Scanning electron microscopy study of the meniscus of the knee in patients with anterior cruciate ligament lesions]

In the aim of bringing a contribution to role of the knee menisci and the functional connection between meniscal lesions and knee joint instability, four patients have been studied, who underwent arthroscopy for sub-acute tear of anterior cruciate ligament without clinical or morphological evidence of meniscal pathology. In these patients biopsies have been taken from one of the menisci and processed for the examination by the SEM. As control, meniscal fragments from two healthy knees have been studied from patients who underwent amputation at thigh for accidental trauma with irreparable lesion of femoral artery. In patients affected by ligamentous tear, on the meniscal surface a lot of deposited material appears, as well as many microlesions as clefts or cracking and distortion of superficial collagenous fibers. All described alteration are more evident in the central portion of menisci than in the anterior or posterior horns. The above mentioned patterns suggest a strong correlation of the knee instability due to ligamentous lesions and meniscal degenerative process which would onset before its clinical or macroscopical evidence.     

Is the meniscus of the knee joint a fibrocartilage?

A histological analysis of the structure of intact knee joint menisci was carried out in adult dogs. By means of specific histochemical methods for the connective tissue and cartilage, it was found that the meniscus as a whole does not have a unique structure. The anterior and posterior horns are populated by round chondroid cells encircled by abundant interstitial substance and branched wavy connective fibers; blood vessels are present. The outer third of the meniscus is constituted of cross bundles of connective fibers, fibrocytes and spindle-like areas of loose connective tissue with blood vessels. The inner avascular two thirds of the meniscus are filled with parallel circumferentially oriented fascicles of connective fibers, ovally elongated chondroid cells, and a small quantity of chondroid interstitial substance. In some menisci, in the inner two thirds of the body, there are isles of typical cartilage, which show metachromasia of the beta type and rarely of the gamma type. The occurrence and way of the manifestation of cartilage are of an individual character. The structural duality of the knee meniscus is accounted for by its functional duality manifested in offering resistance to the forces of traction and pressure, the latter ones favoring the process of evolution of tissue from connective, through chondroid, to cartilaginous.     

Effect of meniscus replacement fixation technique on restoration of knee contact mechanics and stability

The menisci are important biomechanical components of the knee. We developed and validated a finite element model of meniscal replacement to assess the effect of surgical fixation technique on contact behavior and knee stability. The geometry of femoral and tibial articular cartilage and menisci was segmented from magnetic resonance images of a normal cadaver knee using MIMICS (Materialise, Leuven, Belgium). A finite element mesh was generated using HyperWorks (Altair Inc, Santa Ana, CA). A finite element solver (Abaqus v6.9, Simulia, Providence, RI) was used to compute contact area and stresses under axial loading and to assess stability (reaction force generated during anteroposterior translation of the femur). The natural and surgical attachments of the meniscal horns and peripheral rim were simulated using springs. After total meniscectomy, femoral contact area decreased by 26% with a concomitant increase in average contact stresses (36%) and peak contact stresses (33%). Replacing the meniscus without suturing the horns did little to restore femoral contact area. Suturing the horns increased contact area and reduced peak contact stresses. Increasing suture stiffness correlated with increased meniscal contact stresses as a greater proportion of tibiofemoral load was transferred to the meniscus. A small incremental benefit was seen of simulated bone plug fixation over the suture construct with the highest stiffness (50 N/mm). Suturing the rim did little to change contact conditions. The nominal anteroposterior stiffness reduced by 3.1 N/mm after meniscectomy. In contrast to contact area and stress, stiffness of the horn fixation sutures had a smaller effect on anteroposterior stability. On the other hand suturing the rim of the meniscus affected anteroposterior stability to a much larger degree. This model emphasizes the importance of the meniscus in knee biomechanics. Appropriate meniscal replacement fixation techniques are likely to be critical to the clinical success of meniscal replacement. While contact conditions are mainly sensitive to meniscus horn fixation, the stability of the knee under anteroposterior shear loads appeared to be more sensitive to meniscal rim fixation. This model may also be useful in predicting the effect of biomaterial mechanical properties and meniscal replacement shape on knee contact conditions.     

T2 Values of Posterior Horns of Knee Menisci in Asymptomatic Subjects

Purpose

The magnetic resonance (MR) T2 value of cartilage is a reliable indicator of tissue properties and therefore may be used as an objective diagnostic tool in early meniscal degeneration. The purpose of this study was to investigate age, gender, location, and zonal differences in MR T2 value of the posterior horns of knee menisci in asymptomatic subjects.

Methods

Sixty asymptomatic volunteers (30 men and 30 women) were enrolled and divided into three different age groups: 20–34, 35–49 and 50–70 years. The inclusion criteria were BMI<30 kg/cm² _, normalized Western Ontario and McMaster Universities (WOMAC) pain score of zero, and no evidence of meniscal and ligamentous abnormalities on routine knee MR imaging. The T2 values were measured on images acquired with a T2-weighted fat-suppressed turbo spin-echo sequence at 3T.

Results

The mean T2 values in both medial and lateral menisci for the 20–34, 35–49, and 50–70 age groups were 9.94 msec±0.94, 10.73 msec±1.55, and 12.36 msec±2.27, respectively, for women and 9.17 msec±0.74, 9.64 msec±0.67, and 10.95 msec±1.33, respectively, for men. The T2 values were significantly higher in the 50–70 age group than the 20–34 age group (P<0.001) and in women than in men (P = 0.001, 0.004, and 0.049 for each respective age group). T2 values were significantly higher in medial menisci than in lateral menisci only in women age 50–70 (3.33 msec, P = 0.006) and in the white zone and red/white zone of the 50–70 and 35–49 age groups than that of the 20–34 age group (2.47, 1.02; 2.77, 1.16 msec, respectively, all P<0.01).

Conclusion

The MR T2 values of the posterior meniscal horns increase with increasing age in women and are higher in women than in men. The age-related rise of T2 values appears to be more severe in medial menisci than in lateral menisci. Differences exist in the white zone and red/white zone.     

Neuropeptide Y-containing nerves in rat gonads: sex difference and development

The objectives of the present study were 1) to evaluate for a sex difference in innervation of adult rat gonads by neuropeptide Y-immunoreactive (NPY-I) nerves and 2) to examine the development of innervation of rat gonads by NPY-I nerves during the fetal and neonatal periods. With fluorescence immunocytochemistry, NPY-I nerves were profuse in adult ovarian tissues. Ovarian blood vessels were particularly well innervated by NPY-I nerves, and nerves were also detected in interstitial gland tissues. No nerves were found within the testis, and NPY-I nerves were only rarely located within the tunica albuginea. During fetal life, ovaries were devoid of NPY-I nerves; however, nerves were visualized within the connective tissue immediately peripheral to the ovary on fetal Day 22. As early as postnatal Day 2, NPY-I nerves were observed in connective tissue septa of the developing ovary. By postnatal Day 12, NPY-I nerves surrounded developing follicles and blood vessels of the ovarian cortex. In the developing testis after postnatal Day 5, NPY-I nerves were limited to the tunica albuginea and surrounding large subcapsular blood vessels. Structures within the testis lacked innervation by NPY-I nerves. These anatomical studies suggest that NPY-I nerves are absent in the gonads during fetal life and grow into the ovary and not the testis during the perinatal period and that NPY-I nerves may play a role in the functioning of the rat ovary, but may not be important in control of testicular function.     

Analysis of Gene Expression and Ultrastructure of Stifle Menisci from Juvenile and Adult Pigs

The origin of the age-associated degenerative processes in meniscal tissue is poorly understood and may be related to an imbalance of anabolic and catabolic metabolism. The aim of the current study was to compare medial menisci isolated from juvenile pigs and degenerated medial menisci from adult pigs in terms of gene expression profile and ultrastructure. Medial menisci were isolated from the knee joints of juvenile and adult pigs (n = 8 for each group). Degeneration was determined histologically according to a scoring system. In addition, the gene expression profiles of 14 genes encoding extracellular matrix proteins, catabolic matrix metalloproteinases and mediators of inflammation were analyzed. Changes in the ultrastructure of the collagen network of the meniscal tissue were analyzed by using transmission electron microscopy. The histologic analysis of menisci showed significantly higher grade of degeneration in tissue isolated from adult porcine knee joints compared with menisci isolated from juvenile knee joints. In particular, destruction of the collagen network was greater in adult menisci than in juvenile menisci. Degenerated menisci showed significantly decreased gene expression of COL1A1 and increased expression of MMP2, MMP13, and IL8. The menisci from adult porcine knee joints can serve as a model for meniscal degeneration. Degenerative changes were manifested as differences in histopathology, gene expression and ultrastructure of collagen network.Abbreviations: MMP, matrix metalloproteinase; SOX, sex-determining region box; VEGF, vascular endothelial growth factorMuch research is focused on the degeneration of connective tissue, particularly of the cartilage tissue that stabilizes the knee joint. The goal of the current study was to determine whether menisci in adult pigs show patterns of degeneration in the absence of previous major injuries, which might cause a secondary form of degeneration.In the mammalian knee joint, the incongruence between the femoral condyle and the tibial plateau is partially balanced by 2 C-shaped fibrocartilaginous menisci. These menisci absorb impact forces, help to distribute the mechanical load on the tibial plateau, and act as stabilizers of the knee joint.^35,54 Therefore, menisci are thought to play an important role in the development of osteoarthritis in knee joints,³⁹ as indicated by the results following meniscal resection.⁵⁸ Both injuries¹³ and degeneration⁴ of meniscal tissue increase the risk of osteoarthritis of the knee joint. Because the self-repair mechanisms of meniscal tissue appear to be inadequate,⁹ more than 1 million surgical interventions on menisci are performed every year in the United States.²⁷ In addition, degenerative changes in the meniscal tissue are believed to contribute to meniscal lesions.The main component of the meniscus is water (70%); and most of its dry weight is due to the protein collagen, mainly collagen I (98%).⁵⁷ The ultrastructure of meniscal tissue consists of collagen fibers that are orientated circumferentially in the superficial layers. The intermediate layer consists of tangentially orientated collagen fibers.¹⁵ Other proteins involved in the extracellular matrix of meniscal tissue include collagen II and the proteoglycan aggrecan.¹⁵In addition, biglycan and fibromodulin have been found in porcine menisci.⁴²The primary cells in meniscal tissue are chondrocytes which, in concert with fibroblasts, produce the extracellular matrix of fibrocartilage.⁵⁵ In addition, chondrocytes produce the lubricant lubricin,⁵⁶ which also is expressed in meniscal tissue.^24,49Meniscal degeneration can be understood as an imbalance between anabolic and catabolic processes, as it has already been shown for articular cartilage.² Changes in the gene expression of menisci from osteoarthritic knee joints including genes involved in immune and inflammatory responses as well as in tissue development have been previously shown.⁵² Furthermore, the production of the matrix proteins collagen types I, II, and III decreases during the progression of human meniscal degeneration.⁴³ Changes in the gene expression profile of anabolic genes (for example, collagen I, collagen II, aggrecan) and catabolic genes (for example, matrix metalloproteinases) are early indicators of osteoarthritis.^{2,14,22,26,34,51} Therefore, additional studies analyzing the gene expression profiles of healthy and degenerated menisci are warranted to evaluate whether such marker genes also exist for meniscal tissue. As a next step, the ultrastructure of degenerated meniscal tissue should be evaluated to analyze the influence of an altered gene expression profile on the extracellular matrix of menisci.In general, pigs are an appropriate animal model in biomedical research because of their similarities to humans in terms of anatomy and metabolism.^7,19,53 In meniscus research, goats³⁰ and sheeps are well-established animal models.^11,31,58 Nevertheless, the structure of porcine collagen is highly analogous to human collagen.³ This similarity was supported by the results of a comparison of the collagen in the hyaline cartilage among several species. ²⁵ Because pathogenesis for osteoarthritis is similar between human and animal tissue,³⁸ similarities in the pathophysiology of porcine and human meniscal tissue seem likely.The aim of the current study was to compare the medial menisci isolated from juvenile pigs with the degenerated medial menisci from adult pigs to determine whether differences in their gene expression profiles and ultrastructure are present.     

Nanoindentation of human meniscal surfaces

Menisci are crescent shaped fibrocartilaginous structures which support load distribution of the knee. The menisci are specifically designed to fit the contour of the femoral condyles, aiding to disperse the stresses on the tibial plateau and in turn safeguarding the underlying articular cartilage. The importance of the meniscal superficial layer has not been fully revealed and it is suspected that this layer plays a pivotal role for meniscal function. In this study, both femoral (proximal) and tibial (distal) contacting meniscal surfaces were mechanically examined on the nano-level among three distinct regions (anterior, central and posterior) of the lateral and medial menisci. Nanoindentation testing showed no significant differences among regions, surfaces or anatomical locations, possibly elucidating on the homogeneity of the meniscal superficial zone structure (E(instantaneous): 3.17-4.12MPa, E(steady-state): 1.47-1.69MPa). Nanomechanical moduli values were approximately an order of magnitude greater than micro-scale testing derived moduli values. These findings validate the structural homogeneity of the meniscal superficial zone, showing that material properties are statistically similar regardless of meniscal surface and region. Understanding the mechanical behavior of meniscal surfaces is imperative to properly design an effective meniscal replacement.     

Learning outcomes and student-perceived value of clay modeling and cat dissection in undergraduate human anatomy and physiology

Alternatives and/or supplements to animal dissection are being explored by educators of human anatomy at different academic levels. Clay modeling is one such alternative that provides a kinesthetic, three-dimensional, constructive, and sensory approach to learning human anatomy. The present study compared two laboratory techniques, clay modeling of human anatomy and dissection of preserved cat specimens, in the instruction of muscles, peripheral nerves, and blood vessels. Specifically, we examined the effect of each technique on student performance on low-order and high-order questions related to each body system as well as the student-perceived value of each technique. Students who modeled anatomic structures in clay scored significantly higher on low-order questions related to peripheral nerves; scores were comparable between groups for high-order questions on peripheral nerves and for questions on muscles and blood vessels. Likert-scale surveys were used to measure student responses to statements about each laboratory technique. A significantly greater percentage of students in the clay modeling group "agreed" or "strongly agreed" with positive statements about their respective technique. These results indicate that clay modeling and cat dissection are equally effective in achieving student learning outcomes for certain systems in undergraduate human anatomy. Furthermore, clay modeling appears to be the preferred technique based on students' subjective perceptions of value to their learning experience.     

Origins of the neurovascular bundle: interactions between developing nerves and blood vessels in embryonic chick skin

Growth cones of nerves and endothelial cells of blood vessels are closely analogous in their migratory behavior, and they are both set a similar task during the early development of a limb. Both must invade the mesenchyme to form ramifying networks of large nerves and vessels. Both systems must densely pervade certain regions of the developing limb, such as muscle rudiments, and both form dense cutaneous plexuses at precisely the same depth beneath the epidermis. Moreover, adult tissues show many examples of neurovascular bundles in which nerves and blood vessels run closely parallel and branch in a correlated fashion, suggesting some interdependence during development. We have examined the interrelationship between developing nerves and blood vessels in chick wing skin because it allows a particularly convenient two-dimensional analysis of the two systems which can be revealed simultaneously in the same preparation by injection of Indian ink combined with silver-staining. We show that nerves do not use blood vessels as pathways along which to crawl, but that there are two other ways in which neurovascular associations arise: in some situations nerves and blood vessels follow the same route because they are responding independently to the same mesenchymal cues; and in some situations nerves induce blood vessels to remodel around them.     

The development of the connective tissue in the human orbit.

The development of the mesenchymal structures of the human orbit was studied using 10 mu or 60 mu serial sections of orbits of foetuses of 35 to 320 mm stages (C.R. length; 2-9 months). The ontogenesis of the orbital walls, the eye muscles, the blood vessels and the connective tissue was compared. This comparison revealed that the eye muscles and their fasciae together with the adventitial layers of the larger blood vessels and nerves develop first, closely linked in time to the progress of ossification and growth of most of the orbital bones. Only the (endochondral) ossification of the ethmoid starts much later. The orbital connective tissue septa development commences later, i.e. from the third month onwards, concurrently with the development of special mesenchymal condensations, a particular capillary system and adipose tissue. About five months later the adult configuration is attained.     

Effects of pregnancy on the extrinsic innervation of the guinea pig uterus. A histochemical, immunohistochemical and ultrastructural study

Summary The extrinsic innervation of the guinea pig uterus was studied by immunohistochemical, ultrastructural and enzyme histochemical methods.The extrinsic innervation was organized in two major ways. One consisted of nerve trunks and non-varicose nerve fibres running in the suspensory ligament, and the other of a plexus of varicose nerve fibres surrounding vessels, and non-vessel-related non-varicose nerve fibres in the mesouterus. The use of different neuronal and Schwann cell markers showed that the extrinsic innervation was predominantly adrenergic and contained only few peptidergic nerves. Acetylcholinesterase-positive (cholinergic) nerves were only found around the uterine artery.In late pregnancy, the extrinsic nerves of the mesouterus adjacent to foetus-containing uterine horns underwent pronounced degenerative changes comprising both Schwann cell and axonal structures. In comparison, no changes were found in extrinsic nerves of mesouteri adjacent to non-foetus-bearing uterine horns or in extrinsic nerves in the suspensory ligaments. Further, chemical sympathectomy produced axonal degeneration but no changes in the Schwann cells.In conclusion, the pregnancy-induced nerve degeneration is of a very special type different from that following chemical sympathectomy and represents a local phenomenon related to the conceptus. Hypothetically, this could be of importance for counteracting disturbances in placental blood flow.     

Neural apparatus of the wall of the ductus deferens in the dog

While studying the innervation sources of the deferent duct in 10 dogs, 2-3 nerves have been revealed that take their origin at the pelvic neural plexus and approach the duct together with the blood vessels at the pelvic neural plexus and approach the duct together with the blood vessels at the place where it crosses the ureter ("the vascular-neural hilus"). In the experiment performed in 65 dogs the nature of these sources has been revealed. The motor innervation is presented by the nodes of the celiac plexus, of the lumbar and sacral parts of the sympathetic trunk, of the subceliac, gonadal and splanchic pelvic nerves, and the sensitive innervation is multisegmental and is performed by the intravertebral nodes L2-S3. Quantitative investigation of the degenerated neural fibers in the dog deferent duct wall demonstrates that the innervation sources mentioned above participate te a various degree along the course of the organ. In the "hilus" the nerves of the dog deferent duct are divided into the proximal nad distal groups. The proximal group runs towards the prostate and forms a plexus with large loops connected with the neural plexuses of the urinary bladder, the ureter and the prostate. It has small neural nodes. The distally directed nerves run, together with the blood vessels, in the deferent duct towards the epididymis. In the deferent duct wall, adventitila, muscular and mucous neural plexus are found, the cholinergic component prevailing the adrenergic one. The plexuses are somewhat better developed at the beginning of the deferent duct and they are especially pronounced in the ampule. The receptors of the organ's wall are simple, poorly branching.     

Patient-specific knee joint finite element model validation with high-accuracy kinematics from biplane dynamic Roentgen stereogrammetric analysis

Little is known about in vivo menisci loads and displacements in the knee during strenuous activities. A new method that combines high-speed kinematics measured with biplane dynamic Roentgen stereogrammetric analysis (DRSA) and a subject-specific finite element (FE) model for studying in vivo meniscal behavior is presented here. Further model calibration in a very controlled uniaxial low and high-rate compression loading condition is presented by comparing the model behavior against the measured high-accuracy menisci DRSA kinematics and direct tibio-femoral pressure measurement from a K-scan sensor. It is apparent that certain model aspects such as removing of the pressure sensor from the model can result in relatively large errors (14%) in contact parameters that are not reflected in the change of the measured meniscal kinematics. Changing mesh size to 1mm by 1mm elements increased the magnitude of all but one of the contact variables by up to 45%. This local validation using accurate localized patient-specific geometry and meniscal kinematics was needed to enhance model fidelity at the level of contact between menisci and cartilage.     

The effect of kinematic and kinetic changes on meniscal strains during gait

The menisci play an important role in load distribution, load bearing, joint stability, lubrication, and proprioception. Partial meniscectomy has been shown to result in changes in the kinematics and kinetics at the knee during gait that can lead to progressive meniscal degeneration. This study examined changes in the strains within the menisci associated with kinematic and kinetic changes during the gait cycle. The gait changes considered were a 5 deg shift toward external rotation of the tibia with respect to the femur and an increased medial-lateral load ratio representing an increased adduction moment. A finite element model of the knee was developed and tested using a cadaveric specimen. The cadaver was placed in positions representing heel-strike and midstance of the normal gait, and magnetic resonance images were taken. Comparisons of the model predictions to boundaries digitized from images acquired in the loaded states were within the errors produced by a 1 pixel shift of either meniscus. The finite element model predicted that an increased adduction moment caused increased strains of both the anterior and posterior horns of the medial meniscus. The lateral meniscus exhibited much lower strains and had minimal changes under the various loading conditions. The external tibial rotational change resulted in a 20% decrease in the strains in the posterior medial horn and increased strains in the anterior medial horn. The results of this study suggest that the shift toward external tibial rotation seen clinically after partial medial meniscectomy is not likely to cause subsequent degenerative medial meniscal damage, but the consequence of this kinematic shift on the pathogenesis of osteoarthritis following meniscectomy requires further consideration.     

Identification of link proteins and a 116,000-Dalton matrix protein in canine meniscus

The menisci are collagen-rich, fibrocartilagenous structures which are important in protecting the articular cartilage of the knee from some of the impact of weight-bearing. Meniscal proteoglycans have been studied in several mammalian species, including the dog, but very little is known about the noncollagenous proteins of the menisci. In the present study, 4 M guanidinium chloride extracts of meniscal cartilage from normal adult mongrel dogs were studied, and several noncollagenous proteins, including the link proteins and a 116,000-Da subunit protein, which we have recently described in articular cartilage, were found in meniscal cartilage. The 116,000-Da subunit protein represents 3.8% of the total protein extracted from meniscal cartilage. The link proteins sedimented in the bottom of an associative cesium chloride density gradient, where high-buoyant-density proteoglycans sediment.     

The developing neurovascular anatomy of the embryo: a technique of simultaneous evaluation using fluorescent labeling, confocal microscopy, and three-dimensional reconstruction.

The close spatial relationship between peripheral nerves and blood vessels in the adult is well known. However, evidence supporting the congruent development of these structures in embryos remains anecdotal. Neurovascular relationships also have been shown to be conserved in other vertebrates. This homology suggests that either peripheral nerves or blood vessels, or both, might have fundamental morphogenetic roles during embryologic development. Both peripheral nerves and blood vessels have been independently implicated as etiologic agents in the pathogenesis of congenital disabilities, and several congenital anomalies fit their distribution patterns. This article presents a technique for the simultaneous visualization of peripheral nerves and blood vessels at different stages in the developing embryo. The forelimbs of 310 quail embryos were dissected over a 1-year period. Peripheral nerves were labeled with the neural crest and axon antibody, HNK-1, followed by fluorescein-conjugated secondary antibodies. Blood vessels were labeled by a perfusion technique using the fluorescent dye, dioctadecyl-tetramethylindocarbocyanine. Specimens were processed and imaged in whole-mount with confocal microscopy, and images were reconstructed using three-dimensional modeling software. Both nerves and blood vessels seem to undergo a highly stereotypic sequence of development in the embryonic quail forelimb. Furthermore, the existence of a close spatial relationship between nerves and blood vessels suggests either a high degree of developmental interdependence or shared patterning mechanisms. This technique permits further evaluation of the possible role peripheral nerves and blood vessels might play in the pathogenesis of congenital disabilities and provides a starting point for further studies aimed at elucidating the means by which peripheral nerves and blood vessels are patterned in the forelimb of the avian embryo.     

Neuropeptide Y (NPY)-like immunoreactivity in guinea pig uterus is reduced during pregnancy in parallel with noradrenergic nerves

Summary Neuropeptide Y (NPY) is a recently discovered neuropeptide with vasoconstrictor effects when given in vivo. It occurs in many sympathetic neurons, where it appears to coexist with noradrenaline (NA). It is wellknown that profound changes in the levels of uterine NA occur in many species during pregnancy. Therefore we have investigated the distribution of catecholamine neurons and NPY by immunohistochemistry in the pregnant and nonpregnant guinea pig uterus. In the virgin uterus NPY-like immunoreactivity was present in nerve fibres and terminals in the smooth muscle layers of the uterine horns and around blood vessels. The distribution of NPY fibres was very similar to that of noradrenergic nerves visualized with antibodies against the catecholamine synthesizing enzyme tyrosine hydroxylase (TH). In the pregnant uterus, NPY- and TH-like immunoreactivity disappeared almost completely. In the cervix, a slight decrease of immunoreactivity was observed, whereas in the ovaries no changes were noted between the pregnant and nonpregnant condition. The results indicate that NPY and catecholamines coexists in the adrenergic neurons of the guinea pig uterus, cervix and ovary and that they vary together in the myometrium during pregnancy. We suggest that NPY may be of functional importance for the pregnant uterus.     

Neuropeptide Y (NPY)-like immunoreactivity in guinea pig uterus is reduced during pregnancy in parallel with noradrenergic nerves

Neuropeptide Y (NPY) is a recently discovered neuropeptide with vasoconstrictor effects when given in vivo. It occurs in many sympathetic neurons, where it appears to coexist with noradrenaline (NA). It is wellknown that profound changes in the levels of uterine NA occur in many species during pregnancy. Therefore we have investigated the distribution of catecholamine neurons and NPY by immunohistochemistry in the pregnant and nonpregnant guinea pig uterus. In the virgin uterus NPY-like immunoreactivity was present in nerve fibres and terminals in the smooth muscle layers of the uterine horns and around blood vessels. The distribution of NPY fibres was very similar to that of noradrenergic nerves visualized with antibodies against the catecholamine synthesizing enzyme tyrosine hydroxylase (TH). In the pregnant uterus, NPY- and TH-like immunoreactivity disappeared almost completely. In the cervix, a slight decrease of immunoreactivity was observed, whereas in the ovaries no changes were noted between the pregnant and nonpregnant condition. The results indicate that NPY and catecholamines coexists in the adrenergic neurons of the guinea pig uterus, cervix and ovary and that they vary together in the myometrium during pregnancy. We suggest that NPY may be of functional importance for the pregnant uterus.     

Organization of the guinea-pig uterine innervation. Distribution of immunoreactivities for different neuronal markers. Effects of chemical- and pregnancy-induced sympathectomy

Summary The structural organization of the guinea-pig uterine innervation was investigated by an immunofluorescence method using neurofibrillary protein (NF) and neuron-specific enolase (NSE) as general neuronal markers. NF- and NSE-immunoreactive nerve trunks and non-varicose nerves formed continuous networks similar to nerves with analogue morphology and with immunoreactivities for tyrosine hydroxylase (TH; adrenergic nerves) and neuropeptide Y (NPY). NF- and NSE-immunoreactive non-varicose nerves occurred in the myometrium and along vessels, where TH- and NPY-immunoreactive varicose nerves were also comparatively frequent. After chemical sympathectomy all TH- and NPY-immunoreactive varicose nerves and most NF- and NSE-immunoreactive non-varicose nerves disappeared, suggesting colocalization of TH, NPY, NF and NSE immunoreactivities. During pregnancy all NF-, NSE-, TH- and NPY-immunoreactive nerve structures disappeared in the foetus-bearing uterine horns whereas in the cervix and non-foetus-bearing uterine horns only the myometrial TH- and NPY-immunoreactive varicose nerves disappeared. After parturition there was a complete structural restoration of all types of immunoreactive nerves in previously non-foetus-related tissue. The reinnervation of this tissue followed a similar time-course to that after chemical sympathectomy. In contrast, the reinnervation of previously foetus-related tissue was much slower and incomplete.In conclusion, the whole autonomic uterine innervation undergoes overt structural changes during pregnancy and these changes are related to the foetus-bearing regions.     

Sensory nerves determine the pattern of arterial differentiation and blood vessel branching in the skin

Nerves and blood vessels are branched structures, but whether their branching patterns are established independently or coordinately is not clear. Here we show that arteries, but not veins, are specifically aligned with peripheral nerves in embryonic mouse limb skin. Mutations that eliminate peripheral sensory nerves or Schwann cells prevent proper arteriogenesis, while those that disorganize the nerves maintain the alignment of arteries with misrouted axons. In vitro, sensory neurons or Schwann cells can induce arterial marker expression in isolated embryonic endothelial cells, and VEGF(164/120) is necessary and sufficient to mediate this induction. These data suggest that peripheral nerves provide a template that determines the organotypic pattern of blood vessel branching and arterial differentiation in the skin, via local secretion of VEGF.