Design,synthesis and pharmacological evaluation of caffeic acid phenethyl ester acylation as multifunctional neuroprotective agents against oxidative stress injury

4-Acylated or 3,4-diacylated caffeic acid phenethyl ester (CAPE) was prepared as prodrug to improve its stability and lipid solubility. Their neuroprotective activities were assessed by H2O2 model and 6-OHDA model. The results showed that target compounds displayed positive abilities to protect PC12 nerve cells from oxidative stress injury, superior to that of CAPE. Additionally, target compounds showed high blood-brain barrier permeability.     

Synthesis of (1E)-phenyl-2-(3',4'-dihydroxyphenyl)ethenesulfonates as potent neuroprotective agents and evaluation of their activities

A series of (1E)-phenyl-2-(3',4'-dihydroxyphenyl)ethenesulfonate derivatives were designed and synthesized as anti-amyotrophic lateral sclerosis (ALS) agents, based on the lead compound caffeic acid phenethyl ester (CAPE). And their neuroprotective activities were evaluated. The results indicated that replacement of the carboxylic ester by sulfonic ester did not produce better neuroprotective activity in the model of LPS induced inflammation in BV2 cells. However, the results in the model of H2O2 induced damage in PC12 cells showed that the neuroprotective activities of all the target compounds and CAPE were about the same.     

姜黄素对大鼠缺血性脑损伤炎症反应和血脑屏障通透性的影响

目的研究姜黄素对大鼠脑缺血/再灌注损伤炎症反应和血脑屏障通透性的作用,并进一步探讨其潜在的机制。方法利用线栓法制备大鼠脑缺血/再灌注损伤模型,随机分为对照组、模型组和姜黄素治疗组。检测大鼠神经功能损伤评分和脑梗死体积;通过检测脑组织髓过氧物酶的含量说明中性粒细胞浸润和炎症反应的程度;检测脑组织伊文思蓝的含量说明血脑屏障的破坏程度;ELISA检测脑组织肿瘤坏死因子-α(TNF-α)的含量;免疫印记法检测基质金属蛋白酶-9(MMP-9)的表达变化。结果姜黄素治疗组减轻神经功能损伤、脑梗死体积,并减轻中性粒细胞在脑组织的浸润程度、改善血脑屏障完整性。同时姜黄素还可以降低脑组织TNF-α的含量以及MMP-9的表达水平。结论姜黄素通过减轻炎症反应和血脑屏障破坏对大鼠脑缺血/再灌注损伤起脑保护作用。姜黄素的脑保护作用可能与其降低TNF-α含量和MMP-9的表达有关。     

TCP-FA4: A derivative of tranylcypromine showing improved blood-brain permeability

A variety of approaches have been taken to improve the brain penetration of pharmaceutical agents. The amphipathic character of a compound can improve its interaction with the lipid bilayer within cell membranes, and as a result improve permeability. Fatty acid chains or lipoamino acids of various lengths were attached to tranylcypromine (TCP), in an attempt to improve the blood-brain barrier (BBB) permeability by increasing the lipophilicity as well as the amphiphatic character of the drug. TCP-FA4, one of the derivatives containing a four carbon alkyl acid chain, showed the greatest improvement in permeability. This molecule was slightly neuroprotective in a β-amyloid-induced neurodegeneration assay and may also be capable of upregulating brain derived neurotrophic factor (BDNF), as indicated by cell culture assays using human umbilical vein endothelial cells. Since decreased levels of BDNF are observed in many CNS disorders, and direct injection of BDNF is not a viable option due to its poor permeability across the BBB, small molecules capable of regulating BDNF that also cross the BBB may be an interesting treatment option.     

Caffeic acid phenethyl ester protects nigral dopaminergic neurons via dual mechanisms involving haem oxygenase-1 and brain-derived neurotrophic factor

BACKGROUND AND PURPOSE

Caffeic acid phenethyl ester (CAPE) is a component of honey bee propolis that can induce expression of haem oxygenase-1 (HO-1). Because HO-1 induction has been suggested to protect dopaminergic neurons in the substantia nigra, we examined the effect of CAPE in experimental models of dopaminergic neurodegeneration.

EXPERIMENTAL APPROACH

Neuroprotective effect of CAPE was investigated in rat organotypic midbrain slice cultures and in vivo, using a mouse model of dopaminergic neurodegeneration induced by intranigral injection of LPS and intrastriatal injection of 6-hydroxydopamine.

KEY RESULTS

CAPE protected dopaminergic neurons in slice cultures from IFN-γ/LPS-induced injury. The effect of CAPE was inhibited by zinc protoporphyrin IX, an HO-1 inhibitor, and by neutralizing antibody against brain-derived neurotrophic factor (BDNF). A p38 MAPK inhibitor SB203580 prevented activation of NF-E2-related factor 2, attenuated increased expression of HO-1 and BDNF, and blocked the neuroprotective actions of CAPE. In the LPS-injected mouse model, daily intraperitoneal administration of CAPE protected dopaminergic neurons, up-regulated HO-1 and BDNF, and reduced the increase of activated microglia/macrophages. Neuroprotective effects of CAPE against LPS-induced injury was prevented by zinc protoporphyrin IX or anti-BDNF antibody. CAPE protected dopaminergic neurons and alleviated methamphetamine-induced rotational behaviour also in 6-hydroxydopamine hemiparkinsonian mice.

CONCLUSION AND IMPLICATIONS

CAPE is a novel type of neuroprotective agent whose actions are mediated by both HO-1 and BDNF. These findings may provide novel clues to develop neuroprotective agents for treatment of neurodegenerative disorders.     

Endocannabinoids modulate human blood–brain barrier permeability in vitro

Background and Purpose

Endocannabinoids alter permeability at various epithelial barriers, and cannabinoid receptors and endocannabinoid levels are elevated by stroke, with potential neuroprotective effects. We therefore explored the role of endocannabinoids in modulating blood–brain barrier (BBB) permeability in normal conditions and in an ischaemia/reperfusion model.

Experimental Approach

Human brain microvascular endothelial cell and astrocyte co-cultures modelled the BBB. Ischaemia was modelled by oxygen-glucose deprivation (OGD) and permeability was measured by transepithelial electrical resistance. Endocannabinoids or endocannabinoid-like compounds were assessed for their ability to modulate baseline permeability or OGD-induced hyperpermeability. Target sites of action were investigated using receptor antagonists and subsequently identified with real-time PCR.

Key Results

Anandamide (10 μM) and oleoylethanolamide (OEA, 10 μM) decreased BBB permeability (i.e. increased resistance). This was mediated by cannabinoid CB₂ receptors, transient receptor potential vanilloid 1 (TRPV1) channels, calcitonin gene-regulated peptide (CGRP) receptor (anandamide only) and PPARα (OEA only). Application of OEA, palmitoylethanolamide (both PPARα mediated) or virodhamine (all 10 μM) decreased the OGD-induced increase in permeability during reperfusion. 2-Arachidonoyl glycerol, noladin ether and oleamide did not affect BBB permeability in normal or OGD conditions. N-arachidonoyl-dopamine increased permeability through a cytotoxic mechanism. PPARα and γ, CB₁ receptors, TRPV1 channels and CGRP receptors were expressed in both cell types, but mRNA for CB₂ receptors was only present in astrocytes.

Conclusion and Implication

The endocannabinoids may play an important modulatory role in normal BBB physiology, and also afford protection to the BBB during ischaemic stroke, through a number of target sites.     

EMP-induced alterations of tight junction protein expression and disruption of the blood–brain barrier

The blood–brain barrier (BBB) is critical to maintain cerebral homeostasis. In this study, we examined the effects of exposure to electromagnetic pulse (EMP) on the functional integrity of BBB and, on the localization and expression of tight junction (TJ) proteins (occludin and ZO-1) in rats. Animals were sham or whole-body exposed to EMP at 200 kV/m for 400 pulses. The permeability of BBB in rat cerebral cortex was examined by using Evans Blue (EB) and lanthanum nitrate as vascular tracers. The localization and expression of TJ proteins were assessed by western blot and immunofluorescence analysis, respectively. The data indicated that EMP exposure caused: (i) increased permeability of BBB, and (ii) altered localization as well as decreased levels of TJ protein ZO-1. These results suggested that the alteration of ZO-1 may play an important role in the disruption of tight junctions, which may lead to dysfunction of BBB after EMP exposure.     

通窍活血汤对脑缺血再灌注小鼠血脑屏障通透性及脑内单胺类神经递质含量的影响

目的：观察通窍活血汤（TQHXD）对脑缺血再灌注损伤小鼠血脑屏障（BBB）通透性及脑组织中单胺类神经递质的影响。方法将昆明种小鼠随机分为假手术组,模型组,尼莫地平组,脑脉泰组,通窍活血汤低、中、高剂量组（分别为3．85,7．7,15．4 g·kg－1）。于给药第7天采用双侧颈总动脉结扎法制造急性脑缺血再灌注模型,通过测定渗出脑血管外的伊文思蓝含量分析药物对BBB通透性的影响,及采用酶联免疫法（ELISE）测定脑组织中单胺类神经递质去甲肾上腺素（NE）、多巴胺（DA）和5－羟色胺（5-HT）的含量。结果 TQHXD各剂量组均能降低小鼠脑中伊文思蓝的含量,且均能阻止脑损伤后单胺类神经递质的降低。结论 TQHXD对脑缺血再灌注模型小鼠有一定的保护作用。     

Anti-oxidant activity mediated neuroprotective potential of trimetazidine on focal cerebral ischaemia-reperfusion injury in rats

1. The present study was designed to investigate the neuroprotective effect of trimetazidine (TMZ) following focal cerebral ischaemia-reperfusion (I/R) injury in rat forebrain. 2. Cerebral I/R injury was induced in rats by middle cerebral artery occlusion (MCAO) for 2 h, followed by reperfusion for 22 h. Trimetazidine (5 and 25 mg/kg, i.p.) was administered 1 h after induction of MCAO. The effects of TMZ were investigated by measuring neurological deficit, volume of infarct and brain swelling after 22 h reperfusion. Oxidative stress and inflammatory reactivity were assessed by estimating anti-oxidant markers and myeloperoxidase (MPO) activity in brain homogenates. Rectal temperature was measured during the study. The effects of TMZ on blood-brain barrier (BBB) permeability and apoptosis were also investigated in rat brain. Apoptosis was observed by DNA fragmentation studies using agarose gel electrophoresis. 3. Treatment with TMZ significantly (P < 0.01) reduced infarct volume and brain swelling. Superoxide dismutase (SOD) activity was reduced in ipsilateral hemispheres of vehicle (saline)-treated reperfused (RI) animals. Treatment with TMZ significantly restored SOD activity (P < 0.01) and glutathione levels (P < 0.05) after reperfusion compared with RI animals. Lipid peroxidation, MPO activity, BBB permeability and rectal temperature were all significantly (P < 0.01, P < 0.05 and P < 0.001, respectively) reduced in TMZ-treated animals compared with RI animals. 4. These results suggest that TMZ protects the brain against cerebral I/R injury and that this neuroprotective activity could be mediated by its anti-oxidant properties. The reduction in rectal temperature by TMZ treatment may be responsible for maintaining the delicate energy balance during I/R injury in rat brain and could have contributed to the neuroprotective activity of TMZ.     

Effect of caffeic acid phenethyl ester on gastric acid secretion in vitro

Caffeic acid phenethyl ester (CAPE), one of the major components of propolis (honeybee resin), has demonstrated a wide spectrum of activities including suppression of eicosanoids by inhibition of cyclooxygenase-1 and cyclooxygenase-2 enzyme activities. The aim of this study was to investigate the effect of CAPE on basal and secretagogues-stimulated gastric acid secretion in vitro. In the isolated, lumen-perfused, stomach preparation of mouse, CAPE (10-100 microM) did not affect the basal gastric acid secretion nor the secretion stimulated by histamine, pentagastrin, isobutyl methylxanthine and high levels of K+. By contrast, CAPE increased the gastric acid secretion induced by the muscarinic receptor agonist, 5-methylfurmethide (5-MEF). CAPE also inhibited the acetylcholinesterase activity in an in vitro colorimetric assay. Eserine (10 microM), a well known acetylcholinesterase inhibitor, also increased 5-MEF-stimulated acid secretion. Our results show that CAPE increases gastric acid secretion stimulated by an acetylcholine agonist receptor likely through inhibition of acetylcholinesterase activity.     

Correlation between breakdown of the blood-brain barrier and disease outcome of viral encephalitis in mice

Changes in the permeability of the blood-brain barrier (BBB) were evaluated in two mouse models of viral encephalitis. The ability of sodium fluorescein (NaFl) to cross the BBB from the serum into the central nervous system was assayed in animals inoculated with virulent strains of either Banzi or Semliki Forest viruses. To test the hypothesis that increases in BBB permeability were associated with poor disease outcome subsequent experiments measured BBB permeability in conjunction with treatment with the interferon inducer Ampligen (poly I:poly C(12)U). A single intraperitoneal injection of Ampligen (1 mg/kg) administered either 24 h or 4-6 h before, but not 24 h after, virus inoculation with Banzi virus provided significant improvements in survival, viral brain titers, weight change and BBB permeability. In comparison, a similar treatment with Ampligen administered either 24 h or 4-6 h before inoculation with Semliki Forest virus was able to significantly improve weight change, and BBB permeability, but only animals receiving Ampligen 4-6 h pre-virus showed a significantly improved mortality. In general, it was found that evaluation of BBB permeability was a more sensitive indicator of disease outcome and the antiviral efficacy Ampligen than either weight change or brain viral titers.     

Pharmacokinetics and Brain Uptake of Biotinylated Basic Fibroblast Growth Factor Conjugated to a Blood-Brain Barrier Drug Delivery System

Human basic fibroblast growth factor (bFGF) is a potent neuroprotective agent. The clinical efficacy of this neurotrophin, however, is restricted by poor permeability across the blood-brain barrier (BBB). This study was designed to test the hypotheses that bFGF will retain its biological activity and have an enhanced BBB transport after re-formulation and conjugation to a BBB peptide drug delivery vector. The BBB delivery vector is comprised of a conjugate of streptavidin (SA) and the murine OX26 monoclonal antibody against the rat transferrin receptor, and the conjugate of biotinylated bFGF (bio-bFGF) bound to a vector is designated bio-bFGF/OX26-SA. A radioreceptor binding assay shows that the native bFGF, bio-bFGF, and bio-bFGF/OX26-SA conjugate have IC 50 values of 0.12, 0.40, and 0.56 nM, respectively. After an IV bolus injection to the rat, [125 I]-bio-bFGF is avidly taken up by peripheral organs, with low brain uptake at 60 min, 0.010 ± 0.004% of injected dose (ID)/g brain. By contrast, the brain uptake of the [125 I]-bio-bFGF/OX26-SA is increased 5-fold to 0.050 ± 0.011%ID/g, although the uptake of the conjugate by peripheral tissues was decreased relative to the unconjugated bio-bFGF. In conclusion, conjugation of bio-bFGF to a BBB drug delivery vector (a) causes only a minor decrease in affinity for the bFGF receptor, (b) decreases the peripheral organ uptake of the bFGF, and (c) increases the brain uptake of the neurotrophin. The re-formulation of bFGF to enable receptor-mediated transcytosis across the BBB may improve the therapeutic index of this neurotrophin as a neuroprotective agent.     

Pharmacokinetics and brain uptake of biotinylated basic fibroblast growth factor conjugated to a blood-brain barrier drug delivery system

Human basic fibroblast growth factor (bFGF) is a potent neuroprotective agent. The clinical efficacy of this neurotrophin, however, is restricted by poor permeability across the blood-brain barrier (BBB). This study was designed to test the hypotheses that bFGF will retain its biological activity and have an enhanced BBB transport after re-formulation and conjugation to a BBB peptide drug delivery vector. The BBB delivery vector is comprised of a conjugate of streptavidin (SA) and the murine OX26 monoclonal antibody against the rat transferrin receptor, and the conjugate of biotinylated bFGF (bio-bFGF) bound to a vector is designated bio-bFGF/OX26-SA. A radioreceptor binding assay shows that the native bFGF, bio-bFGF, and bio-bFGF/OX26-SA conjugate have IC50 values of 0.12, 0.40, and 0.56 nM, respectively. After an IV bolus injection to the rat, [125I]-bio-bFGF is avidly taken up by peripheral organs, with low brain uptake at 60 min, 0.010+/-0.004% of injected dose (ID)/g brain. By contrast, the brain uptake of the [125I]-bio-bFGF/OX26-SA is increased 5-fold to 0.050+/-0.011%ID/g, although the uptake of the conjugate by peripheral tissues was decreased relative to the unconjugated bio-bFGF. In conclusion, conjugation of bio-bFGF to a BBB drug delivery vector (a) causes only a minor decrease in affinity for the bFGF receptor, (b) decreases the peripheral organ uptake of the bFGF, and (c) increases the brain uptake of the neurotrophin. The re-formulation of bFGF to enable receptor-mediated transcytosis across the BBB may improve the therapeutic index of this neurotrophin as a neuroprotective agent.     

神经生长因子脂质体跨血脑屏障体内外研究神经生长因子脂质体跨血脑屏障体内外研究

目的研究神经生长因子（nerve growth factor,NGF）脂质体在体外血脑屏障（blood-brain barrier,BBB）模型上的通透性以及在体内脑组织的分布，并对体内外结果进行相关性分析。方法用小鼠脑微血管内皮细胞(BMVEC)建立的BBB体外实验模型，研究NGF脂质体在体外模型上的通透率；¹²⁵I-NGF和SDS-PAGE法联合使用研究NGF脂质体在脑组织的分布。结果NGF脂质体的最高包封率为34%，平均粒径小于100 nm。脂质体能够增加NGF在BBB模型上的通透率，以NGF-SSL-T为最大。脑组织药物浓度次序为NGF-SSL-T>NGF-SSL+RMP-7>NGF-SSL。体内外结果具有良好相关性。结论脂质体能够增加NGF跨越BBB的能力，RMP-7偶联在脂质体上（NGF-SSL-T）效果好于RMP-7与脂质体的简单混合（NGF-SSL+RMP-7）。     

Comparison of drug permeabilities across the blood-retinal barrier, blood-aqueous humor barrier, and blood-brain barrier

Drugs vary in their ability to permeate the blood-retinal barrier (BRB), blood-aqueous humor barrier (BAB), and blood-brain barrier (BBB) and the factors affecting the drug permeation remain unclear. In this study, the permeability of various substances across BRB, BAB, and BBB in rats was determined using the brain uptake index (BUI), retinal uptake index (RUI), and aqueous humor uptake index (AHUI) methods. Lipophilic substances showed high permeabilities across BBB and BRB. The RUI values of these substances were approximately four-fold higher than the BUI values. The AHUI versus lipophilicity curve had a parabolic shape with AHUI(max) values at log D(7.4) ranging from -1.0 to 0.0. On the basis of the difference on the lipophilicities, verapamil, quinidine, and digoxin showed lower permeability than predicted from those across BBB and BRB, whereas only digoxin showed a lower permeability across BRB. These low permeabilities were significantly increased by P-glycoprotein inhibitors. Furthermore, anion transporter inhibition increased the absorption of digoxin to permeate into the retina and aqueous humor. In conclusion, this study suggests that efflux transport systems play an important role in the ocular absorption of drugs from the circulating blood after systemic administration.     

Fructose-1,6-bisphosphate ameliorates lipopolysaccharide-induced dysfunction of blood–brain barrier

Fructose-1,6-bisphosphate (FBP), a glycolytic intermediate, has neuroprotective effects in various brain injury models. However, its effects on blood–brain barrier (BBB) are largely unknown. In this study, we investigated the effects of FBP on lipopolysaccharide (LPS)-induced BBB dysfunction in in vitro BBB model comprising co-culture of mouse brain endothelial cell line, bEnd.3 and mouse primary astrocyte and explored its action mechanism therein involved. LPS induced the impairment of endothelial permeability and transendothelial electrical resistance (TEER). The functional changes were confirmed by alterations in immunostaining for junctional proteins occludin, ZO-1 and VE-cadherin, such as the loss of cortical staining pattern and appearance of intercellular gaps in endothelial cells. Co-administration of FBP alleviated the deleterious effects of LPS on BBB permeability and TEER in a dose dependent manner. And also FBP inhibited the LPS-induced changes in the distribution of endothelial junctional proteins, resulting in the better preservation of monolayer integrity. FBP suppressed the production of reactive oxygen species (ROS) but did not affect cyclooxygenase-2 expression and prostaglandin E₂ production in endothelial cells stimulated with LPS. Taken together, these data suggest that FBP could ameliorate LPS-induced BBB dysfunction through the maintenance of junctional integrity, which might be mediated by downregulation of ROS production.     

脑损伤过程中血脑屏障通透性的变化及其调节机制

血脑屏障是由脑微血管内皮细胞、星形神经胶质细胞、外膜细胞、血管周围巨噬细胞和基底膜组成的一个复杂系统,对维持中枢神经系统的正常功能非常重要。脑损伤如脑缺血、脑缺氧、脑外伤和蛛网膜下腔出血过程中伴随血脑屏障通透性的变化。脑缺血及其再灌注后可通过花生四烯酸代谢途径、嘌呤核苷酸代谢途径及一氧化氮途径产生自由基,可能是血脑屏障通透性增加的重要机制。血脑屏障的破坏可加重脑损伤程度;脑血管疾病时,保护血脑屏障的完整性可能是减轻脑损伤的重要措施。     

Predicting blood-brain barrier permeability of drugs: evaluation of different in vitro assays

The ability of a drug to penetrate the blood-brain barrier (BBB) is essential for its use in the pharmaceutical treatment of CNS disorders. Five different in vitro methods to predict BBB permeability of drugs were compared and evaluated in the present study. All assays were performed with a consistent set of seven compounds and in the same physiological buffer to provide a basis for direct comparison of the results. Octanol-buffer and liposome-buffer partition coefficients were most conveniently obtained but failed to predict BBB permeability for certain drugs. The incorporation of drugs into lipid monolayers at the air-buffer interface was found to be a poor predictor of BBB permeability and was furthermore not considered suitable for screening due to the demanding experimental requirements. Permeability studies using Caco-2 cell monolayers provided a good correlation to an in vitro model of the BBB, which was based on primary cultured porcine brain capillary endothelial cells (PBCEC). However, differences in drug permeability between the intestine and brain derived cells were detected, limiting the advantages of the easy handling of the Caco-2 cell line compared to the more time-consuming primary culture of the BCEC.     

Functional role of adenosine receptor subtypes in the regulation of blood-brain barrier permeability: possible implications for the design of synthetic adenosine derivatives.

The objective of this investigation was to determine the functional role of adenosine receptor subtypes in the regulation of blood-brain barrier (BBB) permeability. The presence of the equilibrative es and ei nucleoside transporters at the BBB was also determined. Studies were conducted in an experimental in vitro BBB model comprising bovine brain capillary endothelial cells (BCECs) and rat astrocytes (RAs). The presence of the receptors and transporters was investigated by a combination of RT-PCR and radioligand binding assays. Changes in paracellular permeability were investigated on basis of changes in trans-endothelial-electrical-resistance (TEER) and transport of paracellular markers. In BCECs the presence of A(2A) and A(3) receptors and the es nucleoside transporter was demonstrated. The A(1) receptor was absent, while the presence of the A(2B) receptor and the ei nucleoside transporter remained uncertain. In RAs the presence of all four receptor subtypes and the es and ei nucleoside transporters was demonstrated. Upon application of selective agonists no significant changes in TEER or the transport of the paracellular markers were observed. The functional role of adenosine receptor subtypes in regulating the paracellular permeability of the BBB is probably small. It is unlikely therefore that the BBB transport of synthetic adenosine analogues is modified by permeability changes. The es nucleoside transporter might play a role in the BBB transport of synthetic adenosine analogues.     

Effects of daily dermal application of DEET and epermethrin, alone and in combination, on sensorimotor performance, blood-brain barrier, and blood-testis barrier in rats

DEET and permethrin were implicated in the development of illnesses in some veterans of the Persian Gulf War. This study was designed to investigate the effects of daily dermal application of these chemicals, alone or in combination, on the permeability of the blood-brain barrier (BBB) and blood-testes barrier (BTB) and on sensorimotor performance in male Sprague-Dawley rats. Groups of five rats were treated with a dermal daily dose of 4, 40, or 400 mg/kg DEET in ethanol or 0.013, 0.13, or 1.3 mg/kg permethrin in ethanol for 60 d. A group of 10 rats received a daily dermal dose of ethanol and served as controls. BBB permeability was assessed by injection of an iv dose of the quaternary ammonium compound [3H]hexamethonium iodide. While permethrin produced no effect on BBB permeability, DEET alone caused a decrease in BBB permeability in brainstem. A combination of DEET and permethrin significantly decreased the BBB permeability in the cortex. BTB permeability was decreased by treatment with DEET alone and in combination with permethrin. The same animals underwent a battery of functional behavior tests 30, 45, and 60 d after exposure to evaluate their sensorimotor abilities. All treatments caused a significant decline in sensorimotor performance in a dose- and time-dependent manner. These results show that daily dermal exposure to DEET, alone or in combination with permethrin, decreased BBB permeability in certain brain regions, and impaired sensorimotor performance.