Overcoming Endocytosis Deficiency by Cubosome Nanocarriers.

The use of lipid-based nanoparticles for the delivery of biomacromolecules has attracted considerable attention due to the current interest in protein-based therapeutics. Cubosomes protect the incorporated therapeutics, which are susceptible to degradation by enzymes, thereby improving their bioavailability, and concomitantly enhance cellular uptake. The cubosome nanoparticles presented herein were loaded with bovine serum albumin (BSA) and characterized by small-angle X-ray scattering and dynamic light scattering techniques, while the BSA encapsulation and its release were evaluated in vitro. The ability of this formulation to increase the cellular uptake of albumin by 2-fold was tested on various types of renal tubular cells and confirmed by in vivo renal uptake experiments in mice. The obtained results show that cubosomes are able to deliver BSA inside the cell through distinct uptake and intracellular routing. These data were substantiated, with evidence of a high cubosome-mediated uptake of BSA in Clcn5 knockout mice characterized by defective receptor-mediated endocytosis. The use of cubosomes as a delivery system thus represents a promising approach to overcome the low endocytic uptake in diseased epithelial cells and to treat dysfunctions of the kidney proximal tubule.

The prostaglandin E2 receptor EP3 controls CC-chemokine ligand 2-mediated neuropathic pain induced by mechanical nerve damage.

Prostaglandin (PG) E2 is an important lipid mediator that is involved in several pathophysiological processes contributing to fever, inflammation, and pain. Previous studies have shown that early and continuous application of nonsteroidal anti-inflammatory drugs significantly reduces pain behavior in the spared nerve injury (SNI) model for trauma-induced neuropathic pain. However, the role of PGE2 and its receptors in the development and maintenance of neuropathic pain is incompletely understood but may help inform strategies for pain management. Here, we sought to define the nociceptive roles of the individual PGE2 receptors (EP1-4) in the SNI model using EP knockout mice. We found that PGE2 levels at the site of injury were increased and that the expression of the terminal synthase for PGE2, cytosolic PGE synthase was up-regulated in resident positive macrophages located within the damaged nerve. Only genetic deletion of the EP3 receptor affected nociceptive behavior and reduced the development of late-stage mechanical allodynia as well as recruitment of immune cells to the injured nerve. Importantly, EP3 activation induced the release of CC-chemokine ligand 2 (CCL2), and antagonists against the CCL2 receptor reduced mechanical allodynia in WT but not in EP3 knockout mice. We conclude that selective inhibition of EP3 might present a potential approach for reducing chronic neuropathic pain.

Design and performance of an ultra-flexible two-photon microscope for in vivo research.

We present a cost-effective in vivo two-photon microscope with a highly flexible frontend for in vivo research. Our design ensures fast and reproducible access to the area of interest, including rotation of imaging plane, and maximizes space for auxiliary experimental equipment in the vicinity of the animal. Mechanical flexibility is achieved with large motorized linear stages that move the objective in the X, Y, and Z directions up to 130 mm. 360° rotation of the frontend (rotational freedom for one axis) is achieved with the combination of a motorized high precision bearing and gearing. Additionally, the modular design of the frontend, based on commercially available optomechanical parts, allows straightforward updates to future scanning technologies. The design exceeds the mobility of previous movable microscope designs while maintaining high optical performance.

Prostacyclin regulates spinal nociceptive processing through cyclic adenosine monophosphate-induced translocation of glutamate receptors.

BACKGROUND: Prostacyclin (PGI2) is known to be an important mediator of peripheral pain sensation (nociception) whereas little is known about its role in central sensitization. METHODS: The levels of the stable PGI2-metabolite 6-keto-prostaglandin F1α (6-keto-PGF1α) and of prostaglandin E2 (PGE2) were measured in the dorsal horn with the use of mass spectrometry after peripheral inflammation. Expression of the prostanoid receptors was determined by immunohistology. Effects of prostacyclin receptor (IP) activation on spinal neurons were investigated with biochemical assays (cyclic adenosine monophosphate-, glutamate release-measurement, Western blot analysis) in embryonic cultures and adult spinal cord. The specific IP antagonist Cay10441 was applied intrathecally after zymosan-induced mechanical hyperalgesia in vivo. RESULTS: Peripheral inflammation caused a significant increase of the stable PGI2 metabolite 6-keto-PGF1α in the dorsal horn of wild-type mice (n = 5). IP was located on spinal neurons and did not colocalize with the prostaglandin E2 receptors EP2 or EP4. The selective IP-agonist cicaprost increased cyclic adenosine monophosphate synthesis in spinal cultures from wild-type but not from IP-deficient mice (n = 5-10). The combination of fluorescence-resonance-energy transfer-based cyclic adenosine monophosphate imaging and calcium imaging showed a cicaprost-induced cyclic adenosine monophosphate synthesis in spinal cord neurons (n = 5-6). Fittingly, IP activation increased glutamate release from acute spinal cord sections of adult mice (n = 13-58). Cicaprost, but not agonists for EP2 and EP4, induced protein kinase A-dependent phosphorylation of the GluR1 subunit and its translocation to the membrane. Accordingly, intrathecal administration of the IP receptor antagonist Cay10441 had an antinociceptive effect (n = 8-11). CONCLUSION: Spinal prostacyclin synthesis during early inflammation causes the recruitment of GluR1 receptors to membrane fractions, thereby augmenting the onset of central sensitization.

BKCa channels expressed in sensory neurons modulate inflammatory pain in mice.

Large conductance calcium-activated potassium (BKCa) channels are important regulators of neuronal excitability. Although there is electrophysiological evidence for BKCa channel expression in sensory neurons, their in vivo functions in pain processing have not been fully defined. Using a specific antibody, we demonstrate here that BKCa channels are expressed in subpopulations of peptidergic and nonpeptidergic nociceptors. To test a functional association of BKCa channel activity in sensory neurons with particular pain modalities, we generated mice in which BKCa channels are ablated specifically from sensory neurons and analyzed their behavior in various models of pain. Mutant mice showed increased nociceptive behavior in models of persistent inflammatory pain. However, their behavior in models of neuropathic or acute nociceptive pain was normal. Moreover, systemic administration of the BKCa channel opener, NS1619, inhibited persistent inflammatory pain. Our investigations provide in vivo evidence that BKCa channels expressed in sensory neurons exert inhibitory control on sensory input in inflammatory pain states.

Prostacyclin mediates neuropathic pain through interleukin 1ß-expressing resident macrophages.

Prostacyclin is an important mediator of peripheral pain sensation. Here, we investigated its potential participation in mediating neuropathic pain and found that prostacyclin receptor (IP) knockout mice exhibited markedly decreased pain behavior. Application of an IP antagonist to the injury site or selective IP deficiency in myeloid cells mimicked the antinociceptive effect observed in IP knockout mice. At the site of nerve injury, IP was expressed in interleukin (IL) 1ß-containing resident macrophages, which were less common in IP knockout mice. Local administration of the IP agonist cicaprost inhibited macrophage migration in vitro and promoted accumulation of IP- and IL1ß-expressing cells as well as an increase of IL1ß concentrations at the application site in vivo. Fittingly, the IL1-receptor antagonist anakinra (IL-1ra) decreased neuropathic pain behavior in wild-type mice but not in IP knockout mice. Finally, continuous, but not single administration, of the cyclooxygenase inhibitor meloxicam early after nerve injury decreased pain behavior and the number of resident macrophages. Thus, early synthesis of prostacyclin at the site of injury causes accumulation of IL1ß-expressing macrophages as a key step in neuropathic pain after traumatic injury.

Synthesis of lipid mediators during UVB-induced inflammatory hyperalgesia in rats and mice.

Peripheral sensitization during inflammatory pain is mediated by a variety of endogenous proalgesic mediators including a number of oxidized lipids, some of which serve endogenous modulators of sensory TRP-channels. These lipids are eicosanoids of the arachidonic acid and linoleic acid pathway, as well as lysophophatidic acids (LPAs). However, their regulation pattern during inflammatory pain and their contribution to peripheral sensitization is still unclear. Here, we used the UVB-model for inflammatory pain to investigate alterations of lipid concentrations at the site of inflammation, the dorsal root ganglia (DRGs) as well as the spinal dorsal horn and quantified 21 lipid species from five different lipid families at the peak of inflammation 48 hours post irradiation. We found that known proinflammatory lipids as well as lipids with unknown roles in inflammatory pain to be strongly increased in the skin, whereas surprisingly little changes of lipid levels were seen in DRGs or the dorsal horn. Importantly, although there are profound differences between the number of cytochrome (CYP) genes between mice and rats, CYP-derived lipids were regulated similarly in both species. Since TRPV1 agonists such as LPA 18∶1, 9- and 13-HODE, 5- and 12-HETE were elevated in the skin, they may contribute to thermal hyperalgesia and mechanical allodynia during UVB-induced inflammatory pain. These results may explain why some studies show relatively weak analgesic effects of cyclooxygenase inhibitors in UVB-induced skin inflammation, as they do not inhibit synthesis of other proalgesic lipids such as LPA 18∶1, 9-and 13-HODE and HETEs.

Overdose of the histamine H3 inverse agonist pitolisant increases thermal pain thresholds.

OBJECTIVE AND DESIGN: Pitolisant (BF2.649) is a selective inverse agonist for the histamine H(3) receptor and was developed for the treatment of excessive daytime sleepiness in Parkinson disease, narcolepsy, and schizophrenia. Since H(3)-ligands can decrease inflammatory pain, we tested Pitolisant in inflammatory and neuropathic pain models. MATERIALS AND TREATMENTS: Behavioral effects of pitolisant and the structural different H(3) receptor inverse agonists ciproxifan and ST-889 were tested in zymosan-induced inflammation and the spared nerve injury model for neuropathic pain. METHODS: Responses to mechanical and thermal stimuli were determined. Calcium imaging was performed with primary neuronal cultures of dorsal root ganglions. RESULTS: Clinically relevant doses of pitolisant (10 mg/kg) had no relevant effect on mechanical or thermal pain thresholds in all animal models. Higher doses (50 mg/kg) dramatically increased thermal but not mechanical pain thresholds. Neither ciproxifan nor ST-889 altered thermal pain thresholds. In peripheral sensory neurons high concentrations of pitolisant (30-500 µM), but not ciproxifan, partially inhibited calcium increases induced by capsaicin, a selective activator of transient receptor potential vanilloid receptor 1 (TRPV1). High doses of pitolisant induced a strong hypothermia. CONCLUSION: The data show a dramatic effect of high dosages of pitolisant on the thermosensory system, which appears to be H(3) receptor-independent.

Anti-inflammatory role of microsomal prostaglandin E synthase-1 in a model of neuroinflammation.

A major immunological response during neuroinflammation is the activation of microglia, which subsequently release proinflammatory mediators such as prostaglandin E(2) (PGE(2)). Besides its proinflammatory properties, cyclooxygenase-2 (COX-2)-derived PGE(2) has been shown to exhibit anti-inflammatory effects on innate immune responses. Here, we investigated the role of microsomal PGE(2) synthase-1 (mPGES-1), which is functionally coupled to COX-2, in immune responses using a model of lipopolysaccharide (LPS)-induced spinal neuroinflammation. Interestingly, we found that activation of E-prostanoid (EP)2 and EP4 receptors, but not EP1, EP3, PGI(2) receptor (IP), thromboxane A(2) receptor (TP), PGD(2) receptor (DP), and PGF(2) receptor (FP), efficiently blocked LPS-induced tumor necrosis factor α (TNFα) synthesis and COX-2 and mPGES-1 induction as well as prostaglandin synthesis in spinal cultures. In vivo, spinal EP2 receptors were up-regulated in microglia in response to intrathecally injected LPS. Accordingly, LPS priming reduced spinal synthesis of TNFα, interleukin 1ß (IL-1ß), and prostaglandins in response to a second intrathecal LPS injection. Importantly, this reduction was only seen in wild-type but not in mPGES-1-deficient mice. Furthermore, intrathecal application of EP2 and EP4 agonists as well as genetic deletion of EP2 significantly reduced spinal TNFα and IL-1ß synthesis in mPGES-1 knock-out mice after LPS priming. These data suggest that initial inflammation prepares the spinal cord for a negative feedback regulation by mPGES-1-derived PGE(2) followed by EP2 activation, which limits the synthesis of inflammatory mediators during chronic inflammation. Thus, our data suggest a role of mPGES-1-derived PGE(2) in resolution of neuroinflammation.