Effects of meperidine or saline on thermal, mechanical and electrical nociceptive thresholds in cats

Objective To measure cutaneous electrical nociceptive thresholds in relation to known thermal and mechanical stimulation for nociceptive threshold detection in cats.Study design Prospective, blinded, randomized cross-over study with 1-week washout interval.Animals Eight adult cats [bodyweight 5.1 +/- 1.8 kg (mean + SD)].Methods Mechanical nociceptive thresholds were tested using a step-wise manual inflation of a modified blood pressure bladder attached to the cat's thoracic limb. Thermal nociceptive thresholds were measured by increasing the temperature of a probe placed on the thorax. The electrical nociceptive threshold was tested using an escalating current from a constant current generator passed between electrodes placed on the thoracic region. A positive response (threshold) was recorded when cats displayed any or all of the following behaviors: leg shake, head turn, avoidance, or vocalization. Four baseline readings were performed before intramuscular injection of meperidine (5 mg kg(-1)) or an equal volume of saline. Threshold recordings with each modality were made at 15, 30, 45, 60, 90, and 120 minutes post-injection. Data were analyzed using ANOVA and paired t-tests (significance at p < 0.05).Results There were no significant changes in thermal, mechanical, or electrical thresholds after saline. Thermal thresholds increased at 15-60 minutes (p < 0.01) and mechanical threshold increased at 30 and 45 minutes after meperidine (p < 0.05). Maximum thermal threshold was +4.1 +/- 0.3 degrees C above baseline at 15 minutes while maximum mechanical threshold was 296 +/- 265 mmHg above baseline at 30 minutes after meperidine. Electrical thresholds following meperidine were not significantly different than baseline (p > 0.05). Thermal and electrical thresholds after meperidine were significantly higher than saline at 30 and 45 minutes (p < 0.05), and at 120 minutes (p < 0.05), respectively. Mechanical thresholds were significantly higher than saline treatment at 30 minutes (p <= 0.05).Conclusion and clinical relevance Electrical stimulation did not detect meperidine analgesia whereas both thermal and mechanical thresholds changed after meperidine administration in cats.

The role of the oblique ligament in the mechanical behavior of the medial collateral ligament of the mongrel dog elbow joint - Some biomechanical aspects

This study investigated the oblique ligament mechanical contribution to the medial collateral ligament of the canine elbow joint. Fifteen dogs were used for the study of the failure load, displacement, and energy absorption of the medial collateral and oblique ligaments of the canine elbow joint, associate and separately in the joint. Medial collateral ligament failure load and energy absorption were significantly higher in relation to the isolated oblique ligament. When the ligaments were associated in the joint, they presented an increment in failure load, displacement and energy absorption in relation to the ligaments analyzed separately. It was concluded, therefore, that the oblique ligament could have an important paper in the stability of the canine elbow joint, as it favors the medial collateral ligament resistance to the tensile load, one of the main stabilizer of the elbow joint.

Rufinamide attenuates mechanical allodynia in a model of neuropathic pain in the mouse and stabilizes voltage-gated sodium channel inactivated state

Background: Voltage-gated sodium channels dysregulation is important for hyperexcitability leading to pain persistence. Sodium channel blockers currently used to treat neuropathic pain are poorly tolerated. Getting new molecules to clinical use is laborious. We here propose a drug already marketed as anticonvulsant, rufinamide. Methods: We compared the behavioral effect of rufinamide to amitriptyline using the Spared Nerve Injury neuropathic pain model in mice. We compared the effect of rufinamide on sodium currents using in vitro patch clamp in cells expressing the voltage-gated sodium channel Nav1.7 isoform and on dissociated dorsal root ganglion neurons to amitriptyline and mexiletine. Results: In naive mice, amitriptyline (20 mg/kg) increased withdrawal threshold to mechanical stimulation from 1.3 (0.6–1.9) (median [95% CI]) to 2.3 g (2.2–2.5) and latency of withdrawal to heat stimulation from 13.1 (10.4–15.5) to 30.0 s (21.8–31.9), whereas rufinamide had no effect. Rufinamide and amitriptyline alleviated injury-induced mechanical allodynia for 4 h (maximal effect: 0.10 ± 0.03 g (mean ± SD) to 1.99 ± 0.26 g for rufinamide and 0.25 ± 0.22 g to 1.92 ± 0.85 g for amitriptyline). All drugs reduced peak current and stabilized the inactivated state of voltage-gated sodium channel Nav1.7, with similar effects in dorsal root ganglion neurons. Conclusions: At doses alleviating neuropathic pain, amitriptyline showed alteration of behavioral response possibly related to either alteration of basal pain sensitivity or sedative effect or both. Side-effects and drug tolerance/compliance are major problems with drugs such as amitriptyline. Rufinamide seems to have a better tolerability profile and could be a new alternative to explore for the treatment of neuropathic pain.

Mechanical signals regulating extracellular matrix gene expression in fibroblasts

Mechanical forces are essential for connective tissue homeostasis. The extracellular matrix (ECM) plays a key role in the transmission of forces generated by the organism (e.g. muscle contraction) and externally applied (e.g. gravity). The expression of specific ECM proteins such as collagens and tenascin-C, as well as of matrix metalloproteinases, involved in their turnover, is influenced by mechanical stimuli. The precise mechanisms by which mechanical strains are translated into chemical signals and lead to differential gene expression are however not fully understood. Cell-matrix adhesion sites are good candidates for hosting a "mechanosensory switch", as they transmit forces from the ECM to the cytoskeleton and vice versa by physically linking the cytoskeleton to the ECM. Integrins, transmembrane proteins located to these adhesion sites, have been shown to trigger a set of internal signaling cascades after mechanical stimulation. We have shown that the expression level of tenascin-C directly correlates with externally applied mechanical stress, as well as with RhoA/RhoA-dependent kinase-mediated cytoskeletal tension. Presumably other genes are regulated in a similar manner. The changes in ECM composition and mechanical properties derived from mechanical stress are relevant in medical intervention after ligament and tendon injury.

Effects of mechanical signaling on plant cell cytosolic calcium.

Mechanical signals are important influences on the development and morphology of higher plants. Using tobacco transformed with the Ca(2+)-sensitive luminescent protein aequorin, we recently reported the effects of mechanical signals of touch and wind on the luminescence and thus intracellular calcium of young seedlings. When mesophyll protoplasts are isolated from these transgenic tobacco plants and mechanically stimulated by swirling them in solution, cytoplasmic Ca2+ increases immediately and transiently up to 10 microM, and these transients are unaffected by an excess of EGTA in the medium. The size of the transient effect is related to the strength of swirling. Epidermal strips isolated from transgenic tobacco leaves and containing only viable guard cells and trichomes also respond to the strength of swirling in solution and can increase their cytoplasmic Ca2+ transiently up to 10 microM. Finally, the moss Physcomitrella patens containing recombinant aequorin exhibits transient increases in cytoplasmic Ca2+ up to 5 microM when swirled in solution. This effect is strongly inhibited by ruthenium red. Our data indicate that the effect of mechanical stimulation can be found in a number of different cell types and in a lower plant as well as tobacco and suggest that mechanoperception and the resulting increase in cytoplasmic Ca2+ may be widespread.

Molecular mechanisms involved in cell response to mechanical forces

New devices were designed to generate a localized mechanical vibration of flexible gels where human umbilical vein endothelial cells (HUVECs) were cultured. The stimulation setups were able to apply relatively large strains (30%~50%) at high temporal frequencies (140~207 Hz) in a localized subcellular region. One of the advantages of this technique was to be less invasive to the innate cellular functions because there was no direct contact between the stimulating probe and the cell body. A mechanical vibration induced by the device in the substrate gel where cells were seeded could mainly cause global calcium responses of the cells. This global response was initiated by the influx of calcium across the stretch-activated channels in the plasma membrane. The subsequent production of inositol triphosphate (IP3) via phospholipase C (PLC) activation triggered the calcium release from the endoplasmic reticulum (ER) to cause a global intracellular calcium fluctuation over the whole cell body. This global calcium response was also shown to depend on actomyosin contractility and F-actin integrity, probably controlling the membrane stretch-activated channels. The localized nature of the stimulation is one of the most important features of these new designs as it allowed the observation of the calcium signaling propagation by ER calcium release. The next step was to focus on the calcium influx, more specifically the TRPM7 channels. As TRPM7 expression may modulate cell adhesion, an adhesion assay was developed and tested on HUVECs seeded on gel substrates with different treatments: normal treatment on gels showed highest attachment rate, followed by the partially treated gels (only 5% of usual fibronectin amount) and untreated gels, with the lowest attachment rate. The trend of the attachment rates correlated to the magnitude of the calcium signaling observed after mechanical stimulation. TRPM7 expression inhibition by siRNA caused an increased attachment rate when compared to both control and non-targeting siRNA-treated cells, but resulted in an actual weaker response in terms of calcium signaling. It suggests that TRPM7 channels are indeed important for the calcium signaling in response to mechanical stimulation. A complementary study was also conducted consisting in the mechanical stimulation of a dissected Drosophila embryo. Although ionomycin treatment showed calcium influx in the tissue, the mechanical stimulation delivered as a vertical vibration did not elicited calcium signaling in response. One possible reason is the dissection procedure causing desensitization of the tissue due to the scrapings and manipulations to open the embryo.

Tear Secretion Induced by Selective Stimulation of Corneal and Conjunctival Sensory Nerve Fibers

Purpose. To measure the increase in tear secretion evoked by selective stimulation of the different populations of sensory receptors of the cornea and conjunctiva by using moderate and intense mechanical, chemical, and cold stimuli. Methods. Six healthy subjects participated in the study. Tear secretion was measured in both eyes by the Schirmer’s test conducted under control conditions and after stimulation of the center of the cornea and the temporal conjunctiva with a gas esthesiometer. Mechanical stimulation consisted in three pulses of 3 seconds’ duration of warmed air (at 34°C on the eye surface) applied at moderate (170 mL/min) and high (260 mL/min) flow rates. Cold thermal stimulation was made with cooled air that produced a corneal temperature drop of −1°C or −4.5°C. Chemical (acidic) stimulation was performed with a jet of gas containing a mixture of 80% CO2 in air. Results. The basal volume of tear secretion increased significantly (P < 0.05, paired t-test) after stimulation of the cornea with high-flow mechanical stimuli (260 mL/min), intense cooling pulses (−4.5°C), and chemical stimulation (80% CO2). The same stimuli were ineffective when applied to the conjunctiva. Moderate mechanical (170 mL/min) and cold (−1°C) stimulation of the cornea or the conjunctiva did not change significantly the volume of tear secretion. Conclusions. Reflex tear secretion caused by corneal stimulation seems to be chiefly due to activation of corneal polymodal nociceptors, whereas selective excitation of corneal mechanonociceptors or cold receptors appears to be less effective in evoking an augmented lacrimal secretion. Conjunctival receptors stimulated at equivalent levels do not evoke an increased tear secretion.

Expansion and chondrogenic potential of human articular chondrocytes on macroporous microcarriers

Regenerative medicine techniques are currently being investigated to replace damaged cartilage. Critical to the success of these techniques is the ability to expand the initial population of cells while minimising de-differentiation to allow for hyaline cartilage to form. Three-dimensional culture systems have been shown to enhance the differentiation of chondrocytes in comparison to two-dimensional culture systems. Additionally, bioreactor expansion on microcarriers can provide mechanical stimulation and reduce the amount of cellular manipulation during expansion. The aim of this study was to characterise the expansion of human chondrocytes on microcarriers and to determine their potential to form cartilaginous tissue in vitro. High-grade human articular cartilage was obtained from leg amputations with ethics approval. Chondrocytes were isolated by collagenase digestion and expanded in either monolayers (104 cells/cm2) or on CultiSpher-G microcarriers (104 cells/mg) for three weeks. Following expansion, monolayer cells were passaged and cells on microcarriers were either left intact or the cells were released with trypsin/EDTA. Pellets from these three groups were formed and cultured for three weeks to establish the chondrogenic differentiation potential of monolayer-expanded and microcarrier-expanded chondrocytes. Cell viability, proliferation, glycosaminoglycan (GAG) accumulation, and collagen synthesis were assessed. Histology and immunohistochemistry were also performed. Human chondrocytes remained viable and expanded on microcarriers 10.2±2.6 fold in three weeks. GAG content significantly increased with time, with the majority of GAG found in the medium. Collagen production per nanogram DNA increased marginally during expansion. Histology revealed that chondrocytes were randomly distributed on microcarrier surfaces yet most pores remained cell free. Critically, human chondrocytes expanded on microcarriers maintained their ability to redifferentiate in pellet culture, as demonstrated by Safranin-O and collagen II staining. These data confirm the feasibility of microcarriers for passage-free cultivation of human articular chondrocytes. However, cell expansion needs to be improved, perhaps through growth factor supplementation, for clinical utility. Recent data indicate that cell-laden microcarriers can be used to seed fresh microcarriers, thereby increasing the expansion factor while minimising enzymatic passage.

Adult human articular chondrocytes in a microcarrier-based culture system : expansion and redifferentiation

xpanding human chondrocytes in vitro while maintaining their ability to form cartilage remains a key challenge in cartilage tissue engineering. One promising approach to address this is to use microcarriers as substrates for chondrocyte expansion. While microcarriers have shown beneficial effects for expansion of animal and ectopic human chondrocytes, their utility has not been determined for freshly isolated adult human articular chondrocytes. Thus, we investigated the proliferation and subsequent chondrogenic differentiation of these clinically relevant cells on porous gelatin microcarriers and compared them to those expanded using traditional monolayers. Chondrocytes attached to microcarriers within 2 days and remained viable over 4 weeks of culture in spinner flasks. Cells on microcarriers exhibited a spread morphology and initially proliferated faster than cells in monolayer culture, however, with prolonged expansion they were less proliferative. Cells expanded for 1 month and enzymatically released from microcarriers formed cartilaginous tissue in micromass pellet cultures, which was similar to tissue formed by monolayer-expanded cells. Cells left attached to microcarriers did not exhibit chondrogenic capacity. Culture conditions, such as microcarrier material, oxygen tension, and mechanical stimulation require further investigation to facilitate the efficient expansion of clinically relevant human articular chondrocytes that maintain chondrogenic potential for cartilage regeneration applications.

CD73 and CD29 concurrently mediate the mechanically induced decrease of migratory capacity of mesenchymal stromal cells

The assumption that mesenchymal stromal cell (MSC)-based therapies are capable of augmenting physiological regeneration processes has fostered intensive basic and clinical research activities. However, to achieve sustained therapeutic success in vivo, not only the biological, but also the mechanical microenvironment of MSCs during these regeneration processes needs to be taken into account. This is especially important for e.g., bone fracture repair, since MSCs present at the fracture site undergo significant biomechanical stimulation. This study has therefore investigated cellular characteristics and the functional behaviour of MSCs in response to mechanical loading. Our results demonstrated a reduced expression of MSC surface markers CD73 (ecto-5’-nucleotidase) and CD29 (integrin β1) after loading. On the functional level, loading led to a reduced migration of MSCs. Both effects persisted for a week after the removal of the loading stimulus. Specifi c inhibition of CD73/CD29 demonstrated their substrate dependent involvement in MSC migration after loading. These results were supported by scanning electron microscopy images and phalloidin staining of actin fi laments displaying less cell spreading, lamellipodia formation and actin accumulations. Moreover, focal adhesion kinase and Src-family kinases were identified as candidate downstream targets of CD73/CD29 that might contribute to the mechanically induced decrease in MSC migration. These results suggest that MSC migration is controlled by CD73 CD29, which in turn are regulated by mechanical stimulation of cells. We therefore speculate that MSCs migrate into the fracture site, become mechanically entrapped, and thereby accumulate to fulfil their regenerative functions.

A case for optimising fracture healing through inverse dynamization

The mechanical conditions in the repair tissues are known to influence the outcome of fracture healing. These mechanical conditions are determined by the stiffness of fixation and limb loading. Experimental studies have shown that there is a range of beneficial fixation stiffness for timely healing and that fixation stiffness that is either too flexible or too stiff impairs callus healing. However, much less is known about how mechanical conditions influence the biological processes that make up the sequence of bone repair and if indeed mechanical stimulation is required at all stages of repair. Secondary bone healing occurs through a sequence of events broadly characterised by inflammation, proliferation, consolidation and remodelling. It is our hypothesis that a change in fixation stiffness from very flexible to stiff can shorten the time to healing relative to constant fixation stiffness. Flexible fixation has the benefit of promoting greater callus formation and needs to be applied during the proliferative stage of repair. The greater callus size helps to stabilize the fragments earlier allowing mineralization to occur faster. Together with stable/rigid fixation applied during the latter stage of repair to ensure mineralization of the callus. The predicted benefits of inverse dynamization are shortened healing in comparison to very flexible fixation and healing time comparable or faster than stable fixation with greater callus stiffness.

Development of a novel experimental model to investigate the influence of mechanics on bone healing

This dissertation proposed a novel experimental model combining a defect configuration with an active instrumented fixation device to investigate the influence of mechanics on bone healing. The proposed defect configuration aimed to minimise physiological loading within an experimental fracture gap and the instrumented fixator was used for the application of controlled displacements and in vivo stiffness monitoring of the healing process. This thesis has provided a novel approach to advance current knowledge and understanding of mechanobiology, which has been limited in previous experimental models.

不同生活型植物对机械刺激的反应

机械刺激效应（Effects of mechanical stimulation）是风生态学直接效应的主要表现形式。研究机械刺激对植物的影响，有利于将风的直接与间接效应区别开来，从而精确研究植物对风的反应格局;研究不同生活型植物对机械刺激的反应是植物力学与植物生态学的重要内容，有助于揭示植物对生境的适应机制。 本研究涉及三个实验。第一个实验探讨不同生活型植物对机械刺激和水分互作的响应格局;第二个实验研究匍匐茎草本植物蛇莓对部分机械刺激的反应;最后一个实验揭示不同水分供应条件下，番茄和紫花苜蓿对不同机械刺激频度的响应。在第一个实验中，克隆半灌木羊柴（Hedysarum laeve），一年生草本植物虫实（Corispermum mongolicum），多年生大型禾草沙鞭（Psammochloa villosa）和多年生丛生禾草黑麦草（Lolium perenne）分别接受由两个水平机械刺激（无刺激和刺激60 s d-1）和两个水平水分供应（200 ml d-1和400 ml d-1）组成的处理。在第二个实验中，匍匐茎草本蛇莓（Duchesnea indica）接受4个不同水平的机械刺激：（1）整个克隆不受机械刺激;（2）整个克隆都受机械刺激;（3）除顶端外其余克隆部分受机械刺激;（4）仅克隆顶端受机械刺激，其余部分不受机械刺激。在最后一个实验中，番茄（Lycopersicon esculentum）和紫花苜蓿（Medicago sativa）接受由三个水平机械刺激频度（0，25赫兹和50赫兹）和三个水平水分供应（50ml，150ml和250ml）组成的处理。这些实验主要回答不同生活型植物的生长和（或）机械性状如何响应机械刺激。主要结果如下： （1）在对机械刺激和水分交互效应的实验中，交互效应随物种发生变化。机械刺激和水分的交互效应对羊柴、番茄和紫花苜蓿作用不显著，但对虫实、沙鞭和黑麦草作用显著。 （2）在对机械刺激的研究中，机械刺激对植物的效应有正负之分。如机械刺激降低羊柴和沙鞭的总生物量，表明其是一种胁迫因子。但对于虫实、番茄和紫花苜蓿来说，机械刺激却能不同程度地促进植物的生长。 （3）机械刺激对虫实的机械性状没有显著影响，但对羊柴的机械性状恰好相反。此外，水分对虫实机械性状有显著影响。 （4）不同植物对机械刺激频度的敏感性存在差异。对番茄来而言，50赫兹的机械刺激对其生长具有较强的促进作用;对紫花苜蓿来说，25赫兹的机械刺激对其生长具有较强的促进作用。 （5）蛇莓对局部机械刺激具有显著反应，特别是在顶端进行机械刺激的处理中，整个克隆片段的叶柄长度缩短，根冠比发生改变，将较多的生物量分配到根。 这些结果表明：（1）不同物种对机械刺激和水分互作的反应可能与机械震动方式及物种本身有关;（2）单位植物大小所承受的机械刺激的强度及物种的生长速率是不一样的;（3）不同反应间的相互作用及相互独立可部分解释物种间的效应差异;（4）接触性形态建成的效应不能从一个物种外推到另外一个物种。 以前的研究集中探讨直立茎植物对机械刺激的响应，而对匍匐茎植物的研究极为贫乏。我们对蛇莓部分机械刺激的研究仅仅是一个初步探索。蛇莓的可塑性行为可能是一种适应性策略，因为这类植物常常生长在机械刺激频繁的开阔生境中。上述三个实验仅仅从生长和机械角度探讨了植物的适应性，而要真正揭示植物对多风生境的适应需要对不同物种进行多水平、多层面的研究，以期掌握不同生活型植物对机械刺激响应的一般格局。例如，从激素、细胞、解剖结构等方面探索其内在机制。

Intercellular calcium wave propagation in linear and circuit-like bone cell networks

In the present study, the mechanism of intercellular calcium wave propagation in bone cell networks was identified. By using micro-contact printing and self-assembled monolayer technologies, two types of in vitro bone cell networks were constructed: open-ended linear chains and looped hexagonal networks with precisely controlled intercellular distances. Intracellular calcium responses of the cells were recorded and analysed when a single cell in the network was mechanically stimulated by nano-indentation. The looped cell network was shown to be more efficient than the linear pattern in transferring calcium signals from cell to cell. This phenomenon was further examined by pathway-inhibition studies. Intercellular calcium wave propagation was significantly impeded when extracellular adenosine triphosphate (ATP) in the medium was hydrolysed. Chemical uncoupling of gap junctions, however, did not significantly decrease the transferred distance of the calcium wave in the cell networks. Thus, it is extracellular ATP diffusion, rather than molecular transport through gap junctions, that dominantly mediates the transmission of mechanically elicited intercellular calcium waves in bone cells. The inhibition studies also demonstrated that the mechanical stimulation-induced calcium responses required extracellular calcium influx, whereas the ATP-elicited calcium wave relied on calcium release from the calcium store of the endoplasmic reticulum.

Nociception or pain in a decapod crustacean?

Nociception is the ability to perceive a noxious stimulus and react in a re flexive manner and occurs across a wide range of taxa. However, the ability to experience the associated aversive sensation and feeling, known as pain, is not widely accepted to occur in nonvertebrates. We examined the responses of a decapod crustacean, the prawn, Palaemon elegans, to different noxious stimuli applied to one antenna to assess reflex responses (nociception) and longer-term, specifically directed behavioural responses that might indicate pain. We also examined the effects of benzocaine, a local anaesthetic, on these responses. Noxious stimuli elicited an immediate reflex tail flick response, followed by two prolonged activities, grooming of the antenna and rubbing of the antenna against the side of the tank, with both activities directed specifically at the treated antenna. These responses were inhibited by benzocaine; however, benzocaine did not alter general swimming activity and thus the decline in grooming and rubbing is not due to general anaesthesia. Mechanical stimulation by pinching also resulted in prolonged rubbing, but this was not inhibited by benzocaine. These results indicate an awareness of the location of the noxious stimuli, and the prolonged complex responses indicate a central involvement in their organization. The inhibition by a local anaesthetic is similar to observations on vertebrates and is consistent with the idea that these crustaceans can experience pain.