transduction and increase motor function. Also, NeuN was a good marker for identifying the degree of nerve cell loss after nervous system injury.