*Metabolic Disorders in Patients with Chronic Osteomyelitis: Etiology and Pathogenesis DOI: http://dx.doi.org/10.5772/intechopen.92052*

several authors [14, 31, 32], capable of persisting in osteoblasts and that leads to additional stimulation of inflammatory process that stepwise turns into local hyperergic reaction. It is important to take into account that pathogens also play a role in matrix destruction—particularly *S. aureus* can adhere to matrix elements and destroy them [33].

The hyperergic reaction leads to excessive synthesis of matrix metalloproteases (MMPs). Among them, MMP-2 (gelatinase A) and MMP-9 (gelatinase B) [34] play the main role. Excessive synthesis of MMP is a key point in the process because it has two major effects:

**Figure 4.**

*Pathogenetic stages and processes associated particularly with bone matrix destruction.*

*Clinical Implementation of Bone Regeneration and Maintenance*

OPG is a soluble glycoprotein synthesized by different cell types, including osteoblasts [29]. This compound can be found in monomeric (50 kDa) or dimeric (120 kDa) form (the dimeric form contains disulfide bonds between monomers). Dimeric form has a higher affinity to RANKL than a monomeric one, and the

RANKL (RANK-ligand) is a compound synthesized by several cell types. It can be expressed in three different molecular forms: transmembrane trimer, primary (secreted) form, and cell ectodomain [30]. RANKL serves as a ligand for RANK receptor that activates osteoclasts. RANKL is a target for osteoprotegerin; if OPG is secreted in low amounts, more RANKL remains unbound and the intensity of

Therefore, a "pathogenetic chain" is as follows: infection-osteoblast cell death

The matrix destruction in infection can be explained by several factors. The "starting point" in pathogenesis is the infection itself. *S. aureus* is, as shown by

and proliferation rate decrease-OPG synthesis decrease-RANKL inhibition decrease-osteoclastogenesis activation-bone resorption increase-shift of bone remodeling toward a resorption prevalence-decrease in bone tissue quality and quantity in the infection site. The resulting clinical effect is slowing of the consoli-

dation of the bone tissue defects, pseudarthroses, and related conditions.

chemical analogues of OPG should have the features of dimeric form.

*Pathogenetic stages and processes associated particularly with bone turnover shift.*

**142**

resorption increases.

**Figure 3.**

**5. Matrix destruction**

The matrix destruction leads to increase in following resorption markers: B-cross-laps (С-terminal telopeptide) and desoxypyridinoline/creatinine ratio.

A clinician should note that the destruction of the matrix is combined with lowering of its synthesis by osteoblasts. Therefore, both processes act in a synergic way and occur at the same time, which negatively influences the treatment (**Figure 4**).


**5. Application of EB drawing to formation of nano-Si-dot and nano-polymerpit arrays with a pitch of 25 nm x 25 nm using EB drawing, reactive ion etching (RIE) and nano-imprinting [23, 25]**

The possibility of forming very fine pits or dots with a pitch of less than 25 nm was researched using reactive ion etching (RIE) and nano-imprinting with EB drawn pattern as a mask for the future process. We were able to fabricate ultrahighly packed dot arrays with a dot diameter of less than 15 nm and a dot pitch of 25 nm x 25 nm in negative calixarene resist using EB drawing. We also formed nan-Si dot arrays patterns by CF4 RIE. Furthermore, pit arrays were formed in polymer film through nano-imprinting by the photo-polymer method using a Si dot arrays pattern as the master mold. We demonstrated that the EB-drawn dot arrays resist pattern is very suitable for the fabrication of Si dot arrays and pit arrays with a pitch of 25 nm x 25 nm in this polymer. The Si dot and pit diameters were less than 10 nm.

Fig. 5.1. Process flow for Si nano-dot arrays by RI etching and ashing.


Manipulation of the gut microbiota through the use of probiotic and prebiotics treatments have shown that by augmenting so-called "good bacteria" such as *Bifidobacteria* and *Lactobacillus*, in the gut, visceral hypersensitivity can be reversed in preclinical models. A mixture of eight probiotic bacteria strains (VSL#3) was shown to have protective effects against development of visceral hypersensitivity in the neonatal maternal separation model. Moreover, TPH1, tryptophan hydroxylase 1, the gene for the enzyme responsible for synthesizing serotonin, a key neurotransmitter involved in IBS treatment, was markedly up-regulated by neonatal maternal separation and this effect was reversed by VSL#3 intervention (239). Moreover, the same cocktail of probiotics was shown to prevent visceral hypersensitivity induced by inflammation via intra-colonic instillation of 4% acetic acid when given prophylactically (240). *Bifidobacterium* species particularly, *Bifidobacterium infantis* 35624 has been shown to be particularly effective at ameliorating visceral hyperalgesia in both stressinduced visceral hypersensitivity and colitis (241–243). Moreover, *Lactobacillus* species have also displayed efficacy in visceral pain models (244–247).

Furthermore, antibiotic-induced visceral hypersensitivity again underpins a role of the gut microbiota in the pathophysiology of visceral pain (244, 248). Interestingly, rifaximin, a semisynthetic, non-absorbable antibiotic that demonstrates no clinically relevant bacterial resistance has also shown positive effects in the treatment of IBS (249–257). These findings may seem contradictory, however, rifaximin is particularly efficacious in cases of small bowel bacterial overgrowth found in IBS patients. These findings add to the growing literature that microbiota dysfunction may be a key player in the pathophysiology of IBS and may lead to future novel therapeutic interventions.

## **IMMUNE SYSTEM AND VISCERAL PAIN**

The immune system and thus inflammation have long been associated with psychiatric disorders, in particular, depression (258– 260) and chronic pain disorders (261). Depression is a common comorbidity of visceral pain, as discussed earlier, so it is not surprising that a common mechanism such as neuroinflammation may be at play. The immune system is a critical component of the microbiota–brain–gut axis and plays vital roles in maintaining homeostasis in the nervous systems and GI tract (262). Moreover, direct communication occurs between the immune system and the HPA axis, autonomic nervous system (ANS) and ENS (263– 267). These integrated pathways are all known to be involved in the pathophysiology of visceral pain, and thus it is not unforeseen that the immune system plays a key role in the development of visceral hypersensitivity.

## **MICROGLIA AND VISCERAL PAIN**

Microglia represent the first line of defense for the CNS, acting as a sensor for pathological events (268). The process of central sensitization and the subsequent changes in synaptic plasticity has long been thought to play a major role in nociceptive processes both in the context of chronic pain as well as acute, in both somatic and visceral modalities (269, 270). In the last decade, the role of microglia, both spinal and supra-spinal, has become an area of interest in the context of nociception (271–273). In animal models of both inflammatory and neuropathic pain, activation of microglia is a key step in the onset and maintenance of hypersensitivity and allodynia (274–279).

The role of spinal microglia in visceral pain has only recently been addressed and reviewed nicely by Lu (269). Saab and colleagues were first to report increased activated microglia in a rat model of chronic visceral hyperalgesia, namely the neonatal colon irritation model (280). Moreover, minocycline, a secondgeneration tetracycline compound known to interrupt microglia activation and its associated pro-inflammatory response, reversed the visceral hypersensitivity in adulthood (269, 280).

Furthermore, enhanced microglia activation was reported in the hippocampus, in a model of trinitrobenzene sulfonic acid (TNBS)-induced colitis, concomitant with increased tumor necrosis factor-α (281). However, this study did not assess nociceptive behavior, but one could predict visceral hyperalgesia as observed by others in the TNBS-model (282). More recently, it was shown that the activation of spinal microglia plays a critical role in the initiation and maintenance of visceral hypersensitivity in the TNBSinduced chronic pancreatitis (CP) rat model (283). Intrathecal injection of minocycline attenuated visceral hypersensitivity and phospho-p38 levels in CP rats.

In other non-inflammatory models of visceral hyperalgesia, it has also been shown that chronic psychological stress leads to microglia activation in the lumbar spinal cord (284). Moreover, this stress-induced increase in activated microglia could be blocked by SB203580, a p38 inhibitor or minocycline. Other compounds known to alter the microglia phenotype such as FKN, a microglia activator, was shown to induce visceral hypersensitivity in naïve rats when administered spinally, an effect which was blocked by minocycline (269), thus adding further evidence to support the role of spinal microglia in mediating stress-induced visceral hypersensitivity (269). Taken together, these studies suggest that microglia may play an important role in the pathogenesis of visceral pain.

However, it is also pertinent to note that exact role of inflammation and indeed micro-inflammation still remains controversial in the context of IBS. Indeed, no effect of anti-inflammatory drugs has been shown in IBS such as prednisolone (285).

## **TOLL-LIKE RECEPTORS AS NOVEL THERAPEUTIC TARGETS FOR VISCERAL ANALGESIA**

Toll-like receptors (TLRs) are a family of pattern-recognition receptors of the innate immune system (**Figure 4**). There are 10 human TLRs and 12 mouse TLRs (286). TLR signaling consists of at least two distinct pathways: (1) the MyD88-dependent pathway, which leads to a pro-inflammatory phenotype, and (2) the MyD88-independent pathway, which leads to the production of interferon-β and the maturation of dendritic cells. The MyD88 dependent pathway is common to all TLRs, except TLR3 (287). TLRs are important players in the maintenance of mucosal and commensal homeostasis within the gut via innate host defense mechanisms. Intestinal dysbiosis and inflammation underlie several disease states affecting the GI tract such as IBS and IBD (288, 289). Indeed, reports have shown the involvement of peripheral TLR4 in patients suffering from IBS (290, 291) and in animal models of visceral pain (289, 292, 293). Moreover, the presence of TLR4 in the ENS and in the dorsal root ganglia indicate a role for TLR4 in the transmission of sensory information from the GI tract (294, 295). Furthermore, TLR4 is also expressed within the CNS, predominately in microglia (296), which have been discussed above and their role in visceral pain. Moreover, it is now emerging as a possible therapeutic target for neuropathic pain (297). Taken together, these findings suggest TLR4 as a promising novel target for the treatment of visceral pain.

## **Interaction of TLRs and Opioid Receptors**

In recent years, it has been shown using *in vivo, in vitro*, and *in silico* techniques that members of each structural class of opioids (µ, κ, δ) activate TLR4 (298). Moreover, opioid antagonists such as naloxone and naltrexone non-stereoselectively block TLR4 signaling (299, 300). Modulation of TLR4 expression/function by acute blockade of TLR4, genetic knockout of TLR4, or blockade of TLR4 downstream signaling each lead to a potentiation of the magnitude and duration of opioid analgesia (301). These effects are thought to be mediated at both spinal and supra-spinal sites (299). Given the breadth of opioids now documented to interact with TLR4, many off-target opioid effects previously attributed to unilateral opioid action at classical neuronal opioid receptors might in fact result, at least in part, from the duality of opioid actions at TLR4 (301).

## **GENDER AND VISCERAL PAIN**

Sex differences in pain sensitivity has been a topic of debate for many years and was recently reviewed by Mogil (302).  Moreover, we can now appreciate that gender differences are also apparent in analgesic response (305). Many forms of visceral pain due to their nature are especially prevalent in women, i.e., pain associated with reproductive function (menstruation pains, pain of child birth, or postmenopausal pelvic pain). IBS is a disorder predominated by females (female to male ratio ~ 2:1) which is in parallel with women also being more

susceptible to stress-related disorders (306, 307). Indeed, many studies have reported sex differences in the stress response itself and stress-induced pain modulation (8, 308). However, in the case of animal studies, the picture is not as clear. This is due in part to a bias for using male rodents in most preclinical studies addressing the role of stress modulation on visceral hypersensitivity (134, 135, 289, 309–311).

Sex/gender can influence the brain–gut axis, at an array of sites which logically will effect subsequent clinical outcomes including response to behavioral and drug therapies in patients suffering with visceral hypersensitivity (312). A plethora of factors including mood, stress, gender role, hormones, as well as inflammatory mediators modulate the brain–gut axis (313). As a result, the explanation for gender-related differences in visceral pain is likely multi-factorial involving environmental, psychological, and biological (sex) influences (312, 314, 315).

Studying gender differences in any context comes with significant difficulties, not least in the context of visceral pain disorders. Due to the greater number of women who are diagnosed with these disorders, there is often insufficient power to detect differences in the etiologic factors and treatment response by gender or sex. For example, several studies examining the effectiveness of cognitive behavioral therapy in reducing symptom distress have included only women (316, 317) or were disproportionately composed of women (318). In particular, the smaller number of men in drug trials leaves many studies underpowered to examine gender differences (313).

The variability in reproductive hormones such as estrogen and progesterone, across the menstrual cycle as well as significantly reduced ovarian function during and after menopause may explain changes in GI motility and visceral sensitivity (319). This has been reported in several studies documenting the impact of menstrual cycle on GI symptom reporting including visceral pain (320–323). The most consistent finding is that, many women with and without FGIDs experience an increase in GI symptoms including visceral pain during the late luteal and menses phases of the cycle relative to other cycle phases (313, 323–325). To add to the complexity, is the addition of pharmacological agents known to impact on the menstrual cycle and associated functions, such as oral contraceptive use and hormone replacement therapy. Moreover, the presence of other reproductive organ problems unique to women such as dysmenorrhea can also confound such studies.

It has been well established that adult females have higher basal and stress levels of ACTH and corticosterone than do males (326– 330). Moreover, there is convergent lines of evidence reporting that gonadal hormones, specifically the estrogens, are important regulators of the HPA axis. Indeed, estrogen receptors α and β themselves have been shown to increase CRH expression (8, 331, 332). Females have higher CRH, ACTH, and corticosterone levels during proestrus (333), the phase of the cycle in which estradiol levels are highest, than during other phases of the estrous cycle (334–336). Fluctuations in gonadal hormones specifically, estradiol, during the menstrual cycle can also lead to changes in the neurotransmitter systems, in particular, the serotonergic and glutamatergic systems. Interactions between these systems have implications in the etiology and treatment of stress-related disorders and pain circuitry (142, 307, 337–340). Moreover, gonadal hormones have been shown to significantly affect visceral sensitivity in animal models, however some conflicting results have also shown no effect again highlighting the complexity of sex differences in pain processing both in a preclinical and clinical setting (173, 341–344). Interestingly, the gender of the experimenter was also shown to have a role in pain responses in preclinical models (345). Taken together, it is clear that the role of gender and/or sex in the pathophysiology of visceral pain remains a complex phenomenon which requires further investigation.

## **GLUTAMATE AND VISCERAL PAIN**

### **IONOTROPIC GLUTAMATE RECEPTORS AND VISCERAL PAIN**

N-Methyl-D-aspartate (NMDA) receptors are not only known for their important role in excitatory synaptic transmission but also for their role in pain (346). Glutamate found in vagal and spinal afferents contributes to nociceptive signaling via NMDA and non-NMDA receptors (347). Though evidence for direct links between glutamate and visceral nociception is lacking, NMDA receptor antagonists have been shown to reduce the response of vagal and pelvic afferents in the colon and other viscera to mechanical stimuli (348). A study by Coutinho and colleagues (349) investigated the role of glutamate receptors in the RVM in visceral hyperalgesia using a colonic irritation model. They found that activation of NMDA receptors by colonic inflammation facilitated visceral hyperalgesia while non-NMDA receptors mediate inhibitory effects (349). Moreover, NMDA receptors may also be involved in the transmission of visceral nociception in the non-inflamed gut (350). It was observed that activated peripheral NMDA receptors in the colon caused the release of pro-inflammatory peptides, calcitonin gene-related peptide (CGRP) and substance-P, which have significant roles in the mediation of chronic and severe pain (350).

## **METABOTROPIC GLUTAMATE RECEPTORS AND VISCERAL PAIN**

Metabotropic glutamate receptors with the exception of mGlu6 receptor are expressed in all areas of the pain matrix from the spinal cord to supra-spinal sites. The action of mGlu receptors can be pro-nociceptive or anti-nociceptive depending on the subtype and site of action (351).

In the last decade, the role of mGlu receptors has gained attention in the realm of visceral pain. To our knowledge, the first study explicitly investigating the role of these receptors in visceral nociceptive processes was performed by Chen et al. (352). They found that antagonism of group 1 mGlu receptors with LY393053 reduced nociceptive behaviors in the mouse acetic acid writhing test. Furthermore, administration of a group 1 mGlu receptor agonist (S)-3,5-dihydroxyphenylglycine (DHPG) into the CeA by microdialysis increased the responses to innocuous visceral stimulation; an effect that was reversed by a reactive oxygen species (ROS) scavenger phenyl-*N-t*-butyl nitrone (PBN) and a superoxide dismutase mimetic (TEMPOL). In the same study, mGlu receptor 1 antagonist LY367385 was also found to decrease the responses to visceral stimulation (353).

Moreover, work by Lindstrom and colleagues investigated specifically the role of mGlu5 receptor in a rat model of visceral hypersensitivity (111). Here they found that mGlu5 receptor antagonism with MPEP [2-Methyl-6-(phenylethynyl)-pyridine] and MTEP [3-(2-Methyl-1,3-thiazol-4-yl)ethynyl]pyridine was sufficient to reduce the visceromotor response (VMR) in conscious Sprague-Dawley (SD) rats without altering colonic compliance. Moreover, mGlu5 receptor antagonism reduced colorectal distension (CRD)-evoked increases in heart rate and blood pressure (111). In this study, the effects seen could not be conclusively due to a peripheral or central site of action. Indeed, it has also been found that noxious colonic stimulation increases the number of Fos-positive neurons in the dorsal horn of the thoracic and lumbar spinal cord. Moreover, pre-treatment with MPEP significantly attenuated this (354).

Visceral pain originating from the bladder has also been shown to be mediated via mGlu receptors; specifically mGlu receptor 5 activation in the CeA induces bladder pain sensitization by increasing CeA output, an effect that was reversed by intra-amygdala MPEP treatment and lentivirus-mediated conditional disruption of mGlu5 receptor in the CeA (70).

## **GLUTAMATE TRANSPORTERS AND VISCERAL PAIN**

Due to the negative side effects (psychomimetic activity) seen with modulation of ionotropic receptors, compounds which target these receptors are not suitable for long-term treatment of pain. To redress this and in the drive to develop better analgesics, other mechanisms such as glutamate reuptake may provide more effective treatments in controlling glutamate neurotransmission and thereby exert anti-nociceptive effects (109).

One of the first clear demonstrations that glutamate transport [via excitatory amino-acid transporters (EAATs)] may be implicated in pain processing was performed by Liaw and colleagues where they showed that selective inhibition of glutamate transporters with dl-threo-β-benzyloxyaspartate (TBOA) and dihydrokainate (DHK) produced a dose-dependent spontaneous nociceptive behavior response. These behaviors included licking, shaking, and caudally directed biting (355). Moreover, TBOA administered intrathecally was also found to induce visceral nociceptive behaviors in naïve rats (114). To further unravel the role of glutamate transport in visceral nociception, in particular stress-induced visceral hypersensitivity, the neuroprotective drug riluzole known to activate glutamate reuptake, was investigated to see whether it could attenuate visceral hypersensitivity induced by maternal separation of rats. It was found that riluzole reduces visceral hypersensitivity in stressed animals only, having no effect in non-separated animals. Moreover, EAAT-1 expression was also found to be reduced in the lumbar region of the spinal cord in hyper-sensitive animals. As mentioned previously, riluzole does not affect visceral sensitivity in naïve animals, further emphasizing the role of glutamate transport in pathological pain (114).

EAAT-2 is the main glial transporter for glutamate reuptake and its experimental over-expression in animal models has recently been found to be effective in reducing visceral pain (59, 112, 113). A study by Lin and colleagues (112) found the over-expression of EAAT-2 with the use of cephalosporin antibiotic ceftriaxone, in wildtype mice, attenuated the visceral nociceptive response to CRD. Moreover, similar effects were seen in the EAAT-2 overexpressing transgenic mouse (112). To further support these findings, systemic and intrathecal administration of DHK, a selective EAAT-2 inhibitor blocks the ceftriaxone-induced attenuation of visceral pain (112, 113). In subsequent studies performed by the same group assessing the efficacy of glutamate reuptake in animal models of colitis, it was shown that adeno-associated virusmediated EAAT-2 over-expression was effective to mitigate VMR to 60 mmHg CRD (113). Interestingly, overexpression of EAAT-2 has been shown to reduce bladder nociception (59). However, colon irritation may affect afferents innervating the bladder thus giving a plausible mechanism for cross-organ sensitization (59). In the same study, it was also found that enhanced expression of EAAT-2 by ceftriaxone also reduced the VMR to bladder distension caused by colonic irritation (59).

As stated earlier, stress is one of the main predisposing factors for the development of visceral pain. Interestingly, glutamate transport has also been shown to be altered due to stress, with both early-life stress and stress in adulthood both showing significant alteration in EAAT expression (114, 115). Taken together, these findings provide evidence for an important role of glutamate transport in visceral pain states (271, 356). Moreover, glutamatergic signaling is critical to the process of central sensitization. This term describes the way in which excessive glutamatergic function within neurons and circuits, in particular pain circuits, leads to increases in membrane excitability and synaptic efficacy as well as reduced inhibition. These changes in synaptic function manifest as altered plasticity within the somatosensory nervous system in response to activity, inflammation, and neural injury (357).

## **GABA RECEPTORS AND VISCERAL PAIN**

γ-Aminobutyric acid (GABA), the major inhibitory neurotransmitter in the CNS, plays an important role in anti-nociception. GABA is the key player acting at inhibitory synapses where it exerts its effect by binding to it respective receptors both pre- and post-synaptically. This binding causes conformational change and subsequent opening of the ion channel. The direction of the flow of ions (in/out) and their charge (+/−) result in a negative change in the transmembrane potential, causing hyperpolarization. To date, two classes of GABA receptors are known: (1) GABA<sup>A</sup> receptors are ligand-gated ion channels, and (2) GABA<sup>B</sup> receptors are metabotropic receptors, which are G protein-coupled receptors.

Pharmacological modulation of the GABA<sup>A</sup> receptor through the use of agonists and antagonists indicate that modulation of these circuits within the spinal cord has important implications for pain processing (358–360). However, these analgesic effects particularly seen with benzodiazepines may not be purely anti-nociceptive actions and maybe more non-specific centrally mediated effects.

GABA's anti-nociceptive effects are thought to be mediated by GABA<sup>B</sup> receptors, which are ideally located in both the brain and spinal cord with ubiquitous expression. Moreover, GABA<sup>B</sup> receptors have also been implicated in a whole host of GI functions, such as altering GI motility and visceral sensation (361). The mostly widely used GABA<sup>B</sup> receptor agonist, baclofen has been shown to produce anti-nociception in numerous rat models of visceral pain (67, 362–365). Moreover, subcutaneous injection of baclofen was shown to prevent behavioral responses to bladder pain (362). Likewise, intrathecal administration of baclofen increases the sensitivity threshold to CRD (363). Furthermore, intravenous administration of baclofen attenuated CRD-evoked increases in arterial blood pressure and heart rate (67, 364).

Intraperitoneal injection of baclofen was shown to alter the expression of early immediate genes such as c-fos in the lumbosacral spinal cord after intra-colonic mustard oil-induced inflammation (366, 367). Moreover, it has also been shown via electrophysiological examination that GABA<sup>B</sup> receptor agonists have the ability to modulate responses of vagal mucosal and muscle afferents innervating particular parts of the GI tract, specifically, the esophagus and proximal stomach (368–370).  This data provide evidence that GABA<sup>B</sup> receptor agonists may exert their positive effects by acting at peripheral sites.

However, it is important to note the effects of GABA<sup>B</sup> receptor agonism,other than its anti-nociceptive effect,are undesirable centrally mediated effects, including sedation, respiratory depression, drug tolerance, and motor deficiency, thus, its potential therapeutic use could be significantly curtailed (367). Interestingly, GABA receptor pharmacology is complex with dimerization required for many subunits to form a functional receptor. Moreover, a number of splice variants of the GABAB1 subunit (GABAB1a, GABAB1b, GABAB1c, and GABAB1d) have been identified both in rat and human tissues, and have been found to be differentially expressed depending on the tissue type (372, 373).

Indeed GABA analogs such as gabapentin and pregabalin have also shown efficacy in preclinical models of visceral hypersensitivity (105, 106, 108, 374–377) and moreover, pregabalin has undergone clinical trials for painful CP (107). However, these compounds may exert their effects indirectly in the GABAergic system, as their main mode of action is on α2δ subunits of Ca<sup>V</sup> (375). The emergence of positive allosteric modulators of GABA<sup>B</sup> may provide a novel therapeutic target for treatment of visceral pain disorders (66).

## **GENETIC AND EPIGENETIC REGULATION OF VISCERAL PAIN**

Over the last two decades, the field of pain genetics has explored the influence of genetic make-up on pain perception and processing. Recent work by Camilleri and others has described the role of genetics in IBS (378–384). Genome-wide association (GWA) studies amongst others have led to the elucidation of specific genetic alterations in IBS such as Nav1.5 (385), GPBAR1 (G proteincoupled bile acid receptor 1) (386), KDELR2 (KDEL endoplasmic reticulum protein retention receptor-2) and GRID2IP [glutamate receptor, ionotropic, delta 2 (Grid2) interacting protein] (381), NXPH1 (neurexophilin 1), and CDC42 (cell division control protein 42 homolog) (387). These studies are illustrative examples of how research in the genetic area can contribute to the achievement of better general knowledge in the visceral pain field.

Strain differences have been shown in many behavioral phenotypes including anxiety and depression (388, 389). Moreover, differences due to background strain have also been shown for somatic nociception (390–392). However, there is a dearth of information regarding strain differences in basal visceral sensitivity as opposed to inflammatory-induced visceral sensitivity. To our knowledge, the Wistar Kyoto (WKY) rat strain and the Flinders Sensitive Line rat strain are the only well-validated models of genetic predisposition to visceral hypersensitivity (132, 393, 394). This highlights the need for more comprehensive testing in particular assessment of visceral pain in animal models and elucidating the underlying genetic mechanisms. Moreover, the field of pain epigenetics has progressed our simplistic view of one gene, one protein, one function, to a more complex view of gene–environment interactions.

The term epigenetics refers to processes that lead to stable and/or heritable changes in gene function without any concomitant DNA sequence changes (395). Examples include DNA methylation, histone modification, and chromatin remodeling. The vast majority of work investigating epigenetic mechanisms in pain processing center around histone acetylation and DNA methylation. Pharmacological interference with the process of histone acetylation can affect pain behavior, with both systemic and intrathecal administration of histone deacetylase (HDAC) inhibitors having analgesic effects in models of inflammatory pain (396–398). In one study, this effect was shown to be mediated by expression changes of the mGlu2 receptor in both dorsal root ganglia (DRG) and spinal cord (399). Indeed, histone acetylation has been implicated in stress-induced visceral hypersensitivity and HDAC inhibitors have also shown efficacy in a model of visceral pain (69, 71, 400–402). The hypothesis that IBS could be transferred to future generations has recently been investigated (403) and discussed (404).

Similar influences could be shown in the case of DNA methylation and its reader molecule MeCP2. The methyl binding protein MeCP2 has been shown to promote abnormal up-regulation of a group of genes in inflammatory pain conditions. In rats, its usually repressive function appears to be curtailed through phosphorylation after injection of Complete Freund's adjuvant (CFA) into the ankle joint (405), an effect thought to be partly dependent on intact descending serotonergic input into the spinal dorsal horn (398, 406). Recently, it was shown that chronic stress was associated with increased methylation of the Nr3c1 (glucocorticoid nuclear receptor) promoter and reduced expression of this gene in L6–S2 region of the spinal cord which was associated with visceral hypersensitivity in rat model (401).

Finally, recent speculation has implicated that the mechanisms and indeed pathways by which the gut microbiota may communicate with the CNS may be due in part to epigenetic processes (407).

## **EXPERIMENTAL MODELS OF VISCERAL PAIN**

## **COLORECTAL DISTENSION**

Colorectal distension is the most widely used method to assess visceral sensation both preclinically and clinically. This technique involves insertion of a balloon into the colorectal cavity of the human subject or animal, and with the aid of a barostat, distending in a repeated or ascending phasic manner. Although the main premise of the technique is essentially identical, the way in which it is performed varies between laboratories and researchers. This can be due to numerous factors including the model employed; whether it be human, rat, or mouse, the parameter of interest; baseline visceral sensation, pain threshold, and tolerance to painful stimuli. CRD has been characterized in humans, rats, and mice with the bulk of the work being performed in rats due to ease of use and robust reproducibility (408).

## **MONITORING OF VISCERAL PAIN IN RODENTS**

In 1988, Ness and Gebhart were the first to describe a technique used to assess visceral sensitivity in the preclinical setting. The technique was based on the assessment of pseudoaffective and behavioral responses to controlled isobaric distensions of the GI tract. This has now become the mainstay for assessing visceral pain both clinically and preclinically (409). CRD in rats induces an array of autonomic and behavioral outputs termed pseudoaffective reflexes. These reflexes include; alterations in blood pressure and heart rate, passive avoidance behaviors, and contraction of the abdominal musculature (8), the latter of which is more commonly referred to as the VMR. VMR is the most widely used parameter of the visceral pain response (8, 408).

In the last decade, the use of electromyographic (EMG) signals and its numerous applications have allowed many to assess visceral sensitivity in conscious animals. This procedure allows recording from electrodes which are implanted in the musculature and externalized through the skin, primarily on the abdomen or neck (410–412). They can also be connected to radiotelemetric implants in the abdominal cavity (413, 414). Moreover, the development of manometric recordings measuring changes in the pressure of the balloon inserted into the GI tract, have also allowed for VMR to be assessed in freely moving animals (134, 175, 311, 408, 415, 416). Other groups have implemented other indirect approaches such as manual scoring of the abdominal withdrawal reflex (408, 417), operant behavioral assays (409), and functional brain imaging tools such as functional MRI (44, 418).

## **ANIMAL MODELS OF STRESS-INDUCED VISCERAL PAIN**

Stressful episodes during critical windows of development can have long-lasting effects on the host. Stress occurring during the perinatal period has been linked to the development of psychiatric disorders such as schizophrenia and autism spectrum disorders (419, 420). The early postnatal period is a stress hypo-responsive period during which time there is an intense phase of neuronal growth and myelination (421). Stress during this critical time point has been linked to the development of both somatic and psychiatric phenotypes in preclinical models, including IBS (135, 422, 423). The adolescent period is also a time of neuronal restructuring and is fundamental to precise development of the CNS (215). This developmental period is also the peak time for the onset of numerous psychiatric disorders including schizophrenia, substance abuse, and mood disorders (424). Stress in adulthood can have profound effects on its host. Both acute and chronic stressors can elicit detrimental impacts on physical as well as mental wellbeing. Taken together, it is clear that stress at critical points during our life time can have lifelong effects on the host. Overcoming the effects of these stressors is based on the individual's HPA axis ability to adapt and overcome such insults, however, in the genetically predisposed individual, this feat maybe too high. Aberrant development of the HPA axis can prove detrimental and exhibit itself in the form of both psychiatric and somatic disorders. The importance of animal models in the search for underlying molecular mechanisms and future development of novel therapeutics has never been more pertinent (8, 425–431). Here, we review the current stress-based models in the context of IBS (**Figure 5**).

Experimental models of stress and stressful events have been developed bearing in mind the critical windows for HPA axis development and maturation. Animal models have been specifically developed to target different periods throughout the lifespan to assess (1) vulnerability, (2) triggering, and (3) perpetuating influences of stress and future development of IBS (425). Earlylife stressors in the form of maternal neglect (maternal separation model) or injury (colonic irritation) can increase an individual's risk to develop IBS and other disorders in adulthood (425). During adulthood, life-threatening stressors (rape, warfare), psychological

stressors (acute and chronic stress), or physical stressors (in the form of intestinal dysbiosis due to infection, inflammation, antibiotic usage, and surgery) have all been described and documented as triggering factors to the development of an IBS-type phenotype in rats and mice (425). Finally, the ever expanding catalog of rodent strains and transgenic models has allowed us to use specific strains/knockouts known to exhibit various levels of stress responsivity (Wistar Kyoto and Flinders Sensitive Line) to mimic the influence of genetic and perpetuating factors on the development, severity and duration of IBS symptoms (425).

## **GENETIC STRESS MODELS OF VISCERAL PAIN**

Mood disorders are commonly comorbid with IBS (432–434). Genetic predisposition to such disorders has been implicated in the development of IBS in later life. These genetic factors may not lead directly to the development of IBS *per se* but may indirectly, through heightened stress responsivity, cause altered GI function and IBS symptomatology. The availability of a catalog of rodent strains has led to the advent of genetic studies assessing the exact contribution of genetic factors to disease presentation and progression. Moreover, the use of transgenic rodent strains allows us to specifically investigate single genes implicated in disease pathology.

Using different rat strains of known levels of baseline anxiety: low-anxiety SD and Fisher-344 (F344), and high-anxiety WKY rats, Gunter and colleagues were able to demonstrate a link between anxiety and visceral hypersensitivity. Specifically, highanxiety WKY animals had increased response to CRD compared to low-anxiety strains (132). Moreover, WKY rats exhibited an exaggerated response to acetic acid instillation into the colon, a peripheral sensitization model, compared with low-anxiety strains, SD and F344 (132).

The role of the stress response and the development of IBS was again shown to be intrinsically linked when CRHR1 (CRHR1−/−) knockout animals were shown to have an altered VMR to CRD. VMR to CRD was only observed at the highest distension pressure (60 mmHg). Moreover, pharmacological CRHR1 antagonism decreased the VMR to CRD in CRHR1+/<sup>+</sup> mice (167).

Small interfering RNA (siRNA) technologies have now become one of the most widely used approaches to selectively suppress gene expression and is a powerful tool to assess gene function. This technology has been used extensively to suppress stress-related genes in specific brain areas and models are continuously being developed (435–440). Whilst genetically modified mice have been the mainstay of behavioral genetics to date there is a growing utility for genetically modified rats (441). Moreover, other genetic tools such as optogenetic and Designer Receptor Exclusively Activated by Designer Drugs (DREADD)-based manipulations allows for both species to be used in the future (442).

## **ANIMAL MODELS OF EARLY-LIFE STRESS-INDUCED VISCERAL PAIN Maternal separation stress model**

Stress in early life is a well-established risk factor for the development of psychiatric and somatic disorders in later life. The biopsychosocial model of IBS pathophysiology implicates adverse early-life events and childhood traumas such sexual abuse, neglect,

loss of a family member, or a life-threatening situation. These factors have been linked to enhanced vulnerability of individuals to develop stress-related affective disorders such as depression and anxiety. Moreover, these individuals are also at a higher risk for developing FGIDs such as IBS and visceral pain (181, 443).

To model these environmental factors in rodent models, the maternal separation model was constructed (**Figure 6**) (444). Briefly, this model involves removing new-born pups from the dam, during the critical HPA axis hypo-responsive phase in the early postnatal period. Most commonly, pups are removed for 2– 3 h per day during the first 2 weeks of life from postnatal days 1–2 to postnatal day 12–14 (108, 188, 445, 446). This interruption in the normal maternal environment, leads to a stress response not only in the pups but also in the dams. As a result of this maternal stress response, the maternal care given to the pups is altered. In the last 20 years, the critical importance of maternal care has been researched extensively no more so in the area of epigenetics (447–449). Alterations in maternal care has been shown to have effects on the development and function of the HPA axis. Moreover, the impaired stress response is thought to underlie the deficits seen in different behavioral domains, in particular, both cognitive and emotional modalities (447). This disruption of the normal maternal environment and dam–offspring interaction can subsequently affect the quality of the maternal care received by the pups. Indeed, this model of early-life stress results in longlasting changes to the offspring's CNS, at all levels including altered expression, neurochemistry, electrophysiology, and morphology (8, 450).

When animals are allowed to grow to maturity, a behavioral phenotype is present, characterized by visceral hypersensitivity, increased anxiety and depressive behaviors, altered stress responsivity, altered neurochemistry specifically serotonergic neurotransmission, enhanced immune response, altered gut microbiota profile, and disruption of the intestinal barrier (108). We have previously shown that adult male rats previously subjected to maternal separation exhibit visceral hypersensitivity to CRD (135). Moreover, the effects of MS are exacerbated by exposure to an acute stressor specifically 1 h of water-avoidance stress (WAS) (451, 452).

The maternal separation model is sensitive to many factors such as sex differences, with the abundance of studies being performed in male rodents, however clinically the preponderance of female to male IBS patients is 3:1. It is unclear from the literature that the MS procedure induces the same robust phenotype in female rodents. Moreover, the protocol of separation itself has also proved crucial. Specifically, sex-dependent effects on VMR were evident when the separation was performed by the removal of an entire or half litter from the home cage (446). Males that were exposed to whole litter separation and females that were exposed to both whole litter and half litter separation developed visceral allodynia and hypersensitivity to CRD (446). Moreover, when males underwent an additional acute stress, this did not modify the CRD response. On the other hand, when females were exposed to an additional acute stress, they exhibited an exacerbated response to CRD (8, 446). The rat MS model is the most commonly used rodent model of IBS and has proved crucial in delineating the underlying mechanisms as well as testing novel therapeutic strategies.

Although the maternal separation model is well established and characterized in rats, its utility in mouse strains has proved difficult to replicate. Data from our lab and others suggest that maternal separation stress alone is insufficient to induce a robust, reproducible behavioral phenotype (453, 454). However, when maternal separation stress is combined with unpredictable maternal stress, a more overt behavioral phenotype is exhibited (310, 455).

## **Neonatal Irritation Models**

Early-life stress not only in the form of psychological stress but also in the context of physical stress has been shown to be a valid preclinical model of IBS pathophysiology. The neonatal intestine when exposed to mechanical and chemical stressors results in a pro-inflammatory phenotype with mucosal inflammation and tissue irritation. Daily irritation of the neonatal colon by mechanical irritation (daily noxious CRD) or chemical irritation (daily intra-colonic injection of mustard oil) increases pain behaviors in response to CRD from adolescence to adulthood (417, 456). Moreover, this heightened pain response was accompanied with decreases in exploratory behavior, indicative of an anxietyphenotype. The findings described in these studies implies that irritation of the neonatal gut, be it via mechanical or chemical means, can lead to the process of central sensitization due in part to sensitized peripheral afferents (8, 417, 456). These behavioral outputs are in parallel with IBS in the clinical scenario; visceral hypersensitivity with comorbid anxiety.

## **ANIMAL MODELS OF ADULT STRESS-INDUCED VISCERAL PAIN Acute stress models**

Acute stressors in adulthood can lead to an immediate behavioral phenotype. The most widely used acute stress paradigms to model IBS preclinically are WAS and restraint stress. The WAS paradigm was originally developed by Bonaz and Tache and Enck et al. (457, 458) to assess stress-related alterations in gut motility and motor function. Briefly, rodents are placed on a small platform raised slightly above water level. This stressor is based on the aversive environment of surrounding water. The WAS paradigm has been used extensively to assess the impact of psychological stress on visceral pain modulation (8, 311). Data from the literature show that 1 h WAS was sufficient to induce a delayed visceral hypersensitivity to CRD, in male Wistar rats (459). Moreover, others have shown that chronic WAS-induced effects are thought to be modulated at an epigenetic level, with HDAC inhibitors shown to reverse WAS-induced hyperalgesia (400).

Moreover, other forms of acute stress such as restraint stress for 2 h, was also found to induce heightened VMR to CRD in male (460) and female Wistar rats (446). The time spent in an acute stress situation has proven crucial to the development of the IBS phenotype (461). Acute stressors of longer duration >2 h tend to be susceptible to the animals habituating to the stressful environment and this can confound the behavioral outputs. Moreover, WAS was recently shown to induce both hyperalgesia and analgesia depending on the duration and number of stress sessions (311, 462). Transient stressors generally trigger adaptive responses and this type of model mimics the stress-related hypersensitivity to CRD as reported in IBS patients.

## **Chronic mild stress**

Daily life stressors affect individuals in different ways. Persistent mild stressors can accumulate and potentiate the effects of stress on the host. Modeling chronic daily stress in rodents is achieved through the chronic mild stress (CMS) paradigm. Convergent lines of evidence suggests that high levels of chronic daily stress can impact on the intensity and severity of visceral pain symptoms (181, 463–466). In light of this, an array of rodent models involving unpredictable chronic and intermittent exposure to variety of stressors have recently been developed (8). Following on from the previous section, WAS was in fact one of the first chronic stress models to be adapted to the study of visceral hypersensitivity (467). Early data proved promising with studies showing that a 1 h daily WAS, for 10 consecutive day protocol was sufficient to induce visceral hypersensitivity to CRD in male Wistar rats (410, 468). This effect was long-lasting and persistent up to 30 days after the end of the stress. One of the confounds of this study was based on the methodology used to assess VMR. EMG recordings were performed however this involves surgical implantation of electrodes and ensuing single housing of animals, to avoid injury (410), which in itself could be described as a stressor. Indeed, when VMR was assessed using intraluminal colonic solid-state manometry, both male and female naïve Wistar rats exposed to WAS developed a reduced response to CRD referred to as visceral analgesia (311). This findings appear to be in direct contrast to each other and highlight the challenges of developing models of stress-induced visceral hypersensitivity.

Moreover, the picture becomes even more complex when we consider mouse models. Indeed, chronic WAS in C57Bl/6 mice has shown many varied effects including visceral hyperalgesia (311), visceral analgesia (311), or to have no influence on the VMR (469). Many other factors may be of course at play such as the conditions prior to CRD including surgery and housing environment, and these factors are dependent on the route of VMR assessment (311). Taken together, it becomes clear that the basal environment and state of the animals prior to stress exposure can impact their response to the stressor itself (8).

Furthermore, the nature or type of stressor is also crucial, with habituation known to occur when animals are repeatedly exposed to the same type of stressor, a phenomenon thought to be mediated via oxytocin (470) and endocannabinoid pathways (471). Moreover, these pathways are indeed themselves also important in pain processing (472, 473). Taking this knowledge on board, that homotypic stressors may/do lead to habituation, more recent models employ stressors of different natures. These heterotypic stressors such as cold restraint stress, WAS, or forced swimming were found to induce immediate but not long-term visceral hyperalgesia as monitored by EMG recordings at 8 h, 24 h, and 7 days after the cessation of the stress paradigm in male Wistar rats (474). Developing the model further, with more multifaceted paradigms and longer lasting stress sessions (134, 475, 476) causes a behavioral phenotype in rodents that mimics aspects of depression. This more recently developed model may prove useful when assessing the mechanisms of chronic visceral pain comorbid with depressionlike symptoms (432). Taken together, it is clear that many other factors outside of the stress model can have significant effects on the parameter of interest and thus limit the models utility (477). Factors such as the sex and strain of the animal model, housing conditions before, during and after the stress paradigm and diurnal variation are all known to alter sensitivity to stress (477). From the literature, what we have learned is that variety within the stress paradigm itself (time, type of stressor) appears crucial to inducing stress-induced effects and preventing habituation (470, 478–480). All of these facets could also in themselves potentially alter the influence of CMS paradigms on the visceral pain sensitivity, as recently assessed by Larauche et al. (311).

## **Chronic psychosocial stress**

Daily stressful events are commonplace in modern society. However, when modeling stress in rodent models, the stressors most often occur in a novel environment and not in the animals' home cage. This has been a cause for critique with some of the most widely used stress models. The chronic social defeat and overcrowding paradigm was designed to overcome this obstacle and thus animals undergo stress scenarios in their home cage. The paradigm is based on the unpredictable nature of life's stressors. Sessions of resident intruder social defeat and cage overcrowding are randomized so as to occur at different times of the day, in an unpredictable manner and for a chronic period, 19 days in total. We have recently shown this model to be an effective preclinical model to mimic many of the key phenotypes in the IBS population, specifically a heightened response to CRD (134) and anxietyand depression-related behaviors (481).

Moreover, Reber and colleagues (482) have shown that this model of chronic psychosocial stress robustly enhances GI dysfunction in a mouse model of colitis. Furthermore, other chronic psychosocial stressors such as chronic overcrowding when applied to rats also induces a heightened sensitivity to CRD concomitant with enhanced HPA axis activity and intestinal mucosal inflammation (483). Taken together, these studies demonstrate that this model of chronic psychosocial stress may have multiple effects across the brain–gut axis resulting in an IBS phenotype.

## **Conditioned fear-induced stress model**

There is increasing evidence of augmented prevalence of GI symptoms, including visceral pain and IBS in patients suffering from post-traumatic stress disorder (PTSD) (122, 484–487). Indeed, it has been shown that in some individuals, the experience of stress can prime such individuals to respond differently to subsequent stressful events. Over the last decade, Stam and colleagues have investigated this in preclinical models, particularly rats. They

have shown that short exposures to shocks or a social confrontation environment with a predator or aggressive conspecific animal induces long-lasting conditioned fear-responses to trauma-related cues (425). Moreover, these animals exhibit a generalized behavioral sensitization to novel stressful stimuli that is persistent and may intensify over time (488–491). Furthermore, this group have also shown that 2 weeks after a single session of foot shocks, repeated CRD causes increased cardiovascular reflexes in preshocked rats when compared to their non-shocked controls (489). Remarkably, female rats appear to exhibit an alternative pattern of sensitized behavioral responsiveness to the same challenge, again highlighted the strong role of sex differences in visceral pain processing (492). This specific rodent model mimics clinical features of a subset of IBS patients exhibiting stress-related visceral hypersensitivity due to PTSD. However, it is important to note that the findings presented above are mainly characterized by a single group of investigators, thus the reliability of the model needs further independent confirmation.

## **Physical stressors**

*Post-infectious models of visceral pain.* A significant proportion of IBS cases occur after an illness particularly an infection of the GI tract. This is reported to be as much as 10% of patients with IBS (493). Interestingly, the proportion of people who suffer from an intestinal infection who will then go on to develop IBS ranges from 3 to 36% and appears to be dependent upon the infecting organism. Moreover, the psychological state of the individual at the time of infection appears to also play a critical role in the development of IBS symptoms (494). Infections of bacterial origin when compared to the short-lived effects of viral gastroenteritis appear to be more long-lasting and persistent (494). A transient *Trichinella spiralis* infection was shown to induce sustained visceral hypersensitivity in a mouse model (5, 495). Moreover, similar findings were found in a rat model of *Nippostrongylus brasiliensis* infection (496). Although the vast majority of human post-infectious hypersensitivity symptoms are observed after bacterial infection with *Salmonella, Escherichia coli*, *Shigella*, or *Campylobacter*, there has been limited animal models of this type of visceral hypersensitivity (497, 498).

*Post-inflammatory models of visceral pain.* Inflammation is one of the leading causes/mechanisms thought to underpin IBS and its associated symptomatology (499–502). However, this remains a controversial topic and others have reviewed this previously (503, 504). Moreover, this symptom set appears to be common in patients of inflammatory bowel disorders also such as those in remission from ulcerative colitis (505). Indeed, many clinical cases of IBD switch to IBS cases and this occurs in 30–50% of patients (506). Inflammatory pain models have been well developed in somatic pain assays and to some extinct in visceral pain models. Indeed, an array of chemical irritants have been used in preclinical models to induce colonic inflammation and resultant visceral hypersensitivity (8). In rats, mustard oil (507, 508), acetic acid (509), and zymosan (510, 511) have been shown to induce short-term visceral hypersensitivity due in part to colonic inflammation. Moreover, other compounds such as TNBS induce severe colonic inflammation and visceral hypersensitivity when

administered intra-colonically (512, 513). Interestingly, in some models of inflammatory-induced visceral hypersensitivity, the phenotype can re-emerge days or weeks after the initial inflammatory response. Moreover this re-emergence is not associated with any inflammatory biomarkers (512, 514). Importantly, the experimental design appears to play a major role in the application and effectiveness of such models. Models, such as the dextran sodium sulfate (DSS)-induced colitis model has provided us with some surprising findings where animals exhibited an increased response to CRD on day 5 or day 60 after the induction of colitis in male Balb/c mice. However, chronic DSS colitis was not associated with changes in VMR to CRD (515). However, contrasting findings have also been reported by other groups, whereby DSS colitis failed to induce any alterations in VMR or visceral sensitivity at all time points tested in either C57Bl/6 or Balb/c mice (516). One can speculate on the many possible reasons for these observed differences, however, what they do suggest is that inflammation on its own may not be sufficient to induce visceral hypersensitivity and that the nature and severity of the inflammatory stimulus and their combined effects may determine when/whether post-inflammatory hypersensitivity will result if at all (8, 512). Moreover, as is seen in post-infectious visceral hypersensitivity, psychological state may also play a role (517). Indeed this was investigated by Larsson et al. (469), who demonstrated that prior exposure of animals to a stressor, be it either psychological or psychosocial in origin, revealed an enhanced susceptibility to colitis and an aggravated colonic inflammatory response (482, 518, 519). Furthermore, this was also associated with a heightened susceptibility to recurrence of colonic inflammation even though the colitis had dissipated (520, 521). Similarly, a prior bout of colitis was sufficient to leave the colon in a state more susceptible to the negative effects of stress (522). However, the complex association between colitis, stress, and visceral pain response remains contentious as stress has also been found to both exacerbate or indeed to have no effect on post-inflammatory visceral sensitivity in rats (523) and in mice (8, 469).

## **FUTURE DIRECTIONS**

### **MICROBIOTA MANIPULATIONS**

The role of the microbiota–brain–gut axis on health and disease is an area of biomedicine that is receiving much media attention of late. The complex communication between the gut microbial population and the CNS has far reaching implications not least in the area of visceral pain and psychiatric comorbidities. Modulation of the gut microbiota via prebiotic/probiotic treatment has been shown to have positive effects on visceral pain behaviors as discussed earlier (222, 240–242). Antibiotic treatment has long been known to alter GI function, effects which are reversed upon probiotic treatment (524). Thus, it is logical that antibioticinduced visceral pain may be a future model used to investigate the underlying mechanism of such a phenomenon. Indeed work from our own lab has recently demonstrated that early-life antibioticinduced disruption of the colonizing microbiota is associated with visceral hypersensitivity and altered spinal signaling in adulthood. These results indicate that a temporary alteration in the composition of the GI microbiota during a crucial time-window in the neonatal rat has a long-lasting impact on nociceptive pathways and that the developing pain systems are subject to modulation by the microbiota (248).

Moreover, intriguing studies to assess the exact contribution of the gut microbiota to the development of visceral hypersensitivity and IBS can be achieved through fecal transplantation studies whereby fecal matter from donor IBS patients can be used to inoculate recipient animals and subsequent behaviors can be assessed. This paradigm has already shown its merit in transferring other behaviors specifically anxiety-related behaviors in a mouse model (525).

## **CONCLUSION**

An effective stress response is critical to the survival of any living creature. The ability to sense changes in the environment and adapt accordingly is quintessence to survival. However, the host's reaction to perceived stress can sometimes become disturbed leading to an over-exaggerated response. Stress has been implicated in and associated with a plethora of somatic disorders; ulcers, migraine, hypertension, and psychiatric disorders; anxiety, depression, PTSD. Here, we specifically reviewed the literature in the context of stress as a major risk factor for the development of IBS and visceral pain. The mechanism by which stress impacts on normal physiology to cause such devastating disorders still remains unclear. Numerous mechanisms have been postulated including alterations in plasticity, neurogenesis, catecholaminergic neurotransmission, and more recently gut microbiota. Modern day life exposes us all to stressors of varying types (psychological/physical) and severities (acute/chronic) and as such the search for novel pharmaco-therapeutics has never been more pertinent.

Despite the ever growing body of literature, the exact mechanisms underlying visceral pain still remain less well understood than that of somatic pain. Central sensitization of primary sensory afferents is an important underlying mechanism for both somatic and visceral hypersensitivity and hyperalgesia. One of the hurdles in understanding the mechanisms of stress-induced visceral pain is that visceral pain response is experienced differentially depending on numerous factors, including the time at which stress was applied, duration of stress, sex differences, and genetic background amongst others. Psychological stress at any point during our lifetime can lead to permanent alterations of the HPA axis, the descending pain modulatory system, the immune system, and the gut microbiota, all of which can be manifested as chronic visceral hypersensitivity. Mechanisms by which physical stress such as infections, mediate visceral hypersensitivity are likely to be different to that of psychological stress and may involve altered immune system functioning. Considering the diverse mechanisms of visceral pain, the development of treatment strategies and therapeutic interventions will rely on good animal models, all of which have been reviewed here. It is clear that that the drive to develop clinically relevant models and thus design new novel therapeutics has never been more pertinent.

## **ACKNOWLEDGMENTS**

The authors would like to gratefully acknowledge financial support from Science Foundation Ireland in the form of a center grant (Alimentary Pharmabiotic Centre Grant Number SFI/12/RC/2273). The authors would also like to acknowledge Dr. Yuliya Borre and Dr. Anna Golubeva for assistance in creating the schematic figures.

## **REFERENCES**

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irritable bowel syndrome. *Neurogastroenterol Motil* (2010) **22**(512–9):e114–5. doi:10.1111/j.1365-2982.2009.01427.x

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**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

*Received: 31 October 2014; accepted: 28 January 2015; published online: 16 February 2015.*

*Citation: Moloney RD, O'Mahony SM, Dinan TG and Cryan JF (2015) Stress-induced visceral pain: toward animal models of irritable-bowel syndrome and associated comorbidities. Front. Psychiatry 6:15. doi: 10.3389/fpsyt.2015.00015*

*This article was submitted to Affective Disorders and Psychosomatic Research, a section of the journal Frontiers in Psychiatry.*

*Copyright © 2015 Moloney, O'Mahony, Dinan and Cryan. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.*


**2. Observational studies of dietary vitamin K intake and health outcomes**

**1. Introduction**

170 Vitamin K2 - Vital for Health and Wellbeing

to be useful only for coagulation of the blood.

until the modern research era.

fully interchangeable.

research together.

The story of vitamin K goes back to the 1930s, when Henrik Dam at the University of Copenhagen isolated an antihemorrhagic vitamin that was fat soluble but different from previously isolated vitamins A, D, and E [1]. He found high concentrations of the vitamin in hog liver fat and hemp seed, but found it to be virtually absent in cod liver oil while testing a variety of animal organs, hen eggs, cereals, seeds, vegetables, and various fats and oils including butter fat. The initial quantification was all based on the time required to coagulate blood from a chicken. The term "vitamin K" was used as short for the German term "koagulation." Because of the type of assay used, all vitamin K factors, whether K1 or K2, were thought

At the same time as Henrik Dam was working out the details of the antihemorrhagic vitamin, there was another investigator working in the USA with a vitamin factor not found in cod liver oil, but which worked synergistically with them to promote proper mineralization, bone growth, and to prevent dental caries [2]. Dr. Weston Price found that the amount of the factor in mammalian milk varied with the "nutrition of the animal," with highest levels from milk of cows that were consuming rapidly growing green grass. Because Dr. Price's assay was based on the release of iodine from hydroiodic acid, a test for peroxides, no connection was made between his discovery and any other vitamin activity. What Price found was the activity of vitamin K2, formed in mammary glands from phylloquinone, vitamin K1, found in abundance in rapidly growing green grass [3]. No connection was made between the anticoagulation activity of K1 and the mineral-directing activity of vitamin K2

Because of the way vitamin K was discovered for its coagulation function, it was assumed that this was its only function for many years. The Recommended Daily Allowance (RDA) for vitamin K is based on its coagulation function. Though both phylloquinone (phyllo—from plants) and menaquinones support coagulation, as we explore in this chapter, there are clear differences between the functioning of phylloquinone and the menaquinones, and they are not

This chapter is a review of vitamin K2 research in the area of cardiovascular health, especially dealing with arterial calcification. It is organized in a loose chronological order, following the themes of the research as the field matured. First, there were observations about dietary intake of K1 and K2 and health outcomes, forming hypotheses to be tested further. Associations were discovered at this stage. Harmful observations with warfarin-type drugs, which are vitamin K antagonists, were seen early on in the research cycle as well. Hypotheses were then explored with animal studies, looking for mechanisms and biomarkers—to help get endpoints that were quicker to develop than mortality and disease. Then came the rise of biomarker studies, using markers for vitamin K status (dp-ucMGP), as well as markers for risk factors, such as arterial calcification, pointing to disease outcomes. As the science matured even further, intervention studies related to K2 and biomarker changes emerged. The final stage is now emerging with intervention studies looking not just at biomarkers but disease outcomes, thus tying all of the Observational studies help form hypotheses by finding associations between factors that may or may not be related to the health outcome of interest. One of the early population-based studies in the 1990s examined the vitamin K status of 113 postmenopausal women [4]. Dietary intake of vitamin K was assessed along with examinations for the presence or absence of aortic calcified lesions. Blood samples were assayed for osteocalcin, a vitamin K-dependent protein that is responsible for proper deposition of minerals in bone tissue. It was thought that some forms of osteocalcin might be a marker for vitamin K status. The first type had low affinity for hydroxyapatite, while the second had high affinity. Women with calcified lesions (*n* = 34) had a lower intake of total vitamin K as well as a higher amount of low-affinity osteocalcin. Together these results indicated that these women had impaired vitamin K status that might be related to their atherosclerotic lesions.

A groundbreaking study took place in the Netherlands which changed the way vitamin K2 was thought of afterward. First, a very careful survey of foods eaten in the Netherlands was carried out by interview-based dietary intakes of over 5400 people, guided by a validated food frequency questionnaire (FFQ). For many animal-based foods, the quantitation of vitamin K2 had not been done, so this was carried out and reported as well. The mean intake of K1 and K2, respectively, varied from 124 and 9.3 µg/day in the lowest quartile to 375 and 45 µg/day in the upper quartile [5]. A report of health outcomes from the Rotterdam Study in 2004 from 4800 of these subjects revealed that even though vitamin K2 was a minor part of the total vitamin K intake, only K2 and not K1 intake was associated with a lower risk of disease [6]. For people in the upper third of intakes of K2 compared to the lowest third, there were decreases in relative risks of coronary heart disease (CHD) mortality (57%), all-cause mortality (26%), and severe aortic calcification (52%). Even though intakes of K2 were only about 10% as much as the amounts of K1, their effect on cardiovascular disease was greater. Cheese was the primary source of menaquinones in this cohort in the Netherlands, not exactly highly regarded as a heart healthy food. This fact made confounding by other "healthy" nutrients less likely and made the results more robust.

Similar results have been seen in a second study, from the Prospect-EPIC cohort, also from the Netherlands [7]. About 16,000 women aged 49–70 were followed for 8 years. Vitamin K1 and K2 intakes were estimated from a FFQ . Vitamin K2 intake varied from <20 to >36 µg/day across quartiles. For every increase of 10 µg of K2, there was a 9% reduction in hazard ratio of risk of CHD, with the effects coming mainly from menaquinone subtypes MK-7, MK-8, and MK-9. There was no association between intake of K1 and CHD, as seen in the Rotterdam Study.

Two large cohort studies have been analyzed for associations between vitamin K intake and CHD. When the Nurses' Health Study (NHS) cohort was analyzed for an association between dietary vitamin K intake and cardiovascular outcomes, it was found that K1 intake was associated with a 21% decrease in multivariate relative risk of total CHD [8]. The association was attenuated by adjustments for other dietary factors and lifestyle patterns, so that it was not apparent to the authors whether the results were due to vitamin K1 or that K1 was just a marker for a lifestyle pattern associated with a high intake of K1. Median intakes of K1 for the lowest and highest quintiles were 87 and 300 µg/day for the NHS cohort. In another large cohort, the Health Professionals' Follow-up Study, there was a decrease in the relative risk of total CHD across increasing quintiles of vitamin K1 intake. However, when the results were adjusted for lifestyle and other dietary factors, the trend was no longer significant [9]. Neither of these large cohorts reported dietary intakes of K2, perhaps because the database for menaquinone concentrations in the USA was not complete at that time, nor is it fully available at the time of writing this chapter (only partial data are available on MK-4 but none on higher menaquinones), 17 years after such data were obtained for the Rotterdam Study [5]. The negative results from these cohorts for vitamin K1 and CHD only reinforce just how striking the results were from the Dutch studies for vitamin K2. The findings from the Netherlands were not expected or anticipated by many.

0.64 for cancer mortality and all-cause mortality. It is possible that enough vitamin K1 and other plant-based protective nutrients such as folate, vitamin C, fiber, potassium, and magnesium were supplied by the diet that even those participants who had low intakes of menaquinones were still not at an elevated risk compared to other subjects in the PREDIMED cohort. Participants who increased their intake of either vitamins K1 or K2 or both during the course of the study experienced decreased risk of cardiovascular mortality (K1 only), cancer mortality, and all-cause mortality. The main conclusion from this observational study perhaps is that eating plants is good for you, and the Mediterranean Diet is generally beneficial, as seen in

Vitamin K2: Implications for Cardiovascular Health in the Context of Plant-Based Diets, with Applications for Prostate...

http://dx.doi.org/10.5772/63413

173

Another line of evidence that led to the discovery of the role of menaquinones was the effect of blood thinning drugs such as warfarin and coumarin. While this class of drugs has been very helpful in preventing strokes in the short term, it has also caused damage long term, as people are often prescribed blood thinners for many years. In 1998, it was reported by Price et al. [15] that warfarin caused calcification of the elastic lamellae in rat arteries and heart valves within a period of 2 weeks, with increasing intensity each week. Vitamin K1 was given concurrently to maintain normal blood coagulation. At the time, menaquinone was not even mentioned in the article, not even in the discussion. The discovery of importance was that warfarin had negative side effects for arterial calcification that were not counteracted by

This discovery nearly coincided in time with work on an MGP-deficient mouse model. Matrix γ-carboxylation protein, or matrix Gla protein (MGP), was originally discovered in bone tissue but is actually expressed in many tissues of the body, including vascular smooth muscle cells and chondrocytes in cartilage. MGP requires the activation by vitamin K in order to bind calcium ions and prevent crystallization of calcium. These MGP-deficient mice developed normally to term but died within 2 months as a result of extensive arterial calcification, which led to blood vessel rupture [16]. Also seen was the inappropriate calcification of cartilage, including the growth plate of bones. This MGP-deficient mouse model clearly showed that MGP has a central, active role in preventing calcifications of arterial walls and also of cartilage. This research coupled together with the warfarin-caused calcification pointed to a central role

Further work on the interrelationship between vitamin K1 and K2 was spurred on by feeding studies in rats. When rats were made vitamin K deficient, then fed only K2 as MK-4, they accumulated MK-4 especially in the pancreas, aorta, fatty tissues, and brain. Liver and serum levels of MK-4 were low. When vitamin K–deficient rats were fed only K1, they accumulated K1 in the liver, heart, and fatty tissues, and they also accumulated MK-4 in the same way as the rats that were fed MK-4, indicating that there was conversion from phylloquinone to MK-4 [17]. So, with a warfarin-rat model that was able to induce arterial calcification and knowing that there were differences between K1 and K2 distributions in the rats and that K2 prevented heart disease in the Rotterdam Study, Spronk and coworkers set out to see how to prevent

another report from this cohort [14].

vitamin K1.

**3. Vitamin K antagonist studies**

for MGP in controlling arterial calcification.

The question of whether vitamin K intake is related to arterial calcifications has been probed in two observational studies. In a cross-sectional study of 1689 women, dietary intakes of vitamin K1 and K2 were estimated with an FFQ, and standard screening mammograms were assessed for the presence of breast arterial calcifications [10]. Unadjusted results showed an inverse association between intake of vitamin K2 and breast arterial calcifications, but adjustments for aging, smoking, diabetes, and dietary factors made the association no longer significant. Adjustment for diabetes may have been unwise, as vitamin K2 intake has also been shown to reduce the risk of diabetes among 38,000 Dutch men and women in the Prospect-EPIC cohort mentioned previously [11]. So, this adjustment may have attenuated the results enough to make the association no longer statistically significant.

In another cross-sectional study, 564 postmenopausal women were examined for an association between coronary calcifications and intake of vitamins K1 and K2 [12]. Women were chosen from the Prospect-EPIC cohort study. In this cohort, cheese contributed 54%, milk products contributed 22%, and meat contributed 15% of the K2 intake. The mean intake of K2 ranged from 18.0 ± 4.5 to 48.5 ± 9.0 in the lowest and highest quartiles, respectively. Examinations found that 62% of the women had coronary calcifications. In the model adjusted for age and cardiovascular risk factors, increased menaquinone intakes were associated with a decreased calcification prevalence ratio of 0.80 (95% CI: 0.65–0.98), comparing highest to lowest quartile.

A more recent observational study reported findings contrary to those found in the Prospect-EPIC cohort and the Rotterdam Study. The PREDIMED cohort is a Spanish study to examine the effect of adoption of the Mediterranean Diet on cardiovascular, cancer, and all-cause mortality. The intakes of vitamins K1 and K2 were estimated by FFQ, and endpoints of cardiovascular, cancer, or all-cause mortality were tracked for a median follow-up of 4.8 years. Energy-adjusted intakes of vitamins K1 and K2, respectively, ranged from 170 and 18.4 µg/day in the lowest quartiles for each vitamin to 626 and 57.5 µg/day in the upper quartiles [13]. People in the upper quartile consumed about twice as many vegetables, especially leafy greens, as those in the lowest quartile. The upper quartile of vitamin K intake in this cohort adopting the Mediterranean Diet was substantially higher than seen in the other observational studies of other European or American cohorts. No protective effects for higher intakes of menaquinones were seen in this cohort for cardiovascular mortality, cancer mortality, or all-cause mortality. However, high intakes of phylloquinone lead to a reduced hazard ratio of 0.54 and 0.64 for cancer mortality and all-cause mortality. It is possible that enough vitamin K1 and other plant-based protective nutrients such as folate, vitamin C, fiber, potassium, and magnesium were supplied by the diet that even those participants who had low intakes of menaquinones were still not at an elevated risk compared to other subjects in the PREDIMED cohort. Participants who increased their intake of either vitamins K1 or K2 or both during the course of the study experienced decreased risk of cardiovascular mortality (K1 only), cancer mortality, and all-cause mortality. The main conclusion from this observational study perhaps is that eating plants is good for you, and the Mediterranean Diet is generally beneficial, as seen in another report from this cohort [14].


*2.4. Circular Dichroism (CD) Experiments*

CD experiments were performed using a Chirascan V100 CD spectrometer with a bandwidth of 1 nm at room temperature, unless otherwise specified. The CD samples (~100 μL) were placed in a cuvette of 0.5 mm path length, and the CD spectra were collected from 200 to 350 nm with a step size of 1 nm. For each sample, three sets of scans were acquired, and an average value was taken. CD spectra were background-corrected using the corresponding buffer solution.

#### *2.5. Fluorescence Experiments*

Fluorescence experiments, except for the kinetic study of Ag+ sensing, were performed using a Shimadzu RF-6000 spectrometer at room temperature. The fluorescence samples (~2 mL) were placed in a 10 mm four-sided glazed quartz cuvette, and the fluorescence spectra were collected from 512 to 650 nm with a step size of 1 nm. Fluorescence intensity was recorded at 520 nm with an excitation wavelength of 492 nm. The excitation and emission band widths were 5 nm. For a kinetic study of Ag+ sensing, fluorescence experiments were performed using an Edinburgh FLS1000 photoluminescence spectrometer at room temperature. The sample containing a DNA sensor in the absence of Ag+ (~2.5 mL) was first placed in a 10 mm four-sided glazed quartz cuvette, and the fluorescence intensity at 520 nm was recorded from 0 to 180 s with a step time of 2 s. Ag+ was then added to this sample, and the fluorescence intensity was immediately recorded from 0 to 180 s with a step time of 2 s. The excitation and emission band widths were 2 nm.

The detailed sample conditions for NMR, CD, and fluorescence experiments are stated in the figure legends.

#### **3. Results**


**7. Hydrogen and electric field effect to Iron impurities diffusion in the Zr-Fe melt**

Iron and Zirconium diffusion factor dependence on electric field intensity and Hydrogen presence in the molten Zirconium had been analyzed in the terms of molecular dynamics (MD) method. Model system for research of Iron and Hydrogen ions behavior in the Zr-Fe-H melt at Т=2273К temperature and electric field presence contained 516 Zirconium particles, 60 Iron particles and 1Hydrogen particle in cubic cell with a=2.44195 nm cube edge. Integration of the motion equation was carried out by 1.1·10-15s time steps. Interparticle potentials and its parameters had been taken from (Varaksin & Kozyaichev, 1991, Zhou et.al, 2001). Calculation results of impurities migration in the molten Zirconium are compared to experimental data (Lindt et.al, 1999, Ajaja et.al, 2002, Mimura et.al, 1995). Partial radial distribution functions gij(r) for Zirconium-Iron melt are presented on fig. 12. Most probable inter-atomic distance in first coordination sphere is close to sum of atomic radii for Iron and Zirconium (rZr-Fe= 0.29nm, rFe= 0.130nm, rZr= 0.162nm).

This results comparison to computer simulation data for Ta-Fe melt (Pastukhov et.al, 2010, Vostrjakov et.al, 2010) reveals sufficiently close character of the radial distribution function for large dimension atoms, namely Ta-Ta (0.29 nm, rTa = 0.145nm) and Zr-Zr (0.324nm, rZr=0.162nm). Iron and Zirconium diffusion factors in the Zirconium melt in the presence, as well as absence of electric field and Hydrogen at 2273K had been calculated by means of MD method (fig. 13 and 14). Diffusion factor of Iron (DFe) in the Zirconium melts with Hydrogen linearly depends on electric field intensity (E) and Iron concentration (СFe). Hydrogen diffusion factor negligibly decreases from 2.16·10-4 cm2·s-1 to 1.94·10-4 cm2·s-1, if electric field intensity increases from 900 to 1020 v/m. Hydrogen inducing into system at СFe≈0.1% decreases DFe value from 7.86·10-5 to 6.36·10-5cm2s-1, and electric field 1020 v/m intensity applying decreases DFe to 5.23·10-5cm2·s-1 (fig.14).

Molecular Dynamic Simulation of Short Order and

DZr

0

2

4

D\*10-5,cm2

c

6

8

Hydrogen Diffusion in the Disordered Metal Systems 299

DFe

Fig. 13. Dependencies of DFe and DZr on Iron concentration at 2273К (MD - calculation).

0,00 0,02 0,04 0,06 0,08 0,10

c%, Fe

Fig. 14. Iron (СFe =0.1mas.%) diffusion factor dependence on electric field intensity at 1173K

900 950 1000 1050 1100

E,V/m

Calculated values of Еа for different metals (Flynn et.al, 1970) are in quantity agreement with experimental data. Temperature dependence DH at high temperatures is described in the term of theory (Flynn et.al, 1970). Authors (Shmakov et.al, 1998) calculated DH in Zirconium at 2273K without electric field influence as 3.862·10-4cm2·s-1, which low differ

temperature (MD – calculation).

5,10

5,15

5,20

D\*10-5cm2

c

5,25

5,30

from our calculated value DH=5.01·10-4cm2·s-1.

Calculation results of DFe changing in dependence on E value had been compared to evaporation constant rate for the Fe-ions from Zr, calculated by equation (Pogrebnyak et.al, 1987, Vigov et.al, 1987)

Fig. 12. Partial radial distribution functions gij(r) for Zr-Fe melt at 2273К, calculated in terms of MD model.

$$k = \nu \cdot \mathbb{C}\_{Fe} \exp\left[ -\frac{\mathcal{A} + I - \mathcal{W} - (q^3 \cdot E)^{1/2}}{RT} \right],\tag{12}$$

where ν – ion vibration frequency (1013·s-1), СFe – impurity concentration, λ – Fe evaporation heat, I – first ionization potential (V/Å), E – electric field intensity, W – electron exit work, q – ion charge. Values of λ, I, W, R had taken in electron-volt, E – in volt per angstrom. Diffusion factor D directly depends on rate (*k*) and time (t) evaporation of main metal (Kuznetsov et.al, 1968). The log *k* and log DFe on Е dependences (fig. 15) are relatively similar.

Thus assumption is possible, that limiting factor of Fe removal from the Zr melt is diffusion of Fe. The Hydrogen is considered as light intrusion impurity into metals with different cell type. Therefore Hydrogen diffusion is significant problem in researching of high temperature metals refining. Impurities have less action upon Incoherent diffusion. Therefore this kind of diffusion becomes dominating at high temperature (Maximov et.al, 1975).

We had compared Hydrogen diffusion factors in Ta at 3400K (Pastukhov et.al, 2010) and in Zr at 2273K (Ajaja et.al, 2002). This values are 1,7 10-5 and5,01 10-4 cm2·s-1 respectively. The authors (Maximov et.al, 1975) explain such difference due to Hydrogen diffusion activation energy (Ea) dependence on atomic metal mass, its Debye frequency, modulus of elasticity and volume change at Hydrogen addition.

Calculation results of DFe changing in dependence on E value had been compared to evaporation constant rate for the Fe-ions from Zr, calculated by equation (Pogrebnyak et.al,

Fig. 12. Partial radial distribution functions gij(r) for Zr-Fe melt at 2273К, calculated in terms

r,nm

where ν – ion vibration frequency (1013·s-1), СFe – impurity concentration, λ – Fe evaporation heat, I – first ionization potential (V/Å), E – electric field intensity, W – electron exit work, q – ion charge. Values of λ, I, W, R had taken in electron-volt, E – in volt per angstrom. Diffusion factor D directly depends on rate (*k*) and time (t) evaporation of main metal (Kuznetsov et.al, 1968). The log *k* and log DFe on Е dependences (fig. 15) are relatively similar. Thus assumption is possible, that limiting factor of Fe removal from the Zr melt is diffusion of Fe. The Hydrogen is considered as light intrusion impurity into metals with different cell type. Therefore Hydrogen diffusion is significant problem in researching of high temperature metals refining. Impurities have less action upon Incoherent diffusion. Therefore this kind of diffusion

We had compared Hydrogen diffusion factors in Ta at 3400K (Pastukhov et.al, 2010) and in Zr at 2273K (Ajaja et.al, 2002). This values are 1,7 10-5 and5,01 10-4 cm2·s-1 respectively. The authors (Maximov et.al, 1975) explain such difference due to Hydrogen diffusion activation energy (Ea) dependence on atomic metal mass, its Debye frequency, modulus of elasticity and

*Fe* exp *IW qE k C*

0,0 0,2 0,4 0,6 0,8 1,0

becomes dominating at high temperature (Maximov et.al, 1975).

volume change at Hydrogen addition.

3 1/2 ( )

, (12)

Zr-Zr

Zr-Fe

Fe-Fe

total

*RT*

1987, Vigov et.al, 1987)

of MD model.

0

1

2

3

gij(r)

4

5

6

Fig. 13. Dependencies of DFe and DZr on Iron concentration at 2273К (MD - calculation).

Fig. 14. Iron (СFe =0.1mas.%) diffusion factor dependence on electric field intensity at 1173K temperature (MD – calculation).

Calculated values of Еа for different metals (Flynn et.al, 1970) are in quantity agreement with experimental data. Temperature dependence DH at high temperatures is described in the term of theory (Flynn et.al, 1970). Authors (Shmakov et.al, 1998) calculated DH in Zirconium at 2273K without electric field influence as 3.862·10-4cm2·s-1, which low differ from our calculated value DH=5.01·10-4cm2·s-1.

Fig. 15. Dependence of lgDFe (curve 1) and lgk (curve 2) on electric field intensity.

We have estimated diffusion layer thickness (x) by (13) equation (Flynn et.al, 1970). Calculation were carried out basing on СFe - experimental time – dependence (Mimura et.al, 1995). Value of DFe we calculated by MD – method.

$$\mathbf{C}\_{\{\mathbf{x},t\}} = \mathbf{C}\_0 \text{erfc}\left(\frac{\mathbf{x}}{2 \cdot \sqrt{D\_{\text{Fe}} \cdot t}}\right) \text{\,\,\,\tag{13}$$

Molecular Dynamic Simulation of Short Order and

forces affect, rather than evaporation.

stage of melt.

electric field (fig. 16).

transition to more disordered structure.

corresponds to Ta-Fe without electric field.

Hydrogen Diffusion in the Disordered Metal Systems 301

G/L decreases at temperature increase. Obtained G/L ≈ 1.15 value at СFe= 0.46 and Т = 2450К may indicate, that close to 15% of Iron is being removed due to electro-magnetic

Т,К СFe, mas.% logG logL G/L Remark Issue 2350 0.46 -4.572 -5.066 3.115 L<G [4]

2450 0.46 -4.572 -4.755 1.153 L<G [4]

2900 7.82·10-3 -6.967 -5.406 0.027 L>G [2]

2450 7.82·10-3 -6.967 -6.525 0.361 L >G [2]

2300 7.82·10-3 -6.967 -7.001 1.081 L < G [2]

**8. Hydrogen and electric field effect to Iron impurities diffusion in Ta-Fe melt**  Hydrogen and Iron atoms radial distribution functions and diffusion constants had been found by MD method in the Tantalum melt at 3400K in the presence and absence of outer

The model system was presented by 486 tantalum, 1 iron and 1hydrogen atoms in a cubic cell of 2.13572 nanometers cube edge length. Computer experiment data have found short order of Ta-Fe-H system at 3400K is close to Tantalum structure: first maximum at RTa-Ta ≈ 0.29 nm corresponds to Tantalum atom radius ≈ 0.292 nm. All RDF maxima diffusion is observed at electric field and Hydrogen in the Ta-Fe system. This fact may indicate liquid

Unusual kind of RDF curve for Ta-H atoms pairs obtained at electric field 1020v/m intensity (fig. 17): first maximum of the curve is bifurcated, besides first sub-peak at r1 = 0.22nm corresponds to one of most probable distance Ta-H and second sub-peak at r2 = 0.24nm

Dynamics and local structure of the close to Hydrogen surrounding for the ternary interstitial alloy should depend on Hydrogen concentration, temperature and solvent structure short order. There is no conventional opinion about Hydrogen location in such systems. Since the Hydrogen atom radius is 0.032 nm, it can occupy octahedron (0.0606 nm), as well as tetrahedron (0.0328нм) (Geld et.al, 1985) location. System Ta – H particularity is

Table 3. Dependences of L and G on mean residual Iron content in Zirconium for PAM process. CFe – mean Iron concentration: initial (15min.), middle (90min.), and final (165min.)

 0.01 -6.09 -6.728 4.348 2.2·10-4 -7.902 -8.386 3.047

 0.01 -6.09 -6.418 2.128 2.2·10-4 -7.902 -8.076 1.491

 2.68·10-3 -7.44 -5.871 0.027 0.92·10-3 -7.903 -6.335 0.028

 2.68·10-3 -7.44 -6.990 0.355 0.92·10-3 -7.903 -7.454 0.356

 2.68·10-3 -7.44 -7.466 1.062 0.92·10-3 -7.903 -7.930 1.065

In the equation С0 and С(х,t) are impurity concentrations in initial and refined Zirconium (Flynn et.al, 1970) and t is time of refining. Calculated (*x)* value is equal 7·10-2cm. By the order of value it's close to data on Silicon borating (Filipovski et.al, 1994), which is 1.6-1.8·10- 2cm. It should be noted, that zone thickness of Zirconium shell interaction with molten Uranium is 0.2·10-2cm (Belash et.al, 2006).

We carried out calculation of Iron removal rate (G) from Zirconium by Iron concentration decrease during matched time intervals of plasma-arc-melting (PAM) with Hydrogen basing on the experimental data of (Mimura et.al, 1995) for 9.5 Pa and 50% of Hydrogen concentration in residual Argon. The data are shown in table 3. Mean residual Iron concentration at 15, 90 and 165 minutes of melting compiled 0.46, 0.01 and 2.2·10-4 mas.% respectively. These calculations had been compared with Iron evaporation rate from Zirconium melt obtained from Langmuir equation:

$$L = 0.0583 \gamma C\_{Fe} cp \sqrt{\frac{M}{T}}\tag{14}$$

This equation parameters are following: Iron activity coefficient in Zirconium (γ) is equal to 0.052 from [43], (СFe) - concentration of Iron in Zr, ( *р* ) – Iron vapor pressure at 2273K, (M) – Iron atomic mass, (T) – Kelvin temperature. Temperature of melting [46] is indicated in the 2350 – 2450К limits. Calculation of L by these temperatures founds L < G in both cases. Ratio

Fig. 15. Dependence of lgDFe (curve 1) and lgk (curve 2) on electric field intensity.

1995). Value of DFe we calculated by MD – method.

Uranium is 0.2·10-2cm (Belash et.al, 2006).

Zirconium melt obtained from Langmuir equation:

We have estimated diffusion layer thickness (x) by (13) equation (Flynn et.al, 1970). Calculation were carried out basing on СFe - experimental time – dependence (Mimura et.al,

( ,) 0 , 2 *x t*

In the equation С0 and С(х,t) are impurity concentrations in initial and refined Zirconium (Flynn et.al, 1970) and t is time of refining. Calculated (*x)* value is equal 7·10-2cm. By the order of value it's close to data on Silicon borating (Filipovski et.al, 1994), which is 1.6-1.8·10- 2cm. It should be noted, that zone thickness of Zirconium shell interaction with molten

We carried out calculation of Iron removal rate (G) from Zirconium by Iron concentration decrease during matched time intervals of plasma-arc-melting (PAM) with Hydrogen basing on the experimental data of (Mimura et.al, 1995) for 9.5 Pa and 50% of Hydrogen concentration in residual Argon. The data are shown in table 3. Mean residual Iron concentration at 15, 90 and 165 minutes of melting compiled 0.46, 0.01 and 2.2·10-4 mas.% respectively. These calculations had been compared with Iron evaporation rate from

> 0.0583 *Fe M L C cp T*

This equation parameters are following: Iron activity coefficient in Zirconium (γ) is equal to 0.052 from [43], (СFe) - concentration of Iron in Zr, ( *р* ) – Iron vapor pressure at 2273K, (M) – Iron atomic mass, (T) – Kelvin temperature. Temperature of melting [46] is indicated in the 2350 – 2450К limits. Calculation of L by these temperatures founds L < G in both cases. Ratio

*x C C erfc D t*

*Fe*

(13)

(14)

G/L decreases at temperature increase. Obtained G/L ≈ 1.15 value at СFe= 0.46 and Т = 2450К may indicate, that close to 15% of Iron is being removed due to electro-magnetic forces affect, rather than evaporation.

Table 3. Dependences of L and G on mean residual Iron content in Zirconium for PAM process. CFe – mean Iron concentration: initial (15min.), middle (90min.), and final (165min.) stage of melt.


**Applications of Finite Element Modeling for Mechanical and Mechatronic Systems**

> Edited by Marek Krawczuk and Magdalena Palacz Printed Edition of the Special Issue Published in *Applied Sciences*

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