Form and Size Matter: Increased Risk of Thrombosis in Microvessels with Surgically Created Endothelial Lesions.

Background Atherosclerosis is a known risk factor for flap loss in microsurgery. Several microsurgical techniques, like plaque removal, have been proposed for atherosclerotic vessels, but these techniques often induce intimal injuries. The aim of this study was to investigate the impact of various endothelial defects on the risk of thrombosis in a rat acute intimal injury model. Methods Endothelial defects of various forms and sizes were created in the abdominal aorta of 30 male Wistar rats following a strict protocol. Defect sizes were measured and classified as round, horizontal, or vertical based on their configuration. An hour after reestablishing the blood flow, the abdominal aorta was harvested and the operation site was assessed for signs of thrombosis clinically and using light microscopy. Univariate and multiple linear regression analysis were performed to identify possible influencing factors on thrombosis. Results The mean defect size was 2.65 ± 1.19 mm2. Intimal lesions were classified as round in 36.7%, horizontal in 33.3%, and vertical in 30% of specimens. Thrombus formation was detected in 46.7% clinically and in 50% histologically. Univariate regression analysis revealed that defect size (p = 0.048) and vertical form (p = 0.017) were significantly associated with thrombus formation. Multiple regression analysis corroborated vertical defects as a risk factor for thrombosis (p = 0.03). Conclusion Endothelial injuries are associated with a high risk of thrombosis with highest risks associated with vertical defects. Arteries should be carefully examined for intimal defects before microvascular anastomosis, especially in the atherosclerotic patient.

Analysis of the sensory innervations of the greater trochanter for improving the treatment of greater trochanteric pain syndrome.

In medical practice, greater trochanteric pain syndrome has an incidence of 5.6 per 1,000 adults per year, and affects up to 25% of patients with knee osteoarthritis and low back pain in industrialized nations. It also occurs as a complication after total hip arthroplasty. Different etiologies of the pain syndrome have been discussed, but an exact cause remains unknown. The purpose of this study was to obtain a better understanding of the sensory innervations of the greater trochanter in attempt to improve the treatment of this syndrome. Therefore, we dissected the gluteal region of seven adult and one fetal formalin fixed cadavers, and both macroscopic and microscopic examination was performed. We found a small sensory nerve supply to the periosteum and bursae of the greater trochanter. This nerve is a branch of the n. femoralis and accompanies the arteria and vena circumflexa femoris medialis and their trochanteric branches to the greater trochanter. This nerve enters the periosteum of the greater trochanter directly caudal to the tendon of the inferior gemellus muscle. This new anatomical information may be helpful in improving therapy, such as interventional denervation of the greater trochanter or anatomically guided injections with corticosteroids and local anesthetics.

Mice lacking perforin have improved regeneration of the injured femoral nerve.

The role that the immune system plays after injury of the peripheral nervous system is still not completely understood. Perforin, a natural killer cell- and T-lymphocyte-derived enzyme that mediates cytotoxicity, plays important roles in autoimmune diseases, infections and central nervous system trauma, such as spinal cord injury. To dissect the roles of this single component of the immune response to injury, we tested regeneration after femoral nerve injury in perforin-deficient (Pfp-/-) and wild-type control mice. Single frame motion analysis showed better motor recovery in Pfp-/- mice compared with control mice at 4 and 8 weeks after injury. Retrograde tracing of the motoneuron axons regrown into the motor nerve branch demonstrated more correctly projecting motoneurons in the spinal cord of Pfp-/- mice compared with wild-types. Myelination of regrown axons measured by g-ratio was more extensive in Pfp-/- than in wild-type mice in the motor branch of the femoral nerve. Pfp-/- mice displayed more cholinergic synaptic terminals around cell bodies of spinal motoneurons after injury than the injured wild-types. We histologically analyzed lymphocyte infiltration 10 days after surgery and found that in Pfp-/- mice the number of lymphocytes in the regenerating nerves was lower than in wild-types, suggesting a closed blood-nerve barrier in Pfp-/- mice. We conclude that perforin restricts motor recovery after femoral nerve injury owing to decreased survival of motoneurons and reduced myelination.

Salivary cortisol and psychological mechanisms in patients with acute versus chronic low back pain.

This study was designed to explore whether the basal adrenocortical activity is related with pain-related coping, nonverbal pain behavior, depressive mood, and fatigue in patients with acute and chronic nonspecific low back pain. 19 patients with acute low back pain (ALBP) and 24 with chronic low back pain (CLBP) participated in the study. The adrenocortical activity was assessed through the cortisol awakening response. All participants provided five saliva samples (0, 15, 30, 45, and 60min after waking) on two consecutive days off work. Pain-related coping [fear-avoidance coping (FAC) and endurance coping (EC)], nonverbal pain behavior (NPB), depressive mood, and fatigue were assessed through questionnaires. Among ALPB patients, EC was negatively associated with the cortisol release, whereas fatigue was positively associated with it. Among CLBP patients, FAC, NPB, depressive mood, and fatigue were negatively associated with the cortisol awakening response, whereas EC tended to be positively associated with it. The results indicate that pain-related coping strategies which are expected to be successful appear to lower the adrenocortical activity among ALBP patients, whereas affective distress may enhance the level of cortisol in this group. Among CLBP patients, long-term maladaptive coping strategies might contribute to hypocortisolism.

Assessment of Ultrastructural Neuroplasticity Parameters After In Utero Transduction of the Developing Mouse Brain and Spinal Cord.

The present study combines in utero transduction with transmission electron microscopy (TEM) aiming at a precise morphometrical analysis of ultrastructural parameters in unambiguously identified topographical structures, affected by a protein of interest that is introduced into the organism via viral transfer. This combined approach allows for a smooth transition from macrostructural to ultrastructural identification by following topographical navigation maps in a tissue atlas. High-resolution electron microscopy of the in-utero-transduced tissue reveals the fine ultrastructure of the neuropil and its plasticity parameters, such as cross-sectioned synaptic bouton areas, the number of synaptic vesicles and mitochondria within a bouton profile, the length of synaptic contacts, cross-sectioned axonal areas, the thickness of myelin sheaths, the number of myelin lamellae, and cross-sectioned areas of mitochondria profiles. The analysis of these parameters reveals essential insights into changes of ultrastructural plasticity in the areas of the nervous system that are affected by the viral transfer of the genetic construct. This combined method can not only be used for studying the direct effect of genetically engineered biomolecules and/or drugs on neuronal plasticity but also opens the possibility to study the in utero rescue of neuronal plasticity (e.g., in the context of neurodegenerative diseases).

Sexual characteristics and spermatogenesis in males of the parthenogenetic gecko Lepidodactylus lugubris (Reptilia, Gekkonidae).

Obligately parthenogenetic lizards usually are all-female populations of hybrids producing diploid oocytes by premeiotic endomitosis and quasi-normal meiosis. In an all-female strain of the gekkonid lizard Lepidodactylus lugubris several phenotypic males arose spontaneously. The sexual characteristics of these males were studied using light and electron microscopy and compared with normal males of the bisexual genus Lygodactylus. Emphasis was layed on morphology of seminiferous tubules, occurrence of spermatogenic stages and ultrastructure of spermatozoa. The phenotypic males possessed preanal pores filled with secretions and a sexual nephric segment which were exactly the same as in normal, reproductively active males. In the testes, density and morphology of non-spermatogenic cell types, the Leydig and Sertoli cells, indicate a normal production of testicular testosterone and a normal function of the blood-testis barrier, respectively. Both in the normal and the phenotypic males, all meiotic cell types of spermatogenesis can be recognised in the seminiferous tubules and are apparently identical, indicating a normal meiosis without impairment in the phenotypic males. In contrast, the differentiation process of spermatids is markedly disturbed in the phenotypic males of L. lugubris. In the normal male, spermiogenesis results in mature spermatids and spermatozoa with small elongated nuclei, an acrosomal complex, and a flagellar tail possessing one axoneme. Spermatozoa fill both the lumen of most seminiferous tubules and the lumina of ductus epididymidis and ductus deferens. In the phenotypic male, spermiogenesis results in seemingly normal spermatids and in spermatozoa with large, non-elongated, deformed nuclei and/or irregular tails possessing more than one axoneme. Both the lumen of most seminiferous tubules and the lumina of the ductus epididymidis and the ductus deferens contain relatively few spermatozoa. We suggest that the phenotypic males inherited the ability for a premeiotic endomitosis from their all-female ancestral lineage. While in females this leads to quasi-normal meiosis and diploid oocytes capable of development, the small nuclei of the spermatozoa are unable to contain a diploid set of chromosomes. Because of the high amount of deformed spermatozoa and possibly uncontrolled loss of genetic material in structurally normal, but aneuploid spermatozoa we conclude that these otherwise perfect males are infertile, thus constituting another example of gametic sterility.

Risk of Thromboembolus after Application of Different Tissue Glues during Microvascular Anastomosis.

BACKGROUND: Tissue glues are a useful tool for hemostasis and a potential tool for microvascular anastomosis. The hemostatic power and thrombotic risk of these surgical aids have been the subject of debate, but studies regarding thromboembolic risks of tissue glues are lacking. METHODS: The authors compared the thromboembolic risk of three different tissue glues (six interrupted sutures complemented with fibrin, FloSeal, or TachoSil) against a control group (12 interrupted sutures) in the aorta-filter model in the rat. Each group consisted of 10 rats, examined 4 hours and 14 days postoperatively (total n = 80) using a structured protocol to assess the patency and condition of the filter, amount of thromboembolic material, and histologic appearance of the vessel wall. RESULTS: In total, 160 anastomoses were performed in 80 rats. The overall patency rate for the groups was 90 percent (control), 35 percent (fibrin glue), 25 percent (FloSeal), and 10 percent (TachoSil). All experimental groups had significantly faster mean anastomotic times and less blood loss compared with the control group. Patency rates were reduced and thromboembolic material was significantly increased in all experimental groups. Histologically, the use of tissue glue reduced the incidence of irritation of the internal elastic lamina and was associated with an increased incidence of fibrocyte infiltration of the media, hypercellularity of the adventitia, and adventitial neovascularization and lymphangiogenesis. CONCLUSION: The application of tissue glues in microvascular anastomosis reduces the time needed for anastomosis but was associated with a statistically significant increase in thromboembolism.

Stomatin immunoreactivity in ciliated cells of the human airway epithelium.

Stomatin is a widely distributed 32kD membrane protein of unknown function. In biochemical studies it is associated with cholesterol+sphingomyelin-rich 'rafts' in the cytomembrane. Genetic studies in C. elegans, supported by microscopic studies in mammalian tissue and co-expression studies in oocytes, suggest a functional link with the DEG/ENaC (degenerin/epithelial Na+ channel) superfamily of monovalent ion channels. Since ENaC channels play a prominent role in the physiology of the respiratory epithelium, we have studied the immunolocalization of stomatin in mature and developing human airway epithelium by means of Western blot analysis, immunocytochemistry, and immunoelectron microscopy. Stomatin immunoreactivity (stomatin-IR) was found in the ciliated cells of the conductive airway epithelium in a distinct distribution pattern with the strongest signal along the cilia. Immunogold labelling revealed immunogold particles at the basal bodies, along the cilia, and at the membrane of the microvilli. The presence of stomatin-IR paralleled the stages of ciliogenesis in airway development, and its appearance preceded the elongation of the axoneme and the cilial outgrowth. Due to its presence in the different cellular locations in the ciliated cell, we suggest that stomatin is involved in various cellular functions. From its ultrastructural position, stomatin could be a candidate for a membrane-associated mechanotransducer with a role in the control of ciliary motility. Stomatin as a raft protein might be a microtubule associated protein moving along the outer surface of the microtubules to its terminal site of action in the cilia. Stomatin-IR in microvilli supports the hypothesis of a co-localization with beta- and gamma- ENaC and, in conclusion, their potential functional interaction to control the composition of periciliary mucus electrolytes.

Anatomy of the infrapatellar branch in relation to skin incisions and as the basis to treat neuropathic pain by cryodenervation.

BACKGROUND: Neuropathic knee pain, particularly of the infrapatellar branch, is an important complication of knee replacement surgery, with an incidence as high as 70%. The increasing number of elderly patients requiring knee surgery, including total knee arthroplasty (TKA), has contributed to an increase in the number of patients with this pathology. Treatment includes neurectomy, infiltration therapy, and cryodenervation. Percutaneous cryodenervation of the infrapatellar branch is a promising option. OBJECTIVE: To provide the necessary anatomical analysis to optimize percutaneous cryodenervation of the infrapatellar branch by defining sections of the unbranched ramus infrapatellaris to demonstrate the risk of nerve injury through 3 different skin incisions typically used during TKA. STUDY DESIGN: Anatomical study. METHODS: Cadavers were used for assessment. Exclusion criteria were scars from knee surgery, deep wounds, and a flexion angle of no more than 90°. We compared 3 frequently used skin incisions with the course of the infrapatellar branch and identified sections of the unbranched nerves that were suitable for percutaneous cryodenervation. RESULTS: In total, 18 formalin-fixed cadavers (mean age, 78.9 years) contributed 30 knees (15 pairs) for dissection. We identified the following 4 anatomical variations of the ramus infrapatellaris in relation to the sartorius muscle: anterior, posterior, penetrating, and pes anserinus types. Sections were then found to treat the nerve branch types. The nerve sections were localized using the medial pole of the patella as a palpable landmark and varied in length between 15 mm and 40 mm. The medial parapatellar skin incision showed the highest risk of lesions to the infrapatellar branch (53.3%) followed by the midline skin incision (46.7%) and the lateral parapatellar skin incision (30.0%). LIMITATIONS: This was an observational study, performed using a limited number of cadavers. This therefore precluded generalization and statistical analysis. Significantly more female (13) cadavers were examined compared to male (5). Further studies in human populations, and with larger samples, are necessary to confirm these results. CONCLUSION: Based on our findings, the surgeon can localize the unbranched main nerve. Compared with the current practice, our approach should allow for a lower impact on tissues and should facilitate complete pain relief through a single cryodenervation. Furthermore, we propose that the lateral parapatellar skin incision is an acceptable alternative surgical approach in knee replacement surgery because it is associated with the lowest risk of damage to the infrapatellar branch.

Introduction of a microsurgical in-vivo embolization-model in rats: the aorta-filter model.

Vascular thrombosis with subsequent distal embolization remains a critical event for patients. Prevention of this life-threatening event can be achieved pharmacologically or mechanically with intravascular filter systems. The ability to evaluate the risk of embolization of certain techniques and procedures in vascular and microvascular surgery, such as, tissue glue or fibrin based haemostatic agents lacks convincing models. We performed 64 microvascular anastomoses in 44 rats, including 44 micro-pore polyurethane filter-anastomoses and 20 non-filter anastomoses. The rats were re-anesthetized and the aorta was re-exposed and removed four hours, three, seven, fourteen, thirty-one days, and six months postoperatively. The specimens were examined macro- and microscopically with regard to the appearance of the vessel wall, condition of the filter and the amount of thrombembolic material. Typical postoperative histopathological changes in vessel architecture were observed. Media necrosis was the first significant change three days postoperatively. Localized intimal hyperplasia, media necrosis, increase of media fibromyocytes and adventitial hypercellularity were seen to a significant extent at day seven postoperatively. Significant neovascularization of adventitia adjacent to the filter was seen after 14 days. A significant amount of thrombotic material was seen after four hours, three and 14 days interval. Only three intravascular filters became completely occluded (6.82%). The aorta-filter-anastomosis model appeared to be a valid in-vivo model in situations at risk for thrombembolic events, for microsurgical research and allowed sensitive analysis of surgical procedures and protection of the vascularized tissue. It may be suitable for a wide range of in-vivo microvascular experiments particularly in the rat model.

Organization of peripheral nerves in skin, musculoskeletal system and viscera.

Skin, musculoskeletal system and all organs of the body are supplied by nerve fibers of the somatic and autonomic nervous system, each of the systems with its specific nerve fiber types, fiber composition, fiber density and targets. Experimental data support the hypothesis that tumor tissue might interact with nerve fibers. The peripheral nervous system possesses an extraordinary cellular equipment to protect the axons against pathological stimuli. Only restricted areas lacking a cellular barrier are weak points within the nervous network. Therefore, this article focuses on the functional morphology of the peripheral nervous system and its regional differences.

Stomatin is mis-trafficked in the erythrocytes of overhydrated hereditary stomatocytosis, and is absent from normal primitive yolk sac-derived erythrocytes.

The 32 kD lipid-raft-associated membrane protein 'stomatin' is deficient from the erythrocyte membrane in the Na+-K+ leaky haemolytic anaemia, overhydrated hereditary stomatocytosis (OHSt). To date, no mutation in the gene coding for this protein has so far been found in OHSt. In this study, we have analysed the distribution of stomatin in both cultured erythroid cells from OHSt patients and in normal embryological and fetal erythroid development. In erythroid cell cultures from OHSt patients, stomatin-immunoreactivity (stomatin-IR) was present in progenitor cells but remained restricted to the area of the multivesicular complexes and the nucleus in the developing cells and was not seen in the plasma membrane. This could be consistent with the idea that stomatin is an innocent passenger in a more fundamental trafficking abnormality. In normal embryonic development, we found that, in extraembryonic (yolk sac) erythropoiesis, neither the nucleated red cells nor their enucleated mature derivatives displayed any stomatin-IR. In contrast, all haemangiopoietic progenitor cells of intraembryonic haematopoiesis, starting with the mesodermal precursors in the aorta-gonad-mesonephros region, exhibited strong stomatin-IR. The significance of this observation on these poorly understood cells is currently unclear.

Eukaryotic and prokaryotic stomatins: the proteolytic link.

The 32kD membrane protein stomatin was first studied because it is deficient from the red cell membrane in two forms of the class of haemolytic anaemias known as "hereditary stomatocytosis." The hallmark of these conditions is a plasma membrane leak to the monovalent cations Na+ and K+: the protein is missing only in the most severely leaky of these conditions. No mutation has ever been found in the stomatin gene in these conditions. Stomatin-like proteins have been identified in all three domains of biology, yet their function remains enigmatic. Although the murine knock-out is without phenotype, we have identified a family showing a splicing defect in the stomatin mRNA, in which affected children showed a catastrophic multisystem disease not inconsistent with the now-known wide tissue distribution of stomatin. We report here a study of strongly homologous stomatin-like genes in prokaryotes, which reveals a close connection with a never-studied gene erroneously known as "nfed." This gene codes for a hydrophobic protein with a probable serine protease motif. It is possible that these stomatin-like genes and those which are known as"nfed" form an operon, suggesting that the two protein products are aimed at a common function. The corollary is that stomatin could be a partner protein for a membrane-bound proteolytic process, in both prokaryotes and in eukaryotes generally: this idea is consistent with experimental evidence.

The "stomatin" gene and protein in overhydrated hereditary stomatocytosis.

In overhydrated hereditary stomatocytosis (OHSt), Coomassie- and silver-stained polyacrylamide gels show an apparently complete deficit of the 32-kDa membrane protein, stomatin. We have used an antistomatin antibody to examine peripheral blood films, bone marrow, splenic tissue, and hepatic tissue from these patients by immunocytochemistry. This technique revealed that, in fact, some red cells did show positive stomatin immunoreactivity; and consistent with this result, Western blot analysis of the red cell membranes confirmed that about one twentieth to one fiftieth of the normal amount of stomatin was in fact present. Flow cytometry, combining immunoreactive quantitation of stomatin expression with thiazole orange staining for reticulocytes, showed that in OHSt, it was the young cells that had more stomatin. Magnetic-activated cell separation studies, using beads to which an anti-transferrin receptor antibody was conjugated, confirmed that in OHSt there was a correspondence between expression of stomatin and the transferrin receptor. Immunocytochemistry and Western blotting revealed that in OHSt patients, the protein was present in spleen, liver, neutrophils, platelets, monocytes, and about 50% of the peripheral lymphocytes, with the same distribution as in healthy controls. Neither Southern blots, nor direct sequencing of multiple subclones of the cDNA, nor sequencing of amplicons from genomic DNA revealed any significant abnormality in stomatin gene sequence in these patients. The deficiency of stomatin from red cells appears to be due to a loss of stomatin from these red cells on maturation in the bone marrow and in the circulation.

Homozygous mutations in caveolin-3 cause a severe form of rippling muscle disease.

Heterozygous missense mutations in the caveolin-3 gene (CAV3) cause different muscle disorders. Most patients with CAV3 alterations present with rippling muscle disease (RMD) characterized by signs of increased muscle irritability without muscle weakness. In some patients, CAV3 mutations underlie the progressive limb-girdle muscular dystrophy type 1C (LGMD1C). Here, we report two unrelated patients with novel homozygous mutations (L86P and A92T) in CAV3. Both presented with a more severe clinical phenotype than usually seen in RMD. Immunohistochemical and immunoblot analyses of muscle biopsies showed a strong reduction of caveolin-3 in both homozygous RMD patients similar to the findings in heterozygous RMD. Electron microscopy studies showed a nearly complete absence of caveolae in the sarcolemma in all RMD patients analyzed. Additional plasma membrane irregularities (small plasmalemmal discontinuities, subsarcolemmal vacuoles, abnormal papillary projections) were more pronounced in homozygous than in heterozygous RMD patients. A stronger activation of nitric oxide synthase was observed in both homozygous patients compared with heterozygous RMD. Like in LGMD1C, dysferlin immunoreactivity is reduced in RMD but more pronounced in homozygous as compared with heterozygous RMD. Thus, we further extend the phenotypic variability of muscle caveolinopathies by identification of a severe form of RMD associated with homozygous CAV3 mutations.