Quinze cabeças de tubarões Carcharias taurus (Rafinesque, 1810), pertencentes à Ordem Lamniformes e Família Odontaspididae, foram utilizadas para a realização desse estudo. O material destinado à análise foi obtido junto ao Terminal de Pesca de Santos, localizado em Santos, São Paulo, e descartado pelos pescadores por não apresentar valor comercial. A dissecação das cabeças visou caracterizar os músculos mandibulares, bem como descrever sua inervação realizada pelo ramo mandibular do nervo trigêmeo. Os músculos mandibulares de C. taurus são representados por: músculo pré-orbital, músculo levantador do palatoquadrado, músculo quadrado-mandibular e músculo intermandibular. O nervo trigêmeo de C. taurus origina-se em uma porção lateral da medula oblonga e ramifica-se na órbita para originar o ramo mandibular responsável pela inervação dos músculos derivados do arco mandibular. Os ramos proximais do ramo mandibular do nervo trigêmeo inervam o músculo levantador do palatoquadrado, o músculo pré-orbital e o músculo quadrado-mandibular recebem fibras dos ramos intermediários do ramo mandibular do nervo trigêmeo e a ramificação distal do mesmo é visualizada no músculo intermandibular. / During this study were used fifteen of Carcharias taurus (Rafinesque, 1810) (Odontaspididae, Lamniformes, Elasmobranchii) shark heads. The analised material was obteined in Terminal de Pesca de Santos, located in Santos, São Paulo. The material was descarded by the fishers because it hasn´t none comercial value. The heads dissection is foccated in the caracterization of the mandibular muscles and to discrebed the mandibular branch of the trigeminal nerve innervation as well. The C. taurus mandibular muscles are represented by: muscle pré-orbital, muscle levantador do palatoquadrado, muscle quadrado-mandibular and muscle intermandibular. The origin of the trimeginal nerve of C. taurus is located in a lateral portion of the medula oblonga. In the orbita it ramificates to originate de mandibular branch that inervates the muscles which is derived from the mandibular arch. The mandibular branch of the trigeminal nerve\'s proximal branches inervetes the muscle levantador do palatoquadrado, the muscles pré-orbitall and quadrado-mandibular receives fibers from the intermediate branches of the mandibular branch of the trigeminal nerve and the distal ramification of the mandibular branch are visualised in the muscle intermandibularis.

Bibliography: leaves 184-220.

Tendinosis is a condition of chronic pain that afflicts several human tendons, not least the patellar tendon, in which case it is often clinically referred to as ‘jumper’s knee’. The exact mechanisms behind tendinosis are yet not fully understood. One draw-back in the case of patellar tendinosis has been the lack of knowledge of the innervation patterns of the human patellar tendon. It cannot be excluded that the processes of tendinosis are influenced by nerve mediators, released from nerve endings or from stimulated cells inside the tendon. Thus, the studies of the present thesis aimed to 1) map the general, sensory, cholinergic and sympathetic innervation patterns of the human patellar tendon, in both the tendon tissue proper and the loose paratendinous connective tissue surrounding the tendon, and 2) investigate the possible existence of a production of signal substances, traditionally associated with neurons, in non-neuronal tendon cells, and to see if there are signs of local cholinergic and catecholaminergic signaling pathways. Biopsies of both normal pain-free patellar tendons and patellar tendons from patients with chronic painful tendinosis were collected and investigated. The main method utilized was immunohistochemistry, using antibodies directed against synthesizing enzymes for acetylcholine and catecholamines, against muscarinic and adrenergic receptors, and against markers of general and sensory innervation. In situ hybridization (ISH) to detect mRNA for the cholinergic/catecholaminergic synthesizing enzymes was also used. It was found that the loose paratendinous connective tissue of the patellar tendon was rather richly innervated by nerve structures. These consisted of large nerve fascicles, as well as perivascular innervation in the walls of some of the larger arteries and smaller blood vessels. It was found that part of the nerve structures corresponded to sensory afferents, and that some conformed to cholinergic and, especially, sympathetic nerve fibers. The tendon tissue proper was strikingly less innervated than the paratendinous tissue. The sparse innervation that was found in the tendon tissue proper was seen in narrow zones of loose connective tissue and blood vessels, interspersed between the collagen bundles. The overall impression was that the patterns of distribution of the general, sensory, and autonomic innervations of tendinosis tendon tissue were similar to those of normal tendon tissue proper. The most pioneering findings were the immunohistochemical observations of an expression of enzymes related to production of both acetylcholine and catecholamines within the tendon cells (tenocytes) themselves, as well as of a presence of the receptors for these substances on the same cells; features that were predominantly seen in tendinosis tendons. The observations of the synthesizing enzymes for acetylcholine and catecholamines in tenocytes were confirmed by ISH findings of mRNA for these enzymes in the tenocytes. Immunoreactions for muscarinic and adrenergic receptors were also found in blood vessel walls and in some of the nerve fascicles. In summary, this thesis presents novel information on the innervation patterns of the human patellar tendon, in healthy individuals with pain-free tendons as well as in patients with chronic painful tendinosis. Furthermore, it gives the first evidence of the presence of a local, non-neuronal production in the tendon tissue of signal substances normally seen in neurons, and a basis for these substances to affect the tenocytes as these cells also display muscarinic and adrenergic receptors. Thus, the results indicate an existence of autocrine and/or paracrine cholinergic/catecholaminergic systems in the tendon tissue; systems that seem to be up-regulated in tendinosis. This is of great interest as it is known that stimulation of receptors for both catecholamines and acetylcholine can lead to cell proliferation, interfere with pain sensation, influence collagen production, and take part in vasoregulation, as well as, in the case of adrenergic receptors, promote cell degeneration and apotosis. All these processes represent biological functions/events that are reported to be affected in tendinosis. In conclusion, despite the fact that there is very limited innervation within the patellar tendon tissue proper, it is here shown that effects of signal substances traditionally associated with neurons seem to occur in the tissue, via a local production of these substances in tenocytes.

Pain of the musculoskeletal system is one of the most common reasons for people seeking medical attention, and is also one of the major factors that prevent patients from working. Chronic tendon pain, tendinopathy, affects millions of workers world-wide, and the Achilles tendon is an important structure often afflicted by this condition. The pathogenesis of tendinopathy is poorly understood, but it is thought to be of multifactoral aetiology. It is known that tendon pain is often accompanied not only by impaired function but also by structural tissue changes, like vascular proliferation, irregular collagen organisation, and hypercellularity, whereby the condition is called tendinosis. In light of the poor knowledge of tendinosis pathophysiology and recent findings of a non-neuronal signalling system in tendon tissue, the contributory role of neuropeptides such as substance P (SP) has gained increased interest. SP, known for afferent pain signalling in the nervous system, also has multiple efferent functions and has been described to be expressed by non-neuronal cells. As pain is the most prominent symptom of tendinopathy, the focus of the studies in this thesis was the innervation patterns of the tissue ventral to the Achilles tendon (i.e. the tissue targeted in many contemporary treatment methods) as well as the distribution of SP and its preferred receptor, the neurokinin-1 receptor (NK-1R), in the tendon tissue itself. It was hereby hypothesised that the source of SP affecting the Achilles tendon might be the main cells of the tendon tissue (the tenocytes) as well as paratendinous nerves, and that SP might be involved in tendinosis- development. The studies were conducted, via morphological staining methods including immunohistochemistry and in situ hybridisation, on tendon biopsies from patients suffering from Achilles tendinosis and on those from healthy volunteers. The hypothesis of the thesis was furthermore tested using an experimental animal model (rabbit) of Achilles tendinopathy, which was first validated. The model was based on a previously established overuse protocol of repetitive exercise. In the human biopsies of the tissue ventral to the Achilles tendon, there was a marked occurrence of sympathetic innervation, but also sensory, SP-containing, nerve fibres. NK-1R was expressed on blood vessels and nerve fascicles of the paratendinous tissue, but also on the tenocytes of the tendon tissue proper itself, and notably more so in patients suffering from tendinosis. Furthermore, the human tenocytes displayed not only NK-1R mRNA but also mRNA for SP. The animal model was shown to produce objectively verified tendinosis-like changes, such as hypercellularity and increased vascularity, in the rabbit Achilles tendons, after a minimum of three weeks of the exercise protocol. The contralateral leg of the animals in the model was found to be an unreliable control, as bilateral changes occured. The model furthermore demonstrated that exogenously administered SP triggers an inflammatory response in the paratendinous tissue and accelerates the intratendinous tendinosis-like changes such that they now occur after only one week of the protocol. Injections of saline as a control showed similar results as SP concerning hypercellularity, but did not lead to vascular changes or pronounced paratendinous inflammation. In summary, this thesis concludes that interactions between the peripheral sympathetic and sensory nervous systems may occur in Achilles tendinosis at the level of the ventral paratendinous tissue, a region thought to be of great importance in chronic tendon pain since many successful treatments are directed toward it. Furthermore, the distribution of NK-1R:s in the Achilles tendon described in these studies gives a basis for SP, whether produced by nerves mainly outside the tendon or by tenocytes within the tendon, to affect blood vessels, nerve structures, and/or tendon cells, especially in tendinosis patients. In light of this and of previously known SP-effects, such as stimulation of angiogenesis, pain signalling, and cell proliferation, the proposed involvement of SP in tendinosis development seems likely. Indeed, the animal model of Achilles tendon overuse confirms that SP does induce vascular proliferation and hypercellularity in tendon tissue, thus strengthening theories of SP playing a role in tendinosis pathology.

Graduation date: 1993

Muscle architecture, innervation pattern and fiber type distribution of lumbar multifidus (LMT) throughout its volume was quantified. Musculotendinous (n=10) and neural components (n=3) were dissected and digitized from thirteen embalmed cadaveric specimens. The data were imported into Autodesk® Maya® 2008 to generate 3D neuromuscular models of each specimen. Architectural parameters (fiber bundle length, FBL; fiber bundle angle, FBA; tendon length) were quantified from the models using customized software. The medial branch of the posterior rami (L1-L5) was traced through LMT to determine its distribution. Using immunohistochemistry, Type I/II muscle fibers were identified in 29 muscle biopsies from one fresh frozen specimen. The total area and number of each cell type was calculated using Visiopharm® (image analysis software). Architectural and fiber type data were analyzed using ANOVA with Tukey’s post-hoc test (p ≤ 0.05). From L1-L4, LMT had three architecturally distinct regions: superficial, intermediate and deep. At L5, intermediate LMT was absent. Mean FBL decreased significantly from superficial (5.8 ± 1.6cm) to deep regions (2.9 ± 1.1cm) as did volume (superficial, 5.6 ± 2.3ml; deep, 0.7 ± 0.3ml). In contrast, mean FBA increased from superficial to deep. The medial branch of the posterior ramus (L1-L5) supplied the five bands of LMT. Each medial branch in turn divided to supply the deep, intermediate and superficial regions separately. The area occupied by Type I fibers was significantly less (p< 0.01) in the deep (56%) compared with the superficial regions (75%). Based on architecture and morphology, superficial LMT with the longest FBL and relatively small FBA is well designed for torque production and controlling the lumbar lordosis. Intermediate LMT with significantly longer FBL compared with the deep region and with its caudal to cranial line of action may help to control intersegmental stability. Furthermore, the absence of intermediate LMT at L5 and may contribute to the higher incidence of instability observed at the lumbosacral junction. Deep LMT with its short FBL, large FBA and proximity to the axis of spinal rotation may function to provide proprioceptive input to the CNS rather than a primary stabilizer of the lumbar spine.

Muscle architecture, innervation pattern and fiber type distribution of lumbar multifidus (LMT) throughout its volume was quantified. Musculotendinous (n=10) and neural components (n=3) were dissected and digitized from thirteen embalmed cadaveric specimens. The data were imported into Autodesk® Maya® 2008 to generate 3D neuromuscular models of each specimen. Architectural parameters (fiber bundle length, FBL; fiber bundle angle, FBA; tendon length) were quantified from the models using customized software. The medial branch of the posterior rami (L1-L5) was traced through LMT to determine its distribution. Using immunohistochemistry, Type I/II muscle fibers were identified in 29 muscle biopsies from one fresh frozen specimen. The total area and number of each cell type was calculated using Visiopharm® (image analysis software). Architectural and fiber type data were analyzed using ANOVA with Tukey’s post-hoc test (p ≤ 0.05). From L1-L4, LMT had three architecturally distinct regions: superficial, intermediate and deep. At L5, intermediate LMT was absent. Mean FBL decreased significantly from superficial (5.8 ± 1.6cm) to deep regions (2.9 ± 1.1cm) as did volume (superficial, 5.6 ± 2.3ml; deep, 0.7 ± 0.3ml). In contrast, mean FBA increased from superficial to deep. The medial branch of the posterior ramus (L1-L5) supplied the five bands of LMT. Each medial branch in turn divided to supply the deep, intermediate and superficial regions separately. The area occupied by Type I fibers was significantly less (p< 0.01) in the deep (56%) compared with the superficial regions (75%). Based on architecture and morphology, superficial LMT with the longest FBL and relatively small FBA is well designed for torque production and controlling the lumbar lordosis. Intermediate LMT with significantly longer FBL compared with the deep region and with its caudal to cranial line of action may help to control intersegmental stability. Furthermore, the absence of intermediate LMT at L5 and may contribute to the higher incidence of instability observed at the lumbosacral junction. Deep LMT with its short FBL, large FBA and proximity to the axis of spinal rotation may function to provide proprioceptive input to the CNS rather than a primary stabilizer of the lumbar spine.

Apart from motor and sensory function loss, an injury to the spinal cord can cause sympathetic dysfunction, which has been shown to affect immune responses. In this thesis, data from five experimental studies have been collected to compare physiological and psychophysiological exercise responses between wheelchair athlete subgroups with different disabilities (tetraplegic, paraplegic, and non-spinal cord-injured). In two preparatory studies, physiological exercise responses to exhaustive (Chapter 4) and submaximal exercise (Chapter 5) were investigated in all three disability subgroups. Whilst reliability measures for peak oxygen uptake (VO2peak) were in a range observed previously in able-bodied athletes, the variation in tetraplegic athletes was larger when expressed relative to their VO2peak, questioning the use of this variable to track small changes in aerobic capacity in athletic populations. Submaximal physiological and psychophysiological exercise responses were found to be similar between disability subgroups when expressed as a percentage of VO2peak, justifying the protocol used in the laboratory study on mucosal immune function, which was based on the same percentages of VO2peak for all disability subgroups. The most extensive study of this thesis, detailed in Chapter 6, showed that single laboratory-controlled 60-min exercise sessions increase both salivary secretory immunoglobulin A (sIgA), a marker of mucosal immunity, and α-amylase, a marker of sympathetic activation in all three disability subgroups. However, the impaired sympathetic nervous system in tetraplegic athletes seemed to influence the fine-tuning of their sIgA response when compared with paraplegic and non-spinal cord-injured athletes, resulting in a larger exercise-induced increase of sIgA secretion rate when compared to paraplegic and non-spinal cord-injured athletes. Based on these results, the study detailed in Chapter 7 investigated sIgA responses in tetraplegic athletes during wheelchair rugby court training. Despite their disability, these athletes showed responses thought to be governed by the sympathetic nervous system, such as reductions of saliva flow rate as a result of strenuous exercise. Similarly, the responses observed in Chapter 8 imply a comparable trend of chronic sIgA exercise responses in tetraplegic athletes as found in the able-bodied population, namely a decrease in sIgA secretion rate during periods of heavy training. These are the first studies in wheelchair athlete populations to investigate mucosal immune responses. Interestingly, despite the disruption of their sympathetic nervous system, some responses in tetraplegic athletes are comparable with findings in able-bodied populations. It is possible that due to their highly trained nature, these tetraplegic individuals are able to compensate for their loss of central sympathetic innervation. This may be by way of adapted spinal reflex or parasympathetic nervous system activity, or increased sensitivity of receptors involved in autonomic pathways. Therefore, sympathetic nervous function in tetraplegic athletes may be qualitatively altered, but in parts still be functional.

Bifidobacterium infantis (BI) and other probiotics are non-pathogenic living organisms that have recently gained attention for their possible therapeutic implications on the health of the digestive tract. The mechanisms by which probiotics exert their effects are largely unknown. This study explored the protective and regulatory effect of oral BI on the enteric nervous system (ENS) in the TNBS-induced colitis rats. Electrical field stimulation and chemical stimulation by serotonin (5-HT) were used to elicit changes in the short-circuit current (Isc) response of the colonic rat tissue. BI-fed colitis rats expressed trends of higher secretomotor activity and revealed signs of decreased macroscopic inflammatory damage when compared to sham-fed colitis rats, suggesting a protective and preventative role of oral BI. These findings may provide additional insights for understanding the prophylactic and therapeutic value of specific probiotics in intestinal inflammatory disorders, offering the possibility of a noninvasive alternative to toxic and immune-compromising drugs. / Access to thesis permanently restricted to Ball State community only / Department of Physiology and Health Science

Central mechanisms involving aldosterone - mineralocorticoid receptor (MR) activation mediate the increase in sympathetic tone after myocardial infarction (MI). We hypothesized that an increase in cardiac sympathetic activity (CSA) post MI facilitates cardiac sympathetic axonal sprouting, and that central MR blockade attenuates CSA and reduces cardiac sympathetic hyperinnervation post MI. Western blotting and qRT-PCR were used to assess protein and gene expression, and fluorescent immunohistochemistry was used to study changes in sympathetic innervation. Tyrosine hydroxylase (TH) and Norepinephrine transporter protein content in the non-infarcted base of the heart remained unaltered. In contrast, protein gene product (PGP 9.5) protein was significantly increased 2 fold in the base of the heart, and 6 fold in the peri-infarct area at 1 wk post MI, and associated with increased ubiquitin expression. Cardiac myocytes rather than sympathetic axons were identified as the main source of elevated PGP 9.5 expression. At the infarct border sympathetic hyperinnervation was observed with a 4 fold increase in growth associated protein 43 (GAP 43), a 2 fold increase in TH and a 50% increase in PGP 9.5 positive fibers when compared to the epicardial side of the left ventricle in sham rats. Central infusion of the MR blocker eplerenone at 5 ug/day for 9 days post MI markedly attenuated the increase in TH, GAP 43 and PGP 9.5 nerve densities at the infarct border. Central MR blockade may attenuate sympathetic hyperinnervation by several mechanisms, including decreasing CSA post MI, or affecting expression or function of nerve growth factor protein. Marked PGP 9.5 expression occurs in cardiomyocytes early post MI, which may contribute to the increase in ubiquitin and the early cardiac remodeling post MI.