Spinal repair in immature animals: a novel approach using the South American opossum Monodelphis domestica

1. The adult mammalian central nervous system (CNS) is unable to regenerate following injury and repair has only been seen when implants of peripheral nervous tissue, fetal tissue or Schwann cells are used, or antibodies or trophic molecules applied. However, the immature mammalian CNS has revealed a capacity to repair without extrinsic influence. 2. The marsupial mammal provides a unique opportunity to access the immature CNS without invasive in utero surgery. In particular, the South American opossum Monodelphis domestica is an ideal animal for spinal cord injury studies examining the ability of the immature CNS to repair after injury. 3. The Monodelphis spinal cord may be examined for its response to injury either as an in vitro or in vivo system and, therefore, is a flexible model, allowing many different questions to be addressed by the most suitable approach. 4. The immature Monodelphis CNS was able to support fibre growth that reappeared 4 days after a crush at P3-P8 in vitro. Conduction was also restored at this time, accompanied by synaptic connections. 5. A cut lesion performed in vivo on Monodelphis spinal cords at P7 took longer to repair, with fibres reappearing across the injury site 2 weeks after the lesion; greater disruption to structure was noted both during early stages of repair and in adulthood. 6. Neural pathway tracing with dextran amine from the lumbar cord to the brain in adult Monodelphis, which received spinal lesions at P7, revealed a similar distribution of labelled cells in brainstem and mid-brain nuclei to that of control animals. 7. Studies of the locomotor behaviour of adult Monodelphis that had received either a cut or crush lesion at P7-P8 showed remarkably similar abilities to control animals when performing complex tasks. 8. The results of spinal cord injury studies with the immature Monodelphis CNS may help in the development of treatments for spinal injury patients.     

Spinal administration of the monoacylglycerol lipase inhibitor JZL184 produces robust inhibitory effects on nociceptive processing and the development of central sensitization in the rat

Background and Purpose

The cannabinoid receptor-mediated analgesic effects of 2-arachidonoylglycerol (2-AG) are limited by monoacylglycerol lipase (MAGL). 4-nitrophenyl 4-[bis (1,3-benzodioxol-5-yl) (hydroxy) methyl] piperidine-1-carboxylate (JZL184) is a potent inhibitor of MAGL in the mouse, though potency is reportedly reduced in the rat. Here we have assessed the effects of spinal inhibition of MAGL with JZL184 on nociceptive processing in rats.

Experimental Approach

In vivo spinal electrophysiological assays in anaesthetized rats were used to determine the effects of spinal administration of JZL184 on spinal nociceptive processing in the presence and absence of hindpaw inflammation. Contributions of CB₁ receptors to these effects was assessed with AM251. Inhibition of 2-oleoylglycerol hydrolytic activity and alterations of 2-AG in the spinal cord after JZL 184 were also assessed.

Key Results

Spinal JZL184 dose-dependently inhibited mechanically evoked responses of wide dynamic range (WDR) neurones in naïve anaesthetized rats, in part via the CB₁ receptor. A single spinal administration of JZL184 abolished inflammation-induced expansion of the receptive fields of spinal WDR neurones. However, neither spinal nor systemic JZL184 altered levels of 2-AG, or 2-oleoylglycerol hydrolytic activity in the spinal cord, although JZL184 displayed robust inhibition of MAGL when incubated with spinal cord tissue in vitro.

Conclusions and Implications

JZL184 exerted robust anti-nociceptive effects at the level of the spinal cord in vivo and inhibited rat spinal cord MAGL activity in vitro. The discordance between in vivo and in vitro assays suggests that localized sites of action of JZL184 produce these profound functional inhibitory effects.

Linked Articles

This article is part of a themed section on Cannabinoids. To view the other articles in this section visit http://dx.doi.org/10.1111/bph.2012.167.issue-8     

DEVELOPMENTAL NEUROTOXICITY TO METHAMPHETAMINES

1. To investigate the long-term changes caused by amphetamines in the developing brain, we used both an in vivo and in vitro model of chronic fetal exposure to methamphetamine and related drugs. 2. Offspring of rats, treated with either saline, 2 mg/kg twice a day (b.i.d.) or 10 mg/kg bid. methamphetamine throughout gestation, were examined at 30 days of age for changes in the monoamine system of their brains. 3. At the lower dose methamphetamine was neurotoxic to specific neuronal populations, mostly serotonergic. At the higher dose, methamphetamine retained its neurotoxic properties, but also stimulated the growth of axonal terminals in specific regions as evidenced by an increase in monoamine uptake sites. The neurochemical changes at the higher dose were correlated with deficits in adult behavioural measures. 4. Corresponding in vitro drug treatments of rat neuroblastomas cells also produced a dose-related effect on cellular growth and differentiation patterns. Neurotoxic as well as stimulatory effects of methamphetamine and some related compounds were seen in culture. 5. Our in vivo and in vitro observations demonstrate neurotoxic effects of amphetamines and the remodelling of synaptic morphology in response.     

New approach to treating spinal cord injury using PEG-TAT-modified,cyclosporine-A-loaded PLGA/polymeric liposomes

Cyclosporine-A (CsA) is an immunosuppressant agent that has shown effectiveness as a neuroprotective drug; however, it does not readily cross the blood-spinal cord barrier (BSCB), which constrains the clinical applications of CsA for the treatment of spinal cord injury (SCI). Our group recently tested the ability of novel polyethylene glycol (PEG)-transactivating-transduction protein (TAT)-modified CsA-loaded cationic multifunctional polymeric liposome-poly(lactic-co-glycolic acid) (PLGA) core/shell nanoparticles (PLGA/CsA NPs) to transport and deliver CsA across the BSCB to treat SCI. The PLGA/CsA NPs were successfully constructed. In vitro drug release studies have demonstrated that the sustained release of CsA from PLGA/CsA NPs occurs over ～25?h. The in vivo study presented here showed that injured animals that received PLGA/CsA NPs through the tail vein, exhibited a significant up-regulation of growth-associated protein-43 (GAP-43) expression and an increased number of GAP-43-stained neurons compared with animals that received CsA or the vehicle alone. The improvement in neurological function was also evaluated by the Basso–Beattie–Bresnahan (BBB) open-field test. Moreover, fluorescein isothiocyanate (FITC)-attached PLGA/CsA NPs were successfully aggregated in the intact spinal cord 4?h after injection. Our data suggest that PLGA/CsA NPs have the potential for use as a new treatment method for SCI.     

Effect of mono-(2-ethylhexyl) phthalate on human and mouse fetal testis: In vitro and in vivo approaches

The present study was conducted to determine whether exposure to the mono-(2-ethylhexyl) phthalate (MEHP) represents a genuine threat to male human reproductive function. To this aim, we investigated the effects on human male fetal germ cells of a 10^− 5 M exposure. This dose is slightly above the mean concentrations found in human fetal cord blood samples by biomonitoring studies. The in vitro experimental approach was further validated for phthalate toxicity assessment by comparing the effects of in vitro and in vivo exposure in mouse testes.Human fetal testes were recovered during the first trimester (7-12 weeks) of gestation and cultured in the presence or not of 10^− 5 M MEHP for three days. Apoptosis was quantified by measuring the percentage of Caspase-3 positive germ cells. The concentration of phthalate reaching the fetal gonads was determined by radioactivity measurements, after incubations with ¹⁴C-MEHP.A 10^− 5 M exposure significantly increased the rate of apoptosis in human male fetal germ cells. The intratesticular MEHP concentration measured corresponded to the concentration added in vitro to the culture medium. Furthermore, a comparable effect on germ cell apoptosis in mouse fetal testes was induced both in vitro and in vivo.This study suggests that this 10^− 5 M exposure is sufficient to induce changes to the in vivo development of the human fetal male germ cells.     

Ghrelin and toxicity: recent findings and future challenges

Ghrelin is a novel brain–gut peptide that plays various roles in mammals, including control of food intake and growth hormone release, as well as gastric motility and acid secretion in the gastrointestinal tract. It is mainly secreted by the gastric mucosa, but is also expressed in various other tissues. Different studies confirm the multiple biological roles of and possible protective effects of ghrelin. Multiple in vitro and in vivo studies support the powerful protective action of ghrelin against heart, gastric and liver injury. Moreover, ghrelin has been reported to be beneficial in renal tissue injury and excretory function after ischemia–reperfusion and to exert neuroprotective effects in cerebral ischemic regions. The aim of this review is to summarize and evaluate all the currently available in vivo and in vitro studies regarding the effects of ghrelin on tissue injury induced in different organs and tissues. Copyright © 2012 John Wiley & Sons, Ltd.     

依达拉奉对大鼠坐骨神经损伤后脊髓脂质过氧化的影响

目的:探讨自由基清除剂依达拉奉对坐骨神经损伤后神经功能及脊髓脂质过氧化反应的影响.方法:Wistar大鼠48只,随机分为3组:坐骨神经挤压伤组、依达拉奉治疗组、假手术组.分别于7、14、21、28 d检测各组大鼠坐骨神经功能指数(SFI)、脊髓内过氧化物歧化酶(SOD)和丙二醛(MDA)的变化.结果:伤后各组大鼠SFI均降低,挤压伤组大鼠SFI较依达拉奉治疗组低(P<0.05),神经功能恢复较治疗组缓慢.伤后挤压伤组大鼠脊髓内SOD活性升高,依达拉奉治疗组大鼠脊髓内SOD活性与假手术组相比升高不明显(P>0.05).伤后挤压伤组大鼠脊髓内MDA含量上升明显,依达拉奉治疗组大鼠脊髓内MDA含量在各个时间点均显著低于挤压伤组大鼠脊髓内MDA含量(P相似文献   

Methylglyoxal has injurious effects on maturation of mouse oocytes,fertilization, and fetal development,via apoptosis

Methylglyoxal (MG) is a metabolite of glucose. The serum MG level is increased in diabetic patients, and MG is implicated in diabetic complications related to embryonic development injury. We previously reported cytotoxic effects of MG on mouse embryonic stem cells and blastocysts, and a further association with defects in subsequent development. Here, we further investigate the effects of MG on oocyte maturation and subsequent pre- and post-implantation development, both in vitro and in vivo. Notably, MG induced a significant reduction in the rate of oocyte maturation, fertilization, and in vitro embryonic development. Treatment of oocytes with MG during in vitro maturation (IVM) led to increased resorption of post-implantation embryos and decreased fetal weight. Experiments with an in vivo mouse model disclosed that consumption of drinking water containing 10–20 μM MG led to decreased oocyte maturation and in vitro fertilization, as well as early embryonic developmental injury. Finally, pretreatment with a caspase-3-specific inhibitor effectively prevented MG-triggered injury effects, suggesting that embryo impairment by MG occurs via a caspase-dependent apoptotic process.     

Effects of ochratoxin a on mouse oocyte maturation and fertilization,and apoptosis during fetal development

We previously reported that ochratoxin A (OTA), a mycotoxin found in many foods worldwide, causes nephrotoxicity, hepatotoxicity, and immunotoxicity, and is a risk factor for abnormal embryonic development. More specifically, OTA triggers apoptotic processes in the inner cell mass of mouse blastocysts, decreasing cell viability and embryonic development. In the current study, we investigated the deleterious effects of OTA on mouse oocyte maturation, in vitro fertilization (IVF), and subsequent pre‐ and postimplantation development both in vitro and in vivo. Notably, OTA significantly impaired mouse oocyte maturation, decreased IVF rates, and inhibited subsequent embryonic development in vitro. Preincubation of oocytes with OTA during in vitro maturation increased postimplantation embryonic resorption and decreased mouse fetal weight. In an in vivo animal model, provision of 1–10 μM OTA in the drinking water or intravenous injection of 1 or 2 mg/kg body weight of OTA decreased oocyte maturation and IVF, and had deleterious effects on early embryonic development. Importantly, preincubation of oocytes with a caspase‐3‐specific inhibitor effectively blocked these OTA‐triggered deleterious effects, suggesting that the embryonic injury induced by OTA is mediated via a caspase‐dependent apoptotic mechanism. Furthermore, OTA upregulated the levels of p53 and p21 in blastocyst cells derived from OTA‐pretreated oocytes, indicating that such cells undergo apoptosis via p53‐, p21‐, and caspase‐3‐dependent regulatory mechanisms. This could have deleterious effects on embryonic implantation and fetal survival rates, as seen in our animal models. © 2014 Wiley Periodicals, Inc. Environ Toxicol 31: 724–735, 2016.     

Hazardous effects of sanguinarine on maturation of mouse oocytes,fertilization, and fetal development through apoptotic processes

Previously, we reported that sanguinarine, a phytoalexin with antimicrobial, anti‐oxidant, anti‐inflammatory and pro‐apoptotic effects, is a risk factor for normal embryonic development that triggers apoptotic processes in the inner cell mass of mouse blastocysts, causing decreased embryonic development and cell viability. In the current study, we investigated the deleterious effects of sanguinarine on mouse oocyte maturation, in vitro fertilization (IVF), and subsequent pre‐ and postimplantation development both in vitro and in vivo. Notably, sanguinarine significantly impaired mouse oocyte maturation, decreased IVF rates, and inhibited subsequent embryonic development in vitro. Preincubation of oocytes with sanguinarine during in vitro maturation induced an increase in postimplantation embryo resorption and a decrease in mouse fetal weight. In an in vivo animal model, 1 to 5 μM sanguinarine, provided in drinking water, caused a decrease in oocyte maturation and IVF, and led to deleterious effects on early embryonic development. Importantly, preincubation of oocytes with a caspase‐3‐specific inhibitor effectively blocked sanguinarine‐triggered deleterious effects, clearly implying that embryonic injury induced by sanguinarine is mediated by a caspase‐dependent apoptotic mechanism. © 2014 Wiley Periodicals, Inc. Environ Toxicol 30: 946–955, 2015.     

In vitro neurotoxicity data in human risk assessment of polybrominated diphenyl ethers (PBDEs): Overview and perspectives

Polybrominated diphenyl ethers (PBDEs) are flame retardants routinely detected in samples of cord blood and breast milk. Concerns have been raised with regard to the toxicity of both pre- and postnatal exposures towards the developing nervous system. Although there is an increasing body of literature on the disruption of brain cell functions by certain PBDE congeners in vitro, some challenges have yet to be tackled to enable the translation of in vitro findings into their in vivo counterparts. In this paper, we review findings on the PBDE neurotoxicity in human cells and discuss the research gaps to be addressed. Moreover, we propose a scheme for the incorporation of in vitro data in human risk assessment, namely through (i) the determination of in vitro cell benchmark levels; (ii) the consideration of uncertainties in establishing equivalency between the in vitro and the in vivo tissue benchmark levels (e.g., chronic vs. acute exposure, interactions with other chemicals); and (iii) relating tissue benchmark levels to surrogate levels of internal exposure. Alongside the assessment of brain dosimetry following exposure to PBDEs, in vitro neurotoxicity data provide a unique opportunity to evaluate the risks of prenatal and early life exposures on children neurodevelopment.     

Berberine impairs embryonic development in vitro and in vivo through oxidative stress‐mediated apoptotic processes

Berberine, an isoquinoline alkaloid isolated from several traditional Chinese herbal medicines, has been shown to suppress growth and induce apoptosis in some tumor cell lines. However, berberine has also been reported to attenuate H₂O₂‐induced oxidative injury and apoptosis. The basis for these ambiguous effects of berberine—triggering or preventing apoptosis—has not been well characterized to date. In the current investigation, we examined whether berberine exerts cytotoxic effects on mouse embryos at the blastocyst stage and affects subsequent embryonic development in vitro and in vivo. Treatment of blastocysts with berberine (2.5‐10 μM) induced a significant increase in apoptosis and a corresponding decrease in trophectoderm cell number. Moreover, the implantation success rate of blastocysts pretreated with berberine was lower than that of their control counterparts. Pretreatment with berberine was also associated with increased resorption of postimplantation embryos and decreased fetal weight. In an animal model, intravenous injection of berberine (2, 4, or 6 mg/kg body weight/d) for 4 days resulted in apoptosis of blastocyst cells and early embryonic developmental injury. Berberine‐induced injury of mouse blastocysts appeared to be attributable to oxidative stress‐triggered intrinsic apoptotic signaling processes that impaired preimplantation and postimplantation embryonic development. Taken together, our results clearly demonstrate that berberine induces apoptosis and retards early preimplantation and postimplantation development of mouse embryos, both in vitro and in vivo.     

Apoptotic effects on maturation of mouse oocytes,fertilization and fetal development by puerarin

Previously we identified puerarin, an isoflavone compound, as a risk factor for normal embryonic development that triggers apoptotic processes in the inner cell mass of mouse blastocysts, leading to retardation of embryonic development and cell viability. In the current study, we investigated whether puerarin exerts deleterious effects on mouse oocyte maturation, in vitro fertilization (IVF) and subsequent pre- and post-implantation development, both in vitro and in vivo. Notably, puerarin caused significant impairment of these processes in vitro. Pre-incubation of oocytes with puerarin during in vitro maturation led to increased post-implantation embryo resorption and decreased mouse fetal weight. In an in vivo animal model, intravenous injection with or without puerarin (1, 3 and 5?mg/kg body weight/day) for 4 days caused a decrease in oocyte maturation and IVF, and led to deleterious effects on early embryonic development. Importantly, pre-incubation of oocytes with a caspase-3-specific inhibitor effectively blocked puerarin-triggered deleterious effects, clearly implying that embryonic injury induced by puerarin is mediated by a caspase-dependent apoptotic mechanism. These results clearly demonstrate that puerarin has deleterious effects on mouse oocyte maturation, fertilization and subsequent embryonic development in vitro and in vivo.     

Adult stem cell application in spinal cord injury

The mechanical force incurred by spinal cord injury results in degenerative neural tissue damage beyond the site of initial injury. By nature, the central nervous system (CNS) does not regenerate itself. Cell therapy, in particular, stem cell implantation has become a possible solution for spinal cord injury. Embryonic stem cells and fetal stem cells are the forefathers of the field of stem cell therapy. Isolation and preparation of specific populations of adult stem cells have evolved to the point of stable, long-term culturing with the capability to differentiate into neural phenotypes from all three of the neural lineages: neurons, astrocytes, and oligodendrocytes. Thus, adult stem cells will transcend ethical concerns, technical difficulties, and probably immunorejection. A variety of adult stem cells have been implanted in a rat model of spinal cord injury, ranging from olfactory ensheathing cells, cultured spinal cord stem cells, bone marrow derived stem cells, dermis derived stem cells, and a few others. Although no definite decisions on which adult stem cells are most effective for this CNS injury, their ability to incorporate into the spinal cord, differentiate, and to improve locomotor recovery hold promise for a cure.     

Effect of Cerastes vipera snake venom on muscle spindles,spinal ganglia and spinal cord

Repeated sublethal i.m. doses of Cerastes vipera venom given to mice at weekly intervals for 5 weeks had no effect on the muscle spindles of the injected muscles. Both nuclear chain and nuclear bag intrafusal muscle fibres were histologically normal. Equatorial sensory innervation and the polar motor end plates, those at the ends of the intrafusal muscle fibres, were unaffected. The venom had no effect in vitro on the continuity of the cholinesterase active border of the subneural apparatus of the polar motor end plates. Extrafusal atrophic muscle fibres had central nuclei. Binucleated fibres were common. Other fibres contained 3–4 dense nuclear fragments with no recognizable internal structures.I.p. injection of lethal doses of the venom in the rabbit failed to cause any change in the cell somata of the spinal ganglia. Injection of 0·2 ld₅₀ intrathecally produced marked neurological signs, respiratory failure and death in less than 15 min. The meninges, ganglia and spinal cord above the site of injection showed extensive haemorrhages. Repeated i.p. sublethal injections produced melting out of the Nissl granules at the periphery of some cells. Binucleation of a minority of the nerve cells was observed. The chromatin pattern of the nucleus was changed. It appeared dense with inapparent nuclecoli. No typical mitosis was observed.The spinal ganglia of a 7 days chick embryo treated in vivo with one-fourth of the lethal dose and incubated for 48 hr showed hypertrophy of the cells with acceleration of their differentiation. Cell counts revealed minor differences. The bodies of the vertebrae of envenomated embryos were larger. Earlier fusion of their vertebral arches formed dense spines compared to the faint shadow of the spines seen in the control. Both anteroposterior and transverse diameters of the envenomated spinal cord were larger with more voluminous grey matter than the control. Maximum breadth of both dorsal and ventral horns was greater than the controls. Population density of the cells of the mantle layer was greater in both dorsal and ventral horns of the control embryos indicating the small sized cells of the control. Faster fusion of the cells of the ependymal layer obliterated the dorsal half of the neural cavity with earlier creation of a dorsal median septum in the envenomated embryos. In the control both dorsal horns were separated by a median slit 10–15 μm in breadth.     

HYPOXIC INHIBITION OF BREATHING AND MOTOR ACTIVITY IN THE FOETUS AND NEWBORN

In fetal animals hypoxia of rapid onset causes cessation of breathing movements, electro-ocular activity and decrease of muscle tone. These effects last several hours and are in contrast to the hypernoea and behavioural activation which occurs during hypoxia soon after birth and in the adult. Transection and lesion studies in fetal sheep suggest that hypoxia activates a descending inhibition of respiratory and other motor activities which either originates in the pons or is conveyed to medullary and spinal levels of the neuraxis by fibres through the pons in the region of the Kolliker-Fuse nucleus. Recently, using FOS immunohistochemistry we have identified cells in the medial parabrachial complex which are activated by hypoxia in fetal sheep, but not newborn lambs. It is proposed that these cells have descending inhibitory connections with respiratory and spinal motor pathways, but the precise anatomy and neuro-chemistry of such pathways is unknown. It is not known if the parabrachial cells are directly sensitive to low P o₂ or receive input from other centres or peripheral receptors which monitor arterial P o₂ in the foetus. Nor is it known why these cells are not activated by low P o₂ after birth.     

Strategies to optimize the brain availability of central nervous system drug candidates

Introduction: Access to the CNS is essential for most neurotherapeutics to elicit their effects. Leveraging design strategies incorporating physicochemical properties, in vitro and in vivo assays to predict and measure brain penetration, and brain delivery approaches may enable the drug discovery community to improve access of drug candidates into the CNS compartment.

Areas covered: This article reviews aspects of the most recent molecular design, in vitro and in vivo strategies, and delivery technologies to optimize the unbound brain concentrations (C _b,u) of CNS molecules. Through this, the article provides insight into recent ideas and concepts in CNS drug molecule design, methods for evaluating CNS drug exposures and alternative approaches to maximize drug access to neurocompartments.

Expert opinion: The most pharmacologically relevant measure in assessing a compound's pharmacodynamic response in the CNS is its C _b,u. The utilization of emerging design strategies, together with in vitro and in vivo assays, may enable the design of molecules with optimal C _b,u:C _p,u (C _p,u, unbound plasma concentration) and appropriate C _b,u, to elicit a biological response from the neurotherapeutic target. Where drug properties intrinsically render a compound CNS impaired, using novel CNS delivery approaches may result in sufficient C _b,u to furnish a biological response.     

Ethyl pyruvate promotes spinal cord repair by ameliorating the glial microenvironment

BACKGROUND AND PURPOSE

Spinal cord injury (SCI) triggers a series of endogenous processes, including neuroinflammation and reactive astrogliosis, which may contribute to the failure of neural regeneration and functional recovery. In the present study, the effect of ethyl pyruvate on spinal cord repair was explored.

EXPERIMENTAL APPROACH

Functional assessment and histological analyses of astrogliosis, neuroinflammation, neuronal survival and axonal regeneration were performed to investigate the effects of ethyl pyruvate (0.086, 0.215, 0.431 or 0.646 mmol·kg⁻¹·day⁻¹) on spinal cord repair in a rat model of SCI. The effect of ethyl pyruvate (5, 10 or 15 mM) on astrocytic activation was also evaluated in an in vitro‘scratch-wound’ model.

KEY RESULTS

Functional assessment showed evident improvement of behavioural functions in the ethyl pyruvate-treated rats. Reactive astrogliosis was significantly inhibited in vivo, after injection of ethyl pyruvate (0.431 mmol·kg⁻¹day⁻¹), and in vitro‘scratch-wound’ model in the presence of 10 or 15 mM ethyl pyruvate. The difference between effective concentration in vitro and in vivo suggests that the inhibitory effect of ethyl pyruvate on astrogliosis in damaged spinal cord is indirect. In addition, ethyl pyruvate (0.431 mmol·kg⁻¹day⁻¹) attenuated SCI-induced neuroinflammation; it decreased the Iba-1-, ED-1- and CD11b-positive cells at the lesion site. Importantly, histological analyses showed a significantly greater number of surviving neurons and regenerative axons in the ethyl pyruvate-treated rats.

CONCLUSIONS AND IMPLICATIONS

Ethyl pyruvate was shown to inhibit astrogliosis and neuroinflammation, promote neuron survival and neural regeneration, and improve the functional recovery of spinal cord, indicating a potential neuroprotective effect of ethyl pyruvate against SCI.     

L-type Amino Acid Transporter 1 (SLC7A5)-Mediated Transport of Pregabalin at the Rat Blood-Spinal Cord Barrier and its Sensitivity to Plasma Branched-Chain Amino Acids

Pregabalin is an anti-neuropathic pain drug inhibiting the α2δ subunit of the voltage-dependent calcium channel in the spinal cord. The aim of this study is to characterize the transport mechanism of pregabalin at the blood-spinal cord barrier (BSCB) by means of in vivo experiments in rats and in vitro studies using primary-cultured rat spinal cord endothelial cells. We isolated endothelial cells by culturing rat spinal cord tissue in the presence of puromycin, and confirmed the expression of BSCB markers such as Cd31, Mdr1a, and Claudin-5. The uptake of pregabalin by primary-cultured rat spinal cord endothelial cells was sodium-independent and was significantly inhibited by L-leucine, 2-aminobicyclo-(2,2,1)-heptane-2-carboxylic acid, and JPH203. These results suggest the involvement of L-type amino acid transporter (LAT) 1. LAT1 mRNA and protein was expressed in primary-cultured rat spinal cord endothelial cells, which is consistent with LAT1 expression at the BSCB. In the in vivo study, the transfer of pregabalin to rat spinal cord and brain was significantly decreased by the pre-administration of branched chain amino acids (BCAAs), which are endogenous substrates of LAT1. Our results indicate that pregabalin transport across the BSCB is mediated at least in part by LAT1 and is inhibited by plasma BCAAs.