Marked innervation but also signs of nerve degeneration in between the Achilles and plantaris tendons and presence of innervation within the plantaris tendon in midportion Achilles tendinopathy

Objectives:

The plantaris tendon is increasingly recognised as an important factor in midportion Achilles tendinopathy. Its innervation pattern is completely unknown.

Methods:

Plantaris tendons (n=56) and associated peritendinous tissue from 46 patients with midportion Achilles tendinopathy and where the plantaris tendon was closely related to the Achilles tendon were evaluated. Morphological evaluations and stainings for nerve markers [general (PGP9.5), sensory (CGRP), sympathetic (TH)], glutamate NMDA receptor and Schwann cells (S-100β) were made.

Results:

A marked innervation, as evidenced by evaluation for PGP9.5 reactions, occurred in the peritendinous tissue located between the plantaris and Achilles tendons. It contained sensory and to some extent sympathetic and NMDAR1-positive axons. There was also an innervation in the zones of connective tissue within the plantaris tendons. Interestingly, some of the nerve fascicles showed a partial lack of axonal reactions.

Conclusion:

New information on the innervation patterns for the plantaris tendon in situations with midportion Achilles tendinopathy has here been obtained. The peritendinous tissue was found to be markedly innervated and there was also innervation within the plantaris tendon. Furthermore, axonal degeneration is likely to occur. Both features should be further taken into account when considering the relationship between the nervous system and tendinopathy.     

Presence of substance P and the neurokinin-1 receptor in tenocytes of the human Achilles tendon

Nerve signal substances, such as the tachykinin substance P (SP), may be involved in the changes that occur in response to tendinopathy (tendinosis). It is previously known that the level of SP innervation within tendon tissue is limited, but results of experimental studies have suggested that SP may have stimulatory, angiogenetic and healing effects in injured tendons. Therefore, it would be of interest to know if there is a local SP-supply in tendon tissue. In the present study, the patterns of expression of SP and its preferred receptor, the neurokinin-1 receptor (NK-1 R), in normal and tendinosis human Achilles tendons were analyzed by use of both immunohistochemistry and in situ hybridization. We found that there was expression of SP mRNA in tenocytes, and that tenocytes showed expression of NK-1 R at protein as well as mRNA levels. The observations concerning both SP and NK-1 R were most evident for tenocytes in tendinosis tendons. Our findings suggest that SP is produced in tendinosis tendons, and furthermore that SP has marked effects on the tenocytes via the NK-1 R. It cannot be excluded that the SP effects are of importance concerning the processes of reorganization and healing that occur for tendon tissue in tendinosis. In conclusion, it appears as if SPergic autocrine/paracrine effects occur in tendon tissue during the processes of tendinosis, hitherto unknown effects for human tendons.     

Immunohistochemical and in situ hybridization observations favor a local catecholamine production in the human Achilles tendon

Results of recent studies using immunohistochemistry show evidence of an occurrence of catecholamine production in the cells (tenocytes) of patellar tendons exhibiting tendinopathy (tendinosis). In the present study, antibodies against the catecholamine-synthesizing enzyme tyrosine hydroxylase (TH) and alpha1-adrenoreceptors were applied to sections of specimens of normal and tendinosis Achilles tendons. In situ hybridization using a probe detecting human TH mRNA was also utilized. It was found that sympathetic innervation was very scarce. On the other hand, there were distinct alpha1-adrenoreceptor immunoreactions in blood vessel walls. Interestingly, tenocytes, particularly from tendinosis samples in which the tenocytes showed an abnormal shape (not the typical slender appearance), displayed TH immunoreactions and reactions for TH mRNA. Of further interest was the finding of alpha1-adrenoreceptor immunoreactions in tenocytes. The observations show not only evidence of local catecholamine production at the protein level, which was the case in recent studies for the patellar tendon, but also at the mRNA level. The observations suggest that the tenocytes, especially those with disfigured appearances in tendinosis, can produce catecholamines and also that they can respond to sympathetic transmitters. This is of interest as adrenergic stimulation in other parts of the body is known to induce degenerative/apoptotic and proliferative events, features which are seen in Achilles tendinosis. These observations are completely new findings concerning the human Achilles tendon. It is likely that locally produced catecholamines and the occurrence of autocrine/paracrine effects of these substances are of great relevance during the process of tendinosis.     

Noradrenergic and peptidergic innervation of the mammary gland in the immature pig

The presence and pattern of coexistence of some biologically active substances in nerve fibres supplying the mammary gland in the immature pig were studied using immunohistochemical methods. The substances studied included: protein gene product 9.5 (PGP), tyrosine hydroxylase (TH), somatostatin (SOM), neuropeptide Y (NPY), galanin (GAL), calcitonin gene-related peptide (CGRP) and substance P (SP). The mammary gland was found to be richly supplied by PGP-immunoreactive (PGP-IR) nerve fibres that surrounded blood vessels, bundles of smooth muscle cells and lactiferous ducts. The vast majority of these nerves also displayed immunoreactivity to TH. Immunoreactivity to SOM was observed in a moderate number of nerve fibres which were associated with smooth muscles of the nipple and blood vessels. Immunoreactivity to NPY occurred in many nerve fibres associated with blood vessels and in single nerves supplying smooth muscle cells. Solitary GAL-IR axons supplied mostly blood vessels. Many CGRP-IR nerve fibres were associated with both blood vessels and smooth muscles. SP-IR nerve fibres richly supplied blood vessels only. The colocalization study revealed that SOM, NPY and GAL partly colocalized with TH in nerve fibres supplying the porcine mammary gland.     

Presence of a non-neuronal cholinergic system and occurrence of up- and down-regulation in expression of M2 muscarinic acetylcholine receptors: new aspects of importance regarding Achilles tendon tendinosis (tendinopathy)

Limited information is available concerning the existence of a cholinergic system in the human Achilles tendon. We have studied pain-free normal Achilles tendons and chronically painful Achilles tendinosis tendons with regard to immunohistochemical expression patterns of the M(2) muscarinic acetylcholine receptor (M(2)R), choline acetyltransferase (ChAT), and vesicular acetylcholine transporter (VAChT). M(2)R immunoreactivity was detected in the walls of blood vessels. As evidenced via parallel staining for CD31 and alpha-smooth muscle actin, most M(2)R immunoreactivity was present in the endothelium. M(2)R immunoreactivity also occured in tenocytes, which regularly immunoreact for vimentin. The degree of M(2)R immunoreactivity was highly variable, tendinosis tendons that exhibit hypercellularity and hypervascularity showing the highest levels of immunostaining. Immunoreaction for ChAT and VAChT was detected in tenocytes in tendinosis specimens, particularly in aberrant cells. In situ hybridization revealed that mRNA for ChAT is present in tenocytes in tendinosis specimens. Our results suggest that autocrine/paracrine effects occur concerning the tenocytes in tendinosis. Up-regulation/down-regulation in the levels of M(2)R immunoreactivity possibly take place in tenocytes and blood vessel cells during the various stages of tendinosis. The presumed local production of acetylcholine (ACh), as evidenced by immunoreactivity for ChAT and VAChT and the detection of ChAT mRNA, appears to evolve in response to tendinosis. These observations are of importance because of the well-known vasoactive, trophic, and pain-modulating effects that ACh is known to have and do unexpectedly establish the presence of a non-neuronal cholinergic system in the Achilles tendon.     

Ectatic blood vessels in port-wine stains lack innervation: possible role in pathogenesis.

The innervation pattern of port-wine stains was investigated using indirect immunohistochemistry with antibodies to protein gene product 9.5 (PGP 9.5), neuron-specific enolase (NSE), calcitonin gene-related peptide (CGRP), and neurofilament (NF). The pathologically dilated vessels in the middle and deep dermis were found to have defective innervation with only single or no nerve fibers in their vicinity, while other structures in the skin showed a normal density of fibers. NSE- and PGP-like immunoreactive (-LI) nerve fibers were observed innervating vessels with a normal morphology and other structures in the skin, such as sweat glands and hair follicles, as free nerve endings and in nerve bundles. The nerve bundles were often seen to pass the ectatic vessels without giving off any branches. CGRP-LI nerve fibers were detected running toward epidermis, whereas no fibers were found around the ectatic vessels. NF-LI fibers were seen innervating normal vessels in dermis, while in relation to the dilated vessels, no or only occasional fibers were observed. The lack of innervation may be of importance for the development of the disease as a result of decreased tonus of the vessels and/or a loss of neuronal trophic factors.     

Vascular NK-1 receptor occurrence in normal and chronic painful Achilles and patellar tendons: studies on chemically unfixed as well as fixed specimens

It is not known as to whether the Achilles and patellar tendons contain neurokinin-1 (NK-1) receptors. This is a drawback when considering the fact that pain symptoms are frequent in these and as recent studies show that the pain symptoms might be cured via interference with blood vessel function. In the present study, the human Achilles and patellar tendons were examined concerning immunohistochemical expression of the NK-1 receptor. Chemically unfixed and fixed specimens, TRITC and PAP stainings and a battery of NK-1 receptor antibodies, including antibodies against the C-terminus and the N-terminal region, were utilized. NK-1 receptor immunoreaction could be detected in inner parts of the walls of large blood vessels and in the walls of small blood vessels. To some extent, NK-1 immunoreaction was also detectable in small nerve fascicles and in tenocytes. It was found to be of utmost importance to apply both chemically unfixed and fixed specimens. The use of chemically unfixed tissue was found advantageous in order to depict the immunoreactions in the blood vessel walls. The observations represent new findings and are of relevance as substance P (SP) is known to be of importance where neurogenic angiogenesis contributes to diseases and as SP on the whole has profound effects concerning blood vessel regulation.     

Extensive expression of markers for acetylcholine synthesis and of M2 receptors in tenocytes in therapy-resistant chronic painful patellar tendon tendinosis - a pilot study

We have recently obtained evidence favoring the occurrence of an up-regulation of a non-neuronal cholinergic system in chronic painful patellar tendon tendinosis. It seems possible that this up-regulation to a certain degree may be involved in the manifestations of the disease. Today, there is a new, very successful, line of treatment of patellar tendinosis in the form of Doppler guided sclerosing injections. However, a few patients seem resistant to this therapy. Therefore, we have in this pilot study investigated biopsies from the patellar tendon of three such therapy-resistant patients, using immunohistochemistry. In situ hybridization was also applied. Comparisons were made with a material of specimens from both normal (n=16) and tendinosis (n=7) tendons, also previously examined. The study showed that there were extensive immunoreactions for choline acetyltransferase (ChAT) and vesicular acetylcholine transporter, as well as for the M(2) muscarinic acetylcholine receptor, in the overwhelming majority of the tenocytes. The immunoreactions were more pronounced than those generally obtained in the tendinosis tissue of the previously studied patients and clearly more pronounced than those of patellar tendon tissue of controls. Also, for the first time, we here present findings of mRNA for ChAT within tenocytes. In conclusion, it appears as if there is an excessive local acetylcholine (ACh) production and an occurrence of marked ACh effects in cases of severe tendinosis. An excessive production of local ACh might be related to pain sensation and the processes that occur in tendinosis development, such as cell proliferation. Thus, the results of this pilot study suggest that non-neuronal ACh is highly involved in the pathology of therapy-resistant patellar tendinosis.     

Target-specific innervation by autonomic and sensory nerve fibers in hairy fetal skin transplanted into the anterior eye chamber of adult rat

Summary Pieces of hairy skin tissue of fetal rat were transplanted into the anterior eye chamber of adult rats. The ability of autonomic and sensory nerve fibers from the host iris to innervate the grafted skin tissue was immunohistochemically and enzyme-histochemically examined using antisera against tyrosine hydroxylase (TH), substance P (SP), calcitonin gene-related peptide (CGRP) and vasoactive intestinal peptide (VIP), and a reaction medium for acetylcholinesterase (AchE). The grafted tissue was successfully implanted and connected with the host iris. Epidermis, dermis, subcutaneous tissue, hairs, hair follicles, sebaceous glands, and piloerector muscles developed in the graft. Two weeks after transplantation, TH-, SP-, and CGRP-immunoreactive fibers were observed in association with the blood vessels in the graft. Four weeks after transplantation, TH-immunoreactive fibers were distributed in the piloerector muscles, whereas SP-and CGRP-immunoreactive fibers were present around the hair follicles. VIP-immunoreactive and AchE-positive fibers were restricted to the host iris at all survival times. These results suggest that the outgrowth of autonomic and sensory nerve fibers from the host iris show target specificity for the grafted skin tissue.     

Morphological and neuroanatomical study of the mammary gland in the immature and mature European beaver (Castor fiber)

This study investigated general morphology and immunohistochemical properties of nerve fibres supplying the mammary gland (MG) in the European beaver. The microscopic analysis of the beaver mammary gland revealed the presence of morphological structures which are characteristic for mammals. There were no distinct differences in the morphological features of the mammary gland between the juvenile and non-pregnant mature beaver.The nerve fibres were visualized using antibodies against protein gene product 9.5 (PGP) and biologically active substances including β-hydroxylase tyrosine (DβH), neuropeptide Y (NPY), calcitonine gene-related peptide (CGRP) and substance P (SP). The study has revealed that the MG in the juvenile and mature beaver is richly supplied with PGP-immunoreactive (PGP-IR) nerve fibres. The most abundant innervation was observed in the nipple and less numerous nerve terminals supplied the glandular tissue. Double-labelling immunohistochemistry disclosed that the majority of PGP-IR nerve fibres associated with blood vessels and smooth muscle cells in both the nipple and glandular tissue were also DßH-IR. However, these nerve terminals were less numerous in the glandular tissue than in the nipple. Most of the DßH-IR axons associated with arteries and smooth muscle cells in the entire gland also stained for NPY. Small number of DßH/NPY-IR fibres supplied veins. CGRP-IR fibres were more abundant than those expressing SP. No distinct differences in the distribution and immunohistochemical characteristic of nerve fibres were observed between the juvenile and adult animals. The distribution and immunohistochemical properties of nerve fibres supplying the gland in the beaver remind those previously described in other mammalian species.     

Cutaneous innervation in man visualized with protein gene product 9.5 (PGP 9.5) antibodies

Using antibodies to the neuronal cytoplasmic protein, protein gene product 9.5 (PGP 9.5) the cutaneous innervation in man was investigated. The distribution of PGP 9.5 immunoreactive nerve fibers was compared with the distribution of nerve fibers immunoreactive to neuron specific enolase, neurofilament proteins, calcitonin gene related peptide, vasoactive intestinal polypeptide and neuropeptide Y. PGP 9.5 immunoreactive nerve fibers were found in the epidermis, dermis, in Meissner's corpuscles, innervating Merkel cells, around blood vessels, sweat glands and hair follicles. Merkel cells were also PGP 9.5 positive. The labelled nerve fibers included sensory and autonomic fibers, visualizing the whole innervation of the human skin. The number of positive fibers and the intensity of the fluorescence was greater with PGP 9.5 antibodies than with any of the other markers included. Thus, PGP 9.5 antibodies may serve as a tool for investigations of cutaneous innervation, reinnervation and nerve regeneration in different clinical conditions.     

Cutaneous innervation in man visualized with protein gene product 9.5 (PGP 9.5) antibodies

Summary Using antibodies to the neuronal cytoplasmic protein, protein gene product 9.5 (PGP 9.5) the cutaneous innervation in man was investigated. The distribution of PGP 9.5 immunoreactive nerve fibers was compared with the distribution of nerve fibers immunoreactive to neuron specific enolase, neurofilament proteins, calcitonin gene related peptide, vasoactive intestinal polypeptide and neuropeptide Y. PGP 9.5 immunoreactive nerve fibers were found in the epidermis, dermis, in Meissner's corpuscles, innervating Merkel cells, around blood vessels, sweat glands and hair follicles. Merkel cells were also PGP 9.5 positive. The labelled nerve fibers included sensory and autonomic fibers, visualizing the whole innervation of the human skin. The number of positive fibers and the intensity of the fluorescence was greater with PGP 9.5 antibodies than with any of the other markers included. Thus, PGP 9.5 antibodies may serve as a tool for investigations of cutaneous innervation, reinnervation and nerve regeneration in different clinical conditions.     

Effect of subcutaneous pancreatic tissue transplants on streptozotocin-induced diabetes in rats. III. Distribution of neuropeptides in normal and diabetic (host) pancreas.

This study examines whether there is a change in the pattern of distribution of cholecystokinin-octapeptide (CCK-8), calcitonin-gene-related peptide (CGRP), neuropeptide-Y (NPY), substance P (SP) and vasoactive intestinal polypeptide (VIP) in the pancreas of streptozotocin (STZ)-diabetic (host) rats after subcutaneous pancreatic transplantation. Varicose CCK-8-immunopositive nerve fibres were observed in the wall of blood vessels of both normal and diabetic host pancreata. The density of CCK-8-immunoreactive varicose nerve fibres appeared to have increased in host rat pancreas. CGRP was demonstrated in many nerve fibres located in the wall of blood vessels of both normal and host pancreas. CGRP, however, seemed to be better expressed in the nerves of host pancreas when compared to normal. The pancreata of both normal and diabetic (host) rats contained numerous NPY-immunopositive varicose nerve fibres located in the wall of blood vessels. SP was demonstrated in neurons located in the interlobular areas of normal tissue and in fine varicose nerve fibres of the interacinar region of the pancreas of STZ-induced diabetic rats with SPTG. In normal pancreatic tissue, VIP-immunopositive nerve fibres were observed in all areas of the pancreas. VIP-positive nerve fibres were still discernible especially in the interacinar regions of the pancreas of host rats. In conclusion, the pattern of distribution and density of NPY, SP and VIP in the pancreas of STZ-induced diabetic rats with SPTG is similar to that observed in normal pancreas, but the expression of CGRP and CCK-8 seemed to have increased as a result of transplantation and or diabetes.     

Ontogeny of intrinsic innervation in the human thymus and spleen.

The ontogeny of the innervation of human lymphoid organs has not been studied in detail. Our aim was to assess the nature and distribution of parenchymal nerves in human fetal thymus and spleen. We used the peroxidase immunohistochemical technique with antibodies specific to neuron-specific enolase (NSE), neurofilaments (NF), PGP9.5, S100 protein, and tyrosine hydroxylase (TH) and evaluated our results with image analysis. In human fetal thymus, NSE-, NF-, S100-, PGP9.5-, and TH-positive nerves were identified associated with large blood vessels from 18 gestational weeks (gw) onwards, increasing in density during development. Their branches penetrated the septal areas at 20 gw, reaching the cortex and the corticomedullary junction between 20 and 23 gw. Few nerve fibers were seen in the medulla in close association with Hassall's corpuscles. In human fetal spleen, NSE-, NF-, S100-, PGP9.5-, and TH-positive nerve fibers were localized in the connective tissue surrounding the splenic artery at 18 gw. Perivascular NSE-, NF-, S100-, PGP9.5-, and TH-positive nerve fibers were seen extending into the white pulp, mainly in association with the central artery and its branches, increasing in density during gestation. Scattered NSE-, NF-, S100-, PGP9.5-, and TH-positive nerve fibers and endings were localized in the red pulp from 18 gw onward. The predominant perivascular distribution of most parenchymal nerves implies that thymic and splenic innervation may play an important functional role during intrauterine life.     

In situ hybridization studies favouring the occurrence of a local production of BDNF in the human Achilles tendon

Brain derived neurotrophic factor (BDNF) is a multipotent neurotrophin known for its growth-influencing and apoptosis-modulating functions, as well as for its function to interact with neurotransmitters/neuromodulators. BDNF is reported to be mainly produced in the brain. BDNF can be absorbed into peripheral tissue from the blood stream. Expression of this neurotrophin at the protein level, as well as of the neurotrophin receptor p75, has been previously shown for the principal cells (tenocytes) of the Achilles tendon. However, there is no proof at the mRNA level that BDNF is produced by the tenocytes. As the Achilles tendon tenocytes show "neuronal-like" characteristics, in the form of expressions favouring synthesis of several neuromodulators/neurotransmitters, and as BDNF especially is produced in neurons, it is of interest to confirm this. In the present study, therefore, in situ hybridization for demonstration of BDNF mRNA was performed on biopsies from Achilles tendons of patients with tendinosis and pain-free non-tendinosis individuals. The results showed that the tenocytes of both groups exhibited BDNF mRNA reactions. These observations indeed favour the idea that BDNF is produced by tenocytes in the human Achilles tendon, why Achilles tendon tissue is a tissue in which BDNF can be locally produced. BDNF can have modulatory functions for the tenocytes, including apoptosis-modifying effects via actions on the p75 receptor and interactive effects with neurotransmitters/neuromodulators produced in these cells. This possibility should be further studied for Achilles tendon tissue.     

CGRP-like immunoreactivity in sensory nerve endings of the Golgi tendon organ. A light- and electron-microscopic study in the grey short-tailed opossum (Monodelphis domestica)

Using light- and electron-microscopic immunohistochemistry, it was shown that primary sensory nerve endings in Golgi tendon organs of the grey short-tailed opossum (Monodelphis domestica) contain immunoreactivities to a polyclonal antibody directed against calcitonin gene-related peptide (CGRP). Myelinated afferent axons (6-9 microns in diameter) of the Golgi tendon organs stained moderately for CGRP. Sensory nerve endings within the sensory compartment of the Golgi tendon organs displayed electron-dense accumulations corresponding to dark-brown staining in adjacent semithin sections. On the outer surface of tendon organs C fibre bundles were observed showing CGRP-like immunoreactivity.     

Immunocytochemical evidence of plasticity in the nervous structures of the rat lower lip

In this immunocytochemical study we investigated the distribution of nervous structures in the lower lip of adult rats. The region is characterized by a rich cutaneous and mucosal sensory innervation originating from terminal branches of the trigeminal system. Lower lip innervation was investigated by detection of the general neuronal marker protein gene product 9.5 (PGP 9.5) and the growth-associated protein 43 (GAP-43), a neurochemical marker of neuronal plasticity. The entire neural network of both cutaneous and mucosal aspects was stained by the antibody to PGP 9.5. In particular, nerve fibers were observed in the submucosal and the subepithelial plexuses. Thin immunoreactive fibers were observed within the epithelial layers ending as free fibers or as fibers associated with immunopositive Merkel cells. Well-identified anatomical structures receiving sensory or autonomic innervation were also surrounded by PGP 9.5-ir nerve fibers, in particular, hair follicles, vibrissae, glands, and blood vessels. GAP-43-immunostained nerve fibers were observed in all these structures; however, they were generally less numerous than the PGP 9.5-immunoreactive elements. An equal amount of PGP 9.5 and GAP-43 immunoreactivity occurred, in contrast, in the subepidermal and the submucosal plexuses, or in the epidermis and the mucosal epithelium. The present results show that GAP-43 is normally expressed in the mature trigeminal sensory system of the rat. Skin and oral mucosa are characterized by continuous remodeling that may also involve the sensory nervous apparatus. Continuous neural remodeling, regeneration and sprouting may be the reason for the observed expression of GAP-43.     

Catecholaminergic, cholinergic and peptidergic innervation of gut-associated lymphoid tissue in porcine jejunum and ileum

With its abundance of neurons and immunocytes, the gut is a potentially important site for the study of the interaction between the nervous and immune systems. Using immunohistochemical techniques, we tested the hypothesis that gut-associated lymphoid tissue in the porcine small intestine might receive catecholaminergic, cholinergic and peptidergic innervation. Antibodies against protein gene product (PGP) 9.5 were employed to detect neuronal membranes; antibodies against tyrosine hydroxylase (TH), type 2 vesicular monoamine transporter (VMAT-2) and choline acetyltransferase (ChAT) were used to detect catecholaminergic and cholinergic neurons; and antibodies to neuromedin U-8 (NMU-8), substance P (SP) and vasoactive intestinal peptide (VIP) were also used. PGP9.5-immunoreactive nerve fibers were observed between jejunal Peyer's patch (PP) follicles and in submucosal ganglia localized at the base of continuous ileal PP. Many ChAT-positive and a few TH-/VMAT-2-immunoreactive neurons or axons adjacent to jejunal and ileal PP were observed. Neurons and fibers from ganglia situated between or at the base of PP follicles manifested robust immunoreactivities to VIP and NMU-8; relatively less SP immunoreactivity was observed at these locations. All neuromedin-U 8-positive neurons observed exhibited immunoreactivity to ChAT as did some VIP-positive neurons. The specific chemical coding of enteric neurons in close apposition to jejunal and ileal PP and the differential localization of neuropeptides within the jejunal and ileal PP are indicative of neuroimmunomodulation at these sites.     

Quadriceps tendon allografts as an alternative to Achilles tendon allografts: a biomechanical comparison

Quadriceps tendon with a patellar bone block may be a viable alternative to Achilles tendon for anterior cruciate ligament reconstruction (ACL-R) if it is, at a minimum, a biomechanically equivalent graft. The objective of this study was to directly compare the biomechanical properties of quadriceps tendon and Achilles tendon allografts. Quadriceps and Achilles tendon pairs from nine research-consented donors were tested. All specimens were processed to reduce bioburden and terminally sterilized by gamma irradiation. Specimens were subjected to a three phase uniaxial tension test performed in a custom environmental chamber to maintain the specimens at a physiologic temperature (37 ± 2 °C) and misted with a 0.9 % NaCl solution. There were no statistical differences in seven of eight structural and mechanical between the two tendon types. Quadriceps tendons exhibited a significantly higher displacement at maximum load and significantly lower stiffness than Achilles tendons. The results of this study indicated a biomechanical equivalence of aseptically processed, terminally sterilized quadriceps tendon grafts with bone block to Achilles tendon grafts with bone block. The significantly higher displacement at maximum load, and lower stiffness observed for quadriceps tendons may be related to the failure mode. Achilles tendons had a higher bone avulsion rate than quadriceps tendons (86 % compared to 12 %, respectively). This was likely due to observed differences in bone block density between the two tendon types. This research supports the use of quadriceps tendon allografts in lieu of Achilles tendon allografts for ACL-R.     

Neurochemical pattern of the complex innervation of neuroepithelial bodies in mouse lungs

As best characterized for rats, it is clear that pulmonary neuroepithelial bodies (NEBs) are contacted by a plethora of nerve fiber populations, suggesting that they represent an extensive group of multifunctional intraepithelial airway receptors. Because of the importance of genetically modified mice for functional studies, and the current lack of data, the main aim of the present study was to achieve a detailed analysis of the origin and neurochemical properties of nerve terminals associated with NEBs in mouse lungs. Antibodies against known selective markers for sensory and motor nerve terminals in rat lungs were used on lungs from control and vagotomized mice of two different strains, i.e., Swiss and C57-Bl6. NEB cells were visualized by antibodies against either the general neuroendocrine marker protein gene-product 9.5 (PGP9.5) or calcitonin gene-related peptide (CGRP). Thorough immunohistochemical examination of NEB cells showed that some of these NEB cells also exhibit calbindin D-28 k (CB) and vesicular acetylcholine transporter (VAChT) immunoreactivity (IR). Mouse pulmonary NEBs were found to receive intraepithelial nerve terminals of at least two different populations of myelinated vagal afferents: (1) Immunoreactive (ir) for vesicular glutamate transporters (VGLUTs) and CB; (2) expressing P2X₂ and P2X₃ ATP receptors. CGRP IR was seen in varicose vagal nerve fibers and in delicate non-vagal fibers, both in close proximity to NEBs. VAChT immunostaining showed very weak IR in the NEB-related intraepithelial vagal sensory nerve terminals. nNOS- or VIP-ir nerve terminals could be observed at the base of pulmonary NEBs. While a single NEB can be contacted by multiple nerve fiber populations, it was clear that none of the so far characterized nerve fiber populations contacts all pulmonary NEBs. The present study revealed that mouse lungs harbor several populations of nerve terminals that may selectively contact NEBs. Although at present the physiological significance of the innervation pattern of NEBs remains enigmatic, it is likely that NEBs are receptor–effector end-organs that may host complex and/or multiple functional properties in normal airways. The neurochemical information on the innervation of NEBs in mouse lungs gathered in the present study will be essential for the interpretation of upcoming functional data and for the study of transgenic mice.