Activation of adult rat CNS endothelial cells by opioid-induced toll-like receptor 4 (TLR4) signaling induces proinflammatory, biochemical, morphological, and behavioral sequelae.

CNS immune signaling contributes to deleterious opioid effects including hyperalgesia, tolerance, reward, and dependence/withdrawal. Such effects are mediated by opioid signaling at toll-like receptor 4 (TLR4), presumptively of glial origin. Whether CNS endothelial cells express TLR4 is controversial. If so, they would be well positioned for activation by blood-borne opioids, contributing to opioid-induced pro-inflammatory responses. These studies examined adult primary rat CNS endothelial cell responses to (-)-morphine or its mu opioid receptor (MOR)-inactive metabolite morphine-3-glucuronide (M3G), both known TLR4 agonists. We demonstrate that adult rat CNS endothelial cells express functional TLR4. M3G activated nuclear factor kappaB (NF-κB), increased tumor necrosis factor-α (TNFα) and cyclooxygenase-2 (COX2) mRNAs, and released prostaglandin E2 (PGE2) from these cells. (-)-Morphine-induced upregulation of TNFα mRNA and PGE2 release were unmasked by pre-treatment with nalmefene, a MOR antagonist without TLR4 activity (unlike CTAP, shown to have both MOR- and TLR4-activity), suggestive of an interplay between MOR and TLR4 co-activation by (-)-morphine. In support, MOR-dependent Protein Kinase A (PKA) opposed TLR4 signaling, as PKA inhibition (H-89) also unmasked (-)-morphine-induced TNFα and COX2 mRNA upregulation. Intrathecal injection of CNS endothelial cells, stimulated in vitro with M3G, produced TLR4-dependent tactile allodynia. Further, cortical suffusion with M3G in vivo induced TLR4-dependent vasodilation. Finally, endothelial cell TLR4 activation by lipopolysaccharide and/or M3G was blocked by the glial inhibitors AV1013 and propentofylline, demonstrating endothelial cells as a new target of such drugs. These data indicate that (-)-morphine and M3G can activate CNS endothelial cells via TLR4, inducing proinflammatory, biochemical, morphological, and behavioral sequelae. CNS endothelial cells may have previously unanticipated roles in opioid-induced effects, in phenomena blocked by presumptive glial inhibitors, as well as TLR4-mediated phenomena more broadly.

Distinct mechanisms of relaxation to bioactive components from chamomile species in porcine isolated blood vessels.

German chamomile (Matricaria recutita L.), a widely-used herbal medicine, has been reported to have a wide range of biological effects, including smooth muscle relaxation. The aim of this study was to compare the effects of representative compounds from chamomile (apigenin, luteolin, (-)-α-bisabolol, farnesene, umbelliferone; 3-30 µM) on vascular tone using porcine coronary and splenic arteries mounted for isometric tension recording in isolated tissue baths and precontracted with the thromboxane-mimetic U46619. Apigenin, luteolin, and (-)-α-bisabolol produced slow, concentration-dependent relaxations in both the coronary and splenic arteries that were not blocked by inhibition of nitric oxide synthase or potassium channels. Removal of extracellular calcium inhibited the relaxations to all three compounds, and these compounds also inhibited calcium re-addition-evoked contractions, indicating that the relaxation response may be mediated through inhibition of calcium influx. Apigenin and luteolin, but not (-)-α-bisabolol, enhanced the relaxation to the nitric oxide donor sodium nitroprusside, indicating that apigenin and luteolin may act to regulate cyclic GMP levels. Umbelliferone produced a rapid, transient relaxation in the splenic artery, but not the coronary artery, that was inhibited by L-NAME and removal of the endothelium, suggesting an influence on nitric oxide production. Farnesene, at concentrations up to 30 µM, was without effect in either blood vessel. In conclusion, hydroxylated compounds (apigenin, luteolin and (-)-α-bisabolol) found in chamomile all caused a slow relaxation of isolated blood vessels through an effect on calcium influx. Umbelliferone, on the other hand, produced a rapid, transient relaxation dependent upon release of nitric oxide from the endothelium.

Spinal upregulation of glutamate transporter GLT-1 by ceftriaxone: therapeutic efficacy in a range of experimental nervous system disorders.

Glutamate neurotransmission is highly regulated, largely by glutamate transporters. In the spinal cord, the glutamate transporter GLT-1 is primarily responsible for glutamate clearance. Downregulation of GLT-1 can occur in activated astrocytes, and is associated with increased extracellular glutamate and neuroexcitation. Among other conditions, astrocyte activation occurs following repeated opioids and in models of chronic pain. If GLT-1 downregulation occurs in these states, GLT-1 could be a pharmacological target for improving opioid efficacy and controlling chronic pain. The present studies explored whether daily intrathecal treatment of rats with ceftriaxone, a beta-lactam antibiotic that upregulates GLT-1 expression, could prevent development of hyperalgesia and allodynia following repeated morphine, reverse pain arising from central or peripheral neuropathy, and reduce glial activation in these models. Ceftriaxone pre-treatment attenuated the development of hyperalgesia and allodynia in response to repeated morphine, and prevented associated astrocyte activation. In a model of multiple sclerosis (experimental autoimmune encephalomyelitis; EAE), ceftriaxone reversed tactile allodynia and halted the progression of motor weakness and paralysis. Similarly, ceftriaxone reversed tactile allodynia induced by chronic constriction nerve injury (CCI). EAE and CCI each significantly reduced the expression of membrane-bound, dimerized GLT-1 protein in lumbar spinal cord, an effect normalized by ceftriaxone. Lastly, ceftriaxone normalized CCI- and EAE-induced astrocyte activation in lumbar spinal cord. Together, these data indicate that increasing spinal GLT-1 expression attenuates opioid-induced paradoxical pain, alleviates neuropathic pain, and suppresses associated glial activation. GLT-1 therefore may be a therapeutic target that could improve available treatment options for patients with chronic pain.

Prenatal stress suppresses rat pup ultrasonic vocalization and myoclonic twitching in response to separation.

Prenatally stressed infant rats were separated from their dams and littermates on postnatal Day 14 and their rates of ultrasonic vocalizations (USVs) and myoclonic twitching in response to that separation were recorded. Compared to control pups, prenatally stressed pups vocalized significantly less often and showed significantly less myoclonic twitching in response to this test. Results are interpreted in terms of the ability of prenatal stress to produce infants that are behaviorally inhibited when in a novel or stressful situation.

The effects of two novel objects on the behavior of singly caged adult rhesus macaques.

Six female and six male adult rhesus macaques were given sticks and nylon balls as an attempt at simple cage enrichment. A latin square design was used to compare behavior during separate 4-week periods with each object and during a control period with no object. Frequency and duration of 15 different behaviors were recorded. Resting was the most common activity which decreased slightly in duration when the stick or nylon ball was present (P less than 0.02). The mean duration of stick use was longer than that of the nylon ball (P less than 0.01). No other behaviors changed significantly, including the frequency of abnormal behaviors such as self-abuse, stereotypic acts, and bizarre postures. Generally, these objects were used infrequently and led to few changes in the behavior of singly-caged adult rhesus macaques. However, they did appear to stimulate activity for some individuals.

Influence of cage size on heart rate and behavior in rhesus monkeys.

We studied 6 singly caged adult female rhesus monkeys to determine whether increased cage size had any effect on behavior or heart rate. Two monkeys at a time were placed in cages 40% larger than their standard cage for 1 week on 2 occasions, using a counter-balanced design. Direct behavioral observations were performed 75 minutes/week on each monkey. Heart rate and general activity were monitored 35 hours/week by a telemetry system. Statistically significant differences were not found in aggressive, submissive, abnormal, or self-abusive behavior, nor in time spent in the front half of the cage, duration of grooming, looking at the observer, or stereotyped or nonstereotyped locomotion. Vocalizations increased the first time in the larger cage, but not the second, and decreased upon the second return to the standard cage. Differences with respect to cage size were not found in heart rate or activity level, although there were significant variations at different times of day. We conclude that modest increases in cage size are unlikely to enrich the environment of singly caged laboratory primates.