In commercial flocks of laying hens, keel bone fractures (KBFs) are prevalent and associated with behavioural indicators of pain. However, whether their impact is severe enough to induce a depressive-like state of chronic stress is unknown. As chronic stress downregulates adult hippocampal neurogenesis (AHN) in mammals and birds, we employ this measure as a neural biomarker of subjective welfare state. Radiographs obtained longitudinally from Lohmann Brown laying hens housed in a commercial multi-tier aviary were used to score the severity of naturally-occurring KBFs between the ages of 21–62 weeks. Individual birds’ transitions between aviary zones were also recorded. Focal hens with severe KBFs at 3–4 weeks prior to sampling (n = 15) had lower densities of immature doublecortin-positive (DCX+) multipolar and bipolar neurons in the hippocampal formation than focal hens with minimal fractures (n = 9). KBF severity scores at this time also negatively predicted DCX+ cell numbers on an individual level, while hens that acquired fractures earlier in their lives had fewer DCX+ neurons in the caudal hippocampal formation. Activity levels 3–4 weeks prior to sampling were not associated with AHN. KBFs thus lead to a negative affective state lasting at least 3–4 weeks, and management steps to reduce their occurrence are likely to have significant welfare benefits.

AimTo examine rat molar pulp innervation and identify complex cellular signalling systems involving nerve growth factor (NGF) and its p(75) receptors (NGFR) at different stages of development, maturation and ageing. MethodologyDecalcified mandibular first molar mesial cusps from Wistar rats of ages 0day; 1, 2, 3, 4, 6, 9, 12 and 24weeks (n=5 per group) were sectioned (10m) and incubated with antibodies for NGF, NGFR, calcitonin gene-related peptide (CGRP) and neurofilament. Nerve densities in worn and intact regions of 3- to 24-week-old rats were compared by anova, Bonferroni and t-tests. ResultsDuring odontogenesis, differences in NGF and NGFR expression were observed, with no evidence of nerve fibres, suggesting a signalling mechanism controlling cellular differentiation and dentine formation. Tooth wear in 4-week rats was associated with reduced NGF expression and significantly decreased CGRP axons within affected odontoblast regions. The underlying subodontoblasts started expressing NGF which continued until 9weeks. This may promote a significant increase in CGRP nerve density in affected regions. Nerve density in intact odontoblast regions increased gradually and reached significant levels in 12-week rats. Reduction in nerve densities within worn and intact regions of cusps was observed at 24weeks. ConclusionsAge-related changes and responses to tooth wear may be controlled by the NGF signalling mechanism, with roles in odontoblast/subodontoblast communication and control of sensory innervation at different stages of tooth development, maturation and ageing. Greater understanding of cellular and nerve regulation in the injured pulp may promote therapeutic strategies for pulp survival.

Objective: To re-examine the morphology and potential functions of odontoblasts in intact rat incisors and after cavity preparation into dentine. Design: Intact incisors were fixed, decalcified, snap frozen and sectioned (10 pm), before staining with rhodamine phalloidin or antibodies for cyto-skeletal proteins: vimentin and actin, ion transporter: NaK-ATPase, and dendritic cell marker: OX6. Samples with cavity were processed similarly and stained for actin and vimentin before comparing the lengths of odontoblast processes (OP) at baseline, 3 h and 24 h (n = 5 for each group). Results: Actin was expressed through the full length of OP, while vimentin immunoreactivity was not uniform, with 4 distinct regions. OP showed morphological complexity with fine branches emanating within different regions of dentine. Novel actin-positive tree-like OP were identified within predentine which reduced in intensity and length toward the incisal portion of the tooth. Specimens with cavities showed time-dependant pulpal retraction of OP. Conclusions: Differences in structural antibody expression suggest functional variations in OP within different regions of dentine. The role of actin positive OP in predentine is not known, but could be related to dentine deposition, cellular stability or sensing mechanisms. Cavity preparation into dentine was followed by programmed retraction of OP which could be controlled either mechanically by the spatial limitation of the OP within dentinal tubules or structurally by the presence of vimentin, in addition to actin, in the mid-dentine.

ABSTRACT This study characterizes the complex structural and functional elements of the female rat urethra that may be involved in controlling urethral closure and continence. Urethras were dissected from female Sprague‐Dawley rats (N = 12) euthanized by pentobarbital overdose. Tissues were fixed (4% paraformaldehyde), frozen, and sectioned (8 μm) for light microscopy and immunohistochemistry. Antibodies were used to detect immunoreactivity to calcitonin gene related peptide, nitric oxide synthase, vesicular acetylcholine transporter, and tyrosine hydroxylase. Measurements of urethral wall compliance were taken along its length and in different axes using a closed ended catheter with a circular aperture. The bladder neck and proximal urethra are characterized by a highly folded epithelium and lamina propria. A smooth muscle layer is apparent but not pronounced. Distal to this region the smooth muscle layer thickens and forms the body of the internal sphincter, which has a complex innervation. In the mid urethra, the smooth muscle is thickened resulting in a luminal protrusion, producing an occlusion of the lumen. The structure of the distal urethra is different. The epithelium has few folds and, immediately below the lamina propria large thin walled vascular lacunae can be found. Measurements of the urethral wall compliance demonstrate distinct regional differences with proximal and distal specialisations. These variations, which correlate with muscular and vascular elements, suggest the operation of discrete systems, hence effecting urethral closure during filling. An understanding of these systems may yield insights into urethral pathology and direct approaches to develop pharmacological interventions to promote continence. Anat Rec, 301:1276–1289, 2018. © 2018 Wiley Periodicals, Inc.

ABSTRACTThe aim of this study was to characterize the number, type and distribution of immunochemically identified nerves in epithelium and lamina propria of the female rat urethra. Urethras from female Sprague–Dawley rats (n = 12) were fixed, frozen and sectioned (8 μm). Standard immunohistochemical techniques were used to identify putative nerves using the following antibodies: calcitonin gene related peptide (cgrp), neuronal nitric oxide synthase (nNos), tyrosine hydroxylase (TH) and vesicular acetylcholine transporter (vacht). The number, distribution and characteristics of all immunoreactive (IR) structures adjacent to the urethral epithelium and in the lamina propria was assessed. In the bladder, few cgrp‐IR and vacht‐IR fibers were associated with the urothelium or suburothelium of the lateral wall. In contrast, large numbers of vacht‐IR, nNos‐IR and cgrp‐IR fibers were found close to the epithelium and subepithelium of the bladder neck and throughout the urethra. The number of cgrp‐IR fibers was significantly higher in the urethra in comparison with the bladder neck. A population of undescribed cgrp‐IR cells associated with the bladder neck and proximal urethra has been characterized. Each of these cells appears to be associated with a nerve fiber. In the distal urethra, the number of peptidergic fibers penetrating the epithelium was significantly higher than the rest of the urethra. Clearly, this study has revealed a highly complex and heterogeneous network of putative afferent nerves fibers along the length of the urethra. These structural specializations need to be taken into account when probing the different functions of the urethra. Anat Rec, 302:201–214, 2019. © 2018 Wiley Periodicals, Inc.

The process of tooth formation and development is complex involving many signalling pathways and molecules. The enamel formation is a process controlled entirely by the enamel organ with many cell-cell interaction and signalling. Although this process was studied extensively, the full understanding is still to be achieved. Twenty dental pulps from rat mandibular incisor were dissected, fixed, frozen, sectioned, stained with specific antibodies then carefully examined using fluorescence microscope. The basic findings were cellular heterogeneity, presence of spherical vacuoles which may be blood vessels, and striking differential expression of some very important signalling molecules antigen throughout the enamel organ at different stages of development. This paper revealed some of the complexity associated with enamelogenesis and proved that the previous description of enamel organ is very simplistic.

β3-adrenoceptor agonists influence overactive bladder in humans and animal models. However, data is emerging that the mode of action of these drugs is complex. The present study explored the actions of the β3-adrenergic agonist mirabegron and the non-selective agonist isoprenaline on the contractile systems in the rat and guinea pig bladder. Intravesical pressure was measured in isolated whole bladders from female adult animals. In both species spontaneous contractile activity was observed. The muscarinic agonist arecaidine produced complex responses consisting of an initial transient pressure rise followed by complex phasic activity. Three contractile elements were identified: intrinsic micro-contractile activity, initial transient response and steady state phasic activity. The intrinsic and steady state activity could be further divided into a baseline pressure with superimposed phasic activity. The effects of isoprenaline and mirabegron were investigated on these elements. In the rat, the micro-contractile activity could be completely inhibited by isoprenaline (full agonist). The arecaidine-induced initial and steady state baseline pressures were partially reduced, while the phasic activity was little affected. In the guinea pig, both the arecaidine-induced baseline pressure and the phasic activity were affected by isoprenaline. Mirabegron didn't produce significant inhibitory effects in any of the contractile elements in either species. These results show that complex contractile systems operate in the rat and guinea pig bladder that can be modulated by β1/β2-adrenoceptor mechanisms. No evidence was obtained for any β3-dependent regulation of contraction. These data support similar data in humans. Therefore the primary site of therapeutic action of β3-adrenergic agonists remains unknown.

A growing body of work is describing the absence of a significant sympathetic innervation of the detrusor implying little sympathetic regulation of bladder contractility. However, low doses of adrenergic agonists are capable of relaxing the bladder smooth muscle. If these effects underpin a physiological response then the cellular nature and operation of this system are currently unknown. The present immunohistochemistry study was done to explore the existence of alternative adrenergic signaling elements in the rat bladder wall. Using antibodies to tyrosine hydroxylase (TH) and vesicular mono-amine transporter (vmat), few adrenergic nerves were found in the detrusor although TH immunoreactive (IR) nerves were apparent in the bladder neck. TH-IR and vmat-IR nerves were however abundant surrounding blood vessels. A population of vmat-IR cells was found within the network of interstitial cells that surround the detrusor muscle bundles. These vmat-IR cells were not or only weakly TH-IR. This suggests that these interstitial cells have the capacity to store and release catecholamines that may involve noradrenaline. Cells expressing the β1-adrenoceptor (β1AR-IR) were also detected within the interstitial cell network. Double staining with antibodies to β1AR and vmat suggests that the majority of vmat-IR interstitial cells show β1AR-IR indicative of an autocrine signaling system. In conclusion, a population of interstitial cells has the machinery to store, release and respond to catecholamines. Thus, there might exist a non-neuronal β-adrenergic system operating in the bladder wall possibly linked to one component of motor activity, micro-contractions, a system that may be involved in mechanisms underpinning bladder sensation.

Structural and functional interrelationships between odontoblast (OCL) and sub-odontoblast cell layers (SOCL) are relevant to tissue formation and regulation in health and disease, but are incompletelyunderstood. This study employed contemporary immunohistochemical techniques to gain important new insights on tissue complexity within the apical half of the rodent mandibular incisor. Whole incisors were removed from freshly killedWistar rats, fixed in 4% paraformaldehyde,demineralized, and prepared for standard immunohistochemistry. Tissues were labelled with primary antibodies to vimentin, α actin, NaKATPase, alpha tubulin and COX 1, and secondary antibodies alexafluor 488 and 594, before examination by fluorescence microscopy. Odontoblasts presented in stratified or pseudo-stratified arrangements, rather than simple columnar form. Additional stellate cells,α-actin immunoreactive, were observed predominantly in the distal half of the OCL. Cells in the cell rich zone (crz) also showed complexity, with high cell density, and cells presenting with large nuclei and sparse cytoplasm. These cells showed intense NaKATPase immunoreactivity. Some of these cells sent processes towards and into the OCL, whilst other processes remained within the crz. No processes directed downwards. The OCL of the rat mandibular incisor presents a hitherto undescribed complexity, in terms of cellular arrangement and composition, the functional significance of which is not immediately apparent. The observation that cellular processes from crz tothe OCL and lateral processes within crz suggest functional connectivityand cell to cell communication within the layer. The intense immunoreactivity to NaKATPase suggests functional specification and a high degree of tissue activity. Structural observations will require further investigations.

Hypothesis / aims of study The urethra plays a key role in generating afferent information. Electrophysiological studies have classified afferents, primarily by conduction velocity, as typically Aδ and C fibres. However, the location and nature of these nerves in the urethral wall is uncertain. The present study investigated the location of putative fibres in the proximal urethra of the female rat.

New evidence suggests that the extent and severity of periodontal disease may be a significant risk factor for erectile dysfunction, sperm motility and time to conception. This paper reviews the evidence and informs members of the dental team when dealing with this sensitive issue. As more research is forthcoming the topic of oral and sexual health is likely to be part of regular routine medical screening. Any issue concerning oral health as a risk factor for sexual health is likely to be a sensitive subject, rarely discussed in the dental setting. However, as new evidence emerges, this topic is likely to get into the public domain. All members of the dental team should be aware of such an association. Clinical Relevance: Furthermore, the information in this paper may provide further incentive for certain patients to improve their oral health.

Bladder afferent outflow, linked to sensation, plays a critical role in bladder pathology: abnormal outflow results in altered sensation, leading to increased voiding frequency, urge and often incontinence. β3-adrenoceptor agonists have been suggested to be beneficial in treating these symptoms. However, the absence of a significant sympathetic innervation of the detrusor and only a modest relaxation of bladder muscle by β3 agonists has questioned the therapeutic site of action of β3 agonists in the bladder. The present study was done to explore the possibility that β3-adrenoceptors might be located in the pelvic plexus. Using the rat, where the pelvic plexus is located primarily within a single ganglion, the major pelvic ganglion (MPG), immuno-histochemical approaches were used to identify structures expressing β3-adrenoceptor immuno-reactivity (β3AR-IR). The only structures found to express β3AR-IR were small-diameter tyrosine hydroxylase and vesicular mono-amine transporter immuno-reactive (TH-IR and vmat-IR) neurones. These neurones, found in clusters or singly on the periphery of the ganglion, or dispersed in smaller clumps throughout the MPG, are similar to the small intensely fluorescent (SIF) cells described previously. Not all small cells expressed β3AR-IR. A population of the small cells were also immuno-reactive to the type 3 muscarinic receptor (M3R-IR) and the P2X3 purinergic receptor (P2X3-IR). Clumps of small cells were associated with calcitonin gene-related peptide immuno-reactive (CGRP-IR) nerve fibres (putative sensory fibres) and a small number were contacted by putative cholinergic nerves expressing immuno-reactivity to vesicular acetylcholine transporter (vacht-IR). These observations are consistent with the idea that small cells are interneurons and one of the components making up complex neural circuits within the MPG. The precise physiological role of these neural elements in the MPG is unknown. However, as one therapeutic action of β3-adrenoceptor agonists is to modulate sensation, it is possible that these neural circuits may be involved in the regulation of afferent outflow and sensation.

The arrangement and roles of the odontoblast and its process in sensing and responding to injuries such as tooth wear are incompletely understood. Evidence is presented that dentine exposure by tooth wear triggers structural and functional changes that aim to maintain tooth integrity. Mandibular first molars from freshly culled 8 week Wistar rats were prepared for light microscopy ground-sections (n = 6), or fixed in 4% paraformaldehyde, decalcified in 17% EDTA, sectioned and stained with antibodies to cyto-skeletal proteins (vimentin (vim), α-tubulin (tub) and α-actin), cellular homeostatic elements (sodium potassium ATPase (NaK-ATPase) and sodium hydrogen exchanger (NHE-1)), and sensory nerve fibres (CGRP) (n = 10) for fluorescence microscopy of worn and unworn regions of the mesial cusp. Immunoreactivity (IR) to vim, actin, NaK-ATPase and CGRP was confined to the pulpal third of odontoblast processes (OPs). IR to tub and nhe-1 was expressed by OPs in full dentine thickness. In areas associated with dentine exposure, the tubules contained no OPs. In regions with intact dentine, odontoblasts were arranged in a single cell layer and easily distinguished from the sub-odontoblast cells. In regions with open tubules, the odontoblasts were in stratified or pseudo-stratified in arrangement. Differences in structural antibody expression suggest a previously unreported heterogeneity of the odontoblast population and variations in different regions of the OP. This combined with differences in OPs extension and pulp cellular arrangement in worn and unworn regions suggests active and dynamic cellular responses to the opening of dentinal tubules by tooth wear.

Aims The existence of a motor-sensory system contributing to bladder sensation is now becoming widely accepted. Although it is clear that the motor component of this system appears to be generated within the bladder wall, recent observations suggest that the mechanisms involved in its modulation may lie outside the wall. The present study was undertaken to gain more insights into the peripheral modulation of non-voiding activity and the role of the major pelvic ganglion. Methods Male Sprague-Dawley rats anesthetized with urethane were used. The bladder was filled till 60% of the micturition threshold volume. The baseline pressure and the superimposed non-voiding activity were observed before and after consecutive bilateral transections of the hypogastric and pelvic nerves and bilateral ablation of the major pelvic ganglia. Results Hypogastric and pelvic nerve transection didn't significantly change the baseline pressure and superimposed non-voiding activity. Removal of the major pelvic ganglia resulted into an increased baseline pressure when compared with the control and increased amplitude of the non-voiding contractions when compared with both the decentralized condition (both hypogastric and pelvic nerves transected) and the control. The frequency of the non-voiding contractions wasn't affected. Conclusions Non-voiding activity during the urine storage phase seems to be modulated at the level of the major pelvic ganglion. This suggests the possibility of local circuits between the bladder and the peripheral ganglia that may be responsible for an inhibitory component influencing non-voiding activity.

Periodontal pockets provide an ideal environment for the application of local drug delivery for the management of periodontitis. In this first article, consideration is given to the principles that underlie local drug delivery and the pharmacological challenges. It is essential that such systems are compatible with the periodontal tissues. Potential unwanted effects that can arise from both drug and delivery vehicle are also appraised. Clinical Relevance: Local drug delivery systems still have a place in the management of periodontal diseases. This first article considers the pharmacological principles and unwanted effects that can arise from the use of such systems.

Local drug delivery systems (LDDs) are mainly used as adjuncts to root surface instrumentation (RSI) in the management of periodontal disease. A range of antibacterial agents have been used for this purpose and include the tetracyclines, chlorhexidine, metronidazole, azithromycin, clarithromycin and moxifloxacin. More recently, other non-antibacterial agents have been incorporated into local delivery devices and include simvastatin, metformin and alendronate. This paper will review the effectiveness of such agents when used as an adjunct to conventional non-surgical periodontal therapy. Clinical Relevance: The benefits of adjunctive use of local drug delivery in the management of periodontal disease will be discussed.

Objectives The decision-making process within health care has been widely researched, with shared decision-making, where both patients and clinicians share technical and personal information, often being cited as the ideal model. To date, much of this research has focused on systems where patients receive their care and treatment free at the point of contact (either in government-funded schemes or in insurance-based schemes). Oral health care often involves patients making direct payments for their care and treatment, and less is known about how this payment affects the decision-making process. It is clear that patient characteristics influence decision-making, but previous evidence suggests that clinicians may assume characteristics rather than eliciting them directly. The aim was to explore the influences on how dentists' engaged in the decision-making process surrounding a high-cost item of health care, dental implant treatments (DITs). Methods A qualitative study using semi-structured interviews was undertaken using a purposive sample of primary care dentists (n = 25). Thematic analysis was undertaken to reveal emerging key themes. Results There were differences in how dentists discussed and offered implants. Dentists made decisions about whether to offer implants based on business factors, professional and legal obligations and whether they perceived the patient to be motivated to have treatment and their ability to pay. There was evidence that assessment of these characteristics was often based on assumptions derived from elements such as the appearance of the patient, the state of the patient's mouth and demographic details. The data suggest that there is a conflict between three elements of acting as a healthcare professional: minimizing provision of unneeded treatment, trying to fully involve patients in shared decisions and acting as a business person with the potential for financial gain. Conclusions It might be expected that in the context of a high-cost healthcare intervention for which patients pay the bill themselves, that decision-making would be closer to an informed than a paternalistic model. Our research suggests that paternalistic decision-making is still practised and is influenced by assumptions about patient characteristics. Better tools and training may be required to support clinicians in this area of practice.