Assessment of the potential role of muscle spindle mechanoreceptor afferents in chronic muscle pain in the rat masseter muscle

Low pH leads to changes in several electrical properties of MSA, including initiation of ectopic action potentials which could propagate centrally but could also invade the peripheral endings causing glutamate release and activation of nearby nociceptors within the spindle capsule. This peripheral drive could contribute both to the transition to, and maintenance of, persistent muscle pain as seen in some "functional" pain syndromes

Multiplexed Evaluation of Serum and CSF Pharmacokinetics of Brain-Targeting Single-Domain Antibodies Using a NanoLC–SRM-ILIS Method

FC5 and FC44 are single-domain antibodies (V_HHs), selected by functional panning of phage-display llama V_HH library for their ability to internalize human brain endothelial cells (BEC) and to transmigrate the in vitro BBB model. Quantification of brain delivery of FC5 and FC44 in vivo was challenging using classical methods because of their short plasma half-life and their loss of functionality with radioactive labeling. A highly sensitive (detection limit <2 ng/mL) and specific SRM-ILIS method to detect and quantify unlabeled V_HHs in multiplexed assays was developed and applied to comparatively evaluate brain delivery of FC5 and FC44, and two control V_HHs, EG2 and A20.1. FC5 and FC44 compared to control V_HHs demonstrated significantly (<i>p</i> < 0.01) enhanced transport (50–100-fold) across rat in vitro BBB model as well as in vivo brain targeting assessed by optical imaging. The multiplexed SRM-ILIS analyses of plasma and CSF levels of codosed V_HHs demonstrated that while all 4 V_HHs have similar blood pharmacokinetics, only FC5 and FC44 show elevated CSF levels, suggesting that they are potential novel carriers for delivery of drugs and macromolecules across the BBB

A novel platform for engineering blood-brain barrier-crossing bispecific biologics

The blood-brain barrier (BBB) prevents the access of therapeutic antibodies to central nervous system (CNS) targets. The engineering of bispecific antibodies in which a therapeutic \u201carm\u201d is combined with a BBB-transcytosing arm can significantly enhance their brain delivery. The BBB-permeable single-domain antibody FC5 was previously isolated by phenotypic panning of a naive llama single-domain antibody phage display library. In this study, FC5 was engineered as a mono- and bivalent fusion with the human Fc domain to optimize it as a modular brain delivery platform. In vitro studies demonstrated that the bivalent fusion of FC5 with Fc increased the rate of transcytosis (Papp) across brain endothelial monolayer by 25% compared with monovalent fusion. Up to a 30-fold enhanced apparent brain exposure (derived from serum and cerebrospinal fluid pharmacokinetic profiles) of FC5- compared with control domain antibody-Fc fusions after systemic dosing in rats was observed. Systemic pharmacological potency was evaluated in the Hargreaves model of inflammatory pain using the BBB-impermeable neuropeptides dalargin and neuropeptide Y chemically conjugated with FC5-Fc fusion proteins. Improved serum pharmacokinetics of Fc-fused FC5 contributed to a 60-fold increase in pharmacological potency compared with the single-domain version of FC5; bivalent and monovalent FC5 fusions with Fc exhibited similar systemic pharmacological potency. The study demonstrates that modular incorporation of FC5 as the BBB-carrier arm in bispecific antibodies or antibody-drug conjugates offers an avenue to develop pharmacologically active biotherapeutics for CNS indications.Peer reviewed: YesNRC publication: Ye

Acidic saline injections produce long lasting changes in many of the electrophysiological properties of NVmes cells innervating masseter muscle spindles.

<p>Means ± S.E. of twelve electrical properties are shown for Control (filled circle) and Experimental (open circle) neurons recorded at 7 time periods after the second intramuscular injection. *, p<0.05 for simple contrasts. In panels F, G and J, all ps<0.01.</p

Masseter muscle spindles contain small-calibre afferents that express nociceptor markers.

<p>Left and right photomicrographs have identical frames with different sets of fluorescence filters. In each case, portions of the photomicrographs were digitally merged in boxed areas. A: Nerve fibres immunoreactive for PGP9.5 and containing CGRP (A'). B: A small green CGRP-positive fibre (B', thin arrow) runs across three VGLUT1-positive loops of an annulospiral ending. None of the VGLUT1-positive fibres in B corresponded to the CGRP positive fibres in B'. In the merged image, the fibre to the right appears yellow for most of its length (arrowheads) because it passes over red VGLUT1-positive fibres. C and C': Fibres immunoreactive for both PGP9.5 and SP. D and D': Fibres immunoreactive for PGP9.5 and the capsaicin receptor, TRPV1. c: spindle capsule wall. All scale bars = 10 µm.</p

The putative nociceptors in muscle spindles carry mGluR5 receptors.

<p>In all cases, the photomicrographs from the left and right columns have the exact same frames but with different set of fluorescence filters. Merged portions of these photographs were placed in boxed areas to show the yellow-appearing double-labelled fibres. Left column shows photomicrographs of thin nerve fibres ( pointed by arrows) immunoreactive for P2X3 (A and B) or TRPV1(C). Right column shows immunoreactivity of these same fibers to mGluR5 (A', B', C'). *: fluorescent artefact. c: spindle capsule wall. All scale bars = 10 µm.</p

Methods used to describe membrane properties of NVmes neurons.

<p>A: Hyperpolarizing and depolarizing current injections of 1 s duration were used to construct current voltage relationships. A cursor was used to measure the trans-membrane voltage just before the end of the current step. B: The points of inflection on the I–V curve were taken as the thresholds for inward and outward rectification. C: Data from the depolarizing current pulses was also used to calculate firing threshold, action potential amplitude and half-width, afterhyperpolarization (AHP) amplitude and duration. D: Maximum oscillation amplitude (inset), and thresholds for high-frequency oscillations and for burst firing were also measured with the cursor.</p

The thin axons in muscle spindles are not sympathetic fibers.

<p>Tyrosine hydroxylase (TH)-immunoreactive axons (light green in A, B; yellow in A',B') were seen close to muscle spindles and often over the capsule walls (c), but they were never seen among intrafusal muscle fibres. They were most often associated with blood vessels (*). Scale bars = 25 µm.</p

Long term effects of acidic saline on firing patterns of NVmes neurons.

<p>There was a significant difference in distribution between groups when neurons were distributed into the four categories (Χ² = 9.99, df = 3, p = 0.019), and also when grouped into Adapting and Non-adapting (T+B+TB) (Χ² = 6.85, df = 1, p = 0.009).</p