A comparative study of the epiligament of the medial collateral and the anterior cruciate ligament in the human knee. Immunohistochemical analysis of collagen type I and V and procollagen type III.

BACKGROUND: Recent reports in rat models have shown that fibroblasts in the epiligament, an enveloping tissue of the ligament, are not static cells and play an important role during the early ligament healing of isolated grade III injury of the collateral ligaments of the knee. Fibroblasts produce collagen types I, III and V and infiltrate within the ligament body via the endoligament. In addition, similarities have been reported between the structure of the epiligament of the medial collateral ligament and anterior cruciate ligament of the knee in rat and in human. In line with the ascribed role of the epiligament tissue and the synthesis of these collagens and their role in ligament healing, the aim of this study was to determine their presence in the normal epiligament of the aforementioned ligaments in humans, to compare their differential expression and to present a novel hypothesis about the failure of healing of the anterior cruciate ligament in contrast to the medial collateral ligament. MATERIALS AND METHODS: We used samples from the mid-substance of the medial collateral and the anterior cruciate ligament of the knee joint, acquired from 12 fresh knee joints. Routine histological analysis was performed through hematoxylin and eosin stain, Mallory's trichrome stain and Van Gieson's stain. The immunohistochemical analysis was conducted using monoclonal antibodies against collagen type I and V and procollagen type III. The number of cells in the epiligament, endoligament and the ligament tissue was assessed quantitatively through a computerized system for image analysis NIS-Elements Advanced Research and Statistica software. RESULTS: Our observations revealed certain differences in the morphology of the epiligament, as well as variations in the expression of the investigated molecules. Expression of collagen type I was mostly low-positive (1+) in the epiligament and positive (2+) in the ligament tissue of both ligaments. Expression of procollagen type III was mostly positive (2+) in the epiligament and ligament tissue of the medial collateral ligament, low-positive (1+) in the epiligament and negative (0) in ligament tissue of the anterior cruciate ligament. Expression of collagen type V was predominantly low-positive (1+) in the epiligament and negative (0) in the ligament tissue of both ligaments. The immunoreactivity for all three molecules was always higher in the epiligament of the medial collateral ligament than that of the anterior cruciate ligament. CONCLUSIONS: The results of our study illustrate for the first time that fibroblasts in the human epiligament are indeed responsible for the synthesis of the main types of collagen participating in the early ligament healing, thus corresponding to previous data of the medial collateral ligament healing in animal models. The differences between the epiligament of the investigated ligaments could add a novel explanation for the failed anterior cruciate ligament healing.

Comparative investigation of neuronal nitric oxide synthase immunoreactivity in rat and human claustrum.

We compared the distribution, density and morphological characteristics of nitric oxide synthase-immunoreactive (NOS-ir) neurons in the rat and human claustrum. These neurons were categorized by diameter into three main types: large, medium and small. In the human claustrum, large neurons ranged from 26 to 40µm in diameter, medium neurons from 20 to 25µm and small neurons from 13 to 19µm. In the rat claustrum, large neurons ranged from 19 to 23µm in diameter, medium neurons from 15 to 18µm and small neurons from 10 to 14µm. The cell bodies of large and medium neurons varied broadly in shape - multipolar, elliptical, bipolar and irregular, consistent with a projection neuron phenotype. The small neurons were most seen as being oval or elliptical in shape, resembling an interneuron phenotype. Based on a quantitative comparison of their dendritic characteristics, the NOS-ir neurons of humans and rats displayed a statistically significant difference.

Symptomatic Os Subtibiale Associated with Chronic Pain Around the Medial Malleolus in a Young Athlete.

An os subtibiale is a rare accessory bone located below or behind the medial malleolus. Herein we present a rare case of a painful os subtibiale in a young triathlete who presented with pain, redness and swelling below his left medial malleolus. Plain radiographs and three-dimensional computed tomography revealed a well-defined oval bone distal to the left medial malleolus. After conservative treatment failed, the ossicle was excised in an open surgery with complete resolution of symptoms. This case report emphasizes the need for clinical awareness of different anatomical variations of the bones of the foot.

Topographical distribution and morphology of NADPH-diaphorase-stained neurons in the human claustrum.

We studied the topographical distribution and morphological characteristics of NADPH-diaphorase-positive neurons and fibers in the human claustrum. These neurons were seen to be heterogeneously distributed throughout the claustrum. Taking into account the size and shape of stained perikarya as well as dendritic and axonal characteristics, Nicotinamide adenine dinucleotide phosphate-diaphorase (NADPHd)-positive neurons were categorized by diameter into three types: large, medium and small. Large neurons ranged from 25 to 35 µm in diameter and typically displayed elliptical or multipolar cell bodies. Medium neurons ranged from 20 to 25 µm in diameter and displayed multipolar, bipolar and irregular cell bodies. Small neurons ranged from 14 to 20 µm in diameter and most often displayed oval or elliptical cell bodies. Based on dendritic characteristics, these neurons were divided into spiny and aspiny subtypes. Our findings reveal two populations of NADPHd-positive neurons in the human claustrum-one comprised of large and medium cells consistent with a projection neuron phenotype, the other represented by small cells resembling the interneuron phenotype as defined by previous Golgi impregnation studies.

The role of Willis circle variations during unilateral selective cerebral perfusion: a study of 500 circles.

OBJECTIVES: During unilateral selective cerebral perfusion (uSCP), with right axillary artery or brachiocephalic trunk cannulation, the brain receives blood only via the right common carotid artery and right vertebral artery (VA). The left hemisphere is perfused mainly through the circle of Willis (CW). However, at least 50% of individuals have some variation in the CW. The aim of the present work was to study the variations in CW and VA that could have an impact on haemodynamics during uSCP. METHODS: From May 2005 to March 2012, a total number of 250 circles obtained via routine dissection for medico-legal reasons were examined. The external diameters of all CW segments and both VAs were measured. From January 2008 to March 2012, a total number of 250 patients subjected to computed tomographic angiography of the CW were also examined. RESULTS: Nine evident configurations of the CW that could cause hypoperfusion during uSCP were observed. They were subdivided in to seven types, according to location and the number of major vessels at risk of hypoperfusion. Type IA: hypo/aplasia of left posterior communicating artery (PComA), found in 35.6% of cases; Type IB: hypo/aplasia of anterior communicating artery (AComA), found in 2% of cases; Type IIA: hypo/aplasia of both left PComA and AComA, found in 4.8% of cases; Type IIB: hypo/aplasia of precommunicating (P1) segment of left posterior cerebral artery or right VA, found in 9.2% of cases; Type IIIA: hypo/aplasia of precommunicating (A1) segment of right anterior cerebral artery, found in 6% of cases; Type IIIB: hypo/aplasia of both right VA and AComA, found in 0.2% of cases; Type IV: hypo/aplasia of both right A1 and right VA or both right A1 and left P1, found in 0.8% of cases. All types were present in 58.6% of all examined CWs. CONCLUSIONS: Our results show that CW variations are present in a significant number of patients. Our data support the need for extensive preoperative examination and meticulous intraoperative monitoring of cerebral perfusion during uSCP. Finally, our data support the superiority of bilateral SCP over uSCP, because most of the variations reported do not have haemodynamic significance during bilateral SCP.

Light and electron-microscopic study of leucine enkephalin immunoreactivity in the cat claustrum.

The claustrum is a complex telencephalic structure owing to its reciprocal connectivity with most--if not all--cortical areas. However, there is a paucity of data in the literature concerning its histochemical components, including opioid peptide neurotransmitters. The aim of the present study was to examine the morphology, distribution and ultrastructure of leucine-enkephalin-immunoreactive (Leu-enk-ir) neurons and fibers in the dorsal claustrum (DC) of the cat. Seven healthy, adult male and female cats were used in our study. All animals received humane care. They were irreversibly anesthetized and transcardially perfused with fixative. Brains were removed, postfixed, blocked and sectioned. Sections were incubated with polyclonal anti-Leu-enk antibodies using the Avidin-Biotin-Peroxidase Complex method. Leu-enk-ir neurons and fibers were distributed throughout the DC. Some of the neurons were lightly-stained, while others were darkly-stained. Light-microscopically, they varied in shape: oval, fusiform, multipolar and irregular. With regard to size, they were categorized as small (15 µm or less in diameter), medium (16-20 µm in diameter) and large (21 µm or more in diameter). No specific pattern of regional distribution was found. On the electron microscope level, immunoproduct was observed in neurons, dendrites and terminal boutons. Different types of Leu-enk-ir neurons differ in their ultrastructural features, including two types of synaptic boutons. No gender-specific features were observed. In conclusion, it is our hope that our study will serve to contribute to a better understanding of the functional neuroanatomy of the DC in the cat, and that it can be extrapolated and applied to other mammals, including humans.

Variety of transversus thoracis muscle in relation to the internal thoracic artery: an autopsy study of 120 subjects.

BACKGROUND: The transversus thoracis muscle is a thin muscular layer on the inner surface of the anterior thoracic wall that is always in concern during harvesting of the internal thoracic artery. Because the muscle is poorly described in the surgical literature, the aim of the present study is to examine in details its variations. METHODS: The data was obtained at standard autopsies of 120 Caucasian subjects (Bulgarians) of both sexes (97 males and 23 females), ranging in age from 18 to 91 years (mean age 52.8 ± 17.8 years). The transversus thoracis morphology was thoroughly examined on the inner surface of the chest plates collected after routine incisions. RESULTS: An overall examination revealed that in majority of cases the transversus thoracis slips formed a complete muscular layer (left - 75.8%, right - 83.3%) or some of the slips (left - 22.5%, right - 15%) or all of them (left - 1.7%, right - 1.7%) were quite separated. Rarely (left - 3.3%, right - 5.8%), some fibrous slips of the transversus thoracis were noted. In 55.8% of the cases there was left/right muscle symmetry; 44.2% of the muscles were asymmetrical. Most commonly, the highest muscle attachment was to the second (left - 53.3%, right - 37.5%) or third rib (left - 29.2%, right - 46.7%). The sixth rib was the most common lowest attachment (left - 94.2%, right - 89.2%). Most frequently, the muscle was composed of four (left - 31.7%, right - 44.2%) or fifth slips (left - 53.3%, right - 40.8%). CONCLUSIONS: This study provides detailed basic information on the variety of the transversus thoracic muscle. It also defines the range of the clearly visible, uncovered by the muscle part of the internal thoracic artery and the completeness of the muscular layer over it. The knowledge of these peculiar muscle-arterial relations would definitely be beneficial to cardiac surgeon in performing fast and safe arterial harvesting.

Neuronal nitric oxide synthase immunopositive neurons in cat claustrum--a light and electron microscopic study.

Nitric oxide is a unique neurotransmitter, which participates in many physiological and pathological processes in the organism. Nevertheless there are little data about the neuronal Nitric Oxide Synthase immunoreactive (nNOS-ir) neurons and fibers in the dorsal claustrum (DC) of a cat. In this respect the aims of this study were: (1) to demonstrate nNOS-ir in the neurons and fibers of the DC; (2) to describe their light microscopic morphology and distribution; (3) to investigate and analyze the ultrastructure of the nNOS-ir neurons, fibers and synaptic terminals; (4) to verify whether the nNOS-ir neurons consist a specific subpopulation of claustral neurons; (5) to verify whether the nNOS-ir neurons have a specific pattern of organization throughout the DC. For demonstration of the nNOS-ir the Avidin-Biotin-Peroxidase Complex method was applied. Immunopositive for nNOS neurons and fibers were present in all parts of DC. On the light microscope level nNOS-ir neurons were different in shape and size. According to the latter they were divided into three groups-small (with diameter under 15 microm), medium-sized (with diameter from 16 to 20 microm) and large (with diameter over 21 microm). Some of nNOS-ir neurons were lightly-stained while others were darkly-stained. On the electron microscope level the immunoproduct was observed in neurons, dendrites and terminal boutons. Different types of nNOS-ir neurons differ according to their ultrastructural features. Three types of nNOS-ir synaptic boutons were found. As a conclusion we hope that the present study will contribute to a better understanding of the functioning of the DC in cat and that some of the data presented could be extrapolated to other mammals, including human.

Electron microscopic 3D-reconstruction of dendritic spines in cultured hippocampal neurons undergoing synaptic plasticity.

Dendritic spines are assumed to constitute the locus of neuronal plasticity, and considerable effort has been focused on attempts to demonstrate that new memories are associated with the formation of new spines. However, few studies that have documented the appearance of spines after exposure to plasticity-producing paradigms could demonstrate that a new spine is touched by a bona fida presynaptic terminal. Thus, the functional significance of plastic dendritic spine changes is not clearly understood. We have used quantitative time lapse confocal imaging of cultured hippocampal neurons before and after their exposure to a conditioning medium which activates synaptic NMDA receptors. Following the experiment the cultures were prepared for 3D electron microscopic reconstruction of visually identified dendritic spines. We found that a majority of new, 1- to 2-h-old spines was touched by presynaptic terminals. Furthermore, when spines disappeared, the parent dendrites were sometime touched by a presynaptic bouton at the site where the previously identified spine had been located. We conclude that new spines are most likely to be functional and that pruned spines can be transformed into shaft synapses and thus maintain their functionality within the neuronal network.

Some variations of the circle of Willis, important for cerebral protection in aortic surgery--a study in Eastern Europeans.

BACKGROUND: During unilateral selective cerebral perfusion (SCP), via cannulation of the brachiocephalic trunk, the brain receives blood only through the right common carotid artery and the right vertebral artery. For perfusion of the contralateral (left) hemisphere it is counted on the competence of the circle of Willis (CoW). It is well known that variations of CoW are present in more than 50% of the people. Furthermore, these variations usually affect more than one vessel of the circle. The aim of the present work was to study the variations of CoW, which could have an impact on cerebral blood supply during unilateral SCP. METHODS AND MATERIALS: We study 112 CoWs obtained from cadavers via routine dissection in the Department of Forensic Medicine of Medical University, Sofia. The external diameter of both vertebral arteries and all arteries that form CoW was measured with a caliper-gauge. RESULTS: We identify the variations of CoW such as significant hypoplasy and/or lack of a branch of the circle. Bearing in mind the characteristics of the blood flow during unilateral SCP some of these variations were classified as significant during unilateral SCP. They were subdivided into groups according to most probable stroke site after unilateral SCP. CONCLUSIONS: Because of the high percent of the variations, hemodynamically significant during unilateral SCP, a suggestion for routine preoperative CT-angio of CoW could be made. Furthermore, an intraoperative follow-up with NIRO, transcranial Doppler, EEG, and so forth could also be recommended.

Parvalbumin in the cat claustrum: ultrastructure, distribution and functional implications.

The presence of the calcium-binding protein (CaBP) parvalbumin (PV) in the neuronal elements of the cat's dorsal claustrum was studied by immunohistochemistry at the light- and electron-microscopic level. PV-immunoreactive neurons and fibers were detected in all parts of the claustrum. The PV-immunoreactive neurons were divided into several subtypes according to their size and shape. Approximately 7% of all PV-immunoreactive neurons were classified as large, while approximately half of the labeled neurons were medium-sized. The small PV-immunoreactive neurons were 45% of the total PV-immunoreactive neuronal population. Ultrastructurally, many spiny and aspiny dendrites were heavily immunolabeled, and the reaction product was present in dendritic spines as well. Several types of synaptic boutons containing reaction product were also found. These boutons terminated on both labeled and unlabeled postsynaptic targets (soma, dendrites, etc.), forming asymmetric or symmetric synapses. Approximately 70% of all PV-immunoreactive terminals contained round synaptic vesicles and formed asymmetric synapses. The majority of these boutons were of the ''large round'' type. A lesser percentage were of the ''small round'' type. This paper represents the first study demonstrating the existence of PV, a CaBP, in the cat claustrum, and its distribution at the light and electron microscope level. Beyond the relevance of this research from the standpoint of adding to the paucity of literature on PV immunoreactivity in the claustrum of various other mammals (e.g. monkey, rabbit, rat, mouse), it is of particular significance that the cat claustrum is more similar to the rabbit claustrum than to any other mammalian species studied thus far, noted by the existence of four distinct morphologic subtypes. We also demonstrate a lack of intrinsic, and possibly functional, heterogeneity as evidenced by the uniform distribution of PV throughout the cat claustrum, across the four cell subtypes (i.e. inhibitory interneurons as well as projection neurons). Indeed, the association with, and influence of, the cat claustrum on diverse multisensory mechanisms may have more to do with its afferent than efferent relationships, which speaks strongly for its importance in the sensory hierarchy. Exactly what role PV plays in the claustrum is subject to discussion, but it can be postulated that, since CaBP is associated with GABAergic interneurons, synaptogenesis and neuronal maturation, it may also serve as a neuroprotectant, particularly with regard to pathologies associated with the aging process, such as in Alzheimer's disease.

Lack of paternal care affects synaptic development in the anterior cingulate cortex.

Exposure to enriched or impoverished environmental conditions, experience and learning are factors which influence brain development, and it has been shown that neonatal emotional experience significantly interferes with the synaptic development of higher associative forebrain areas. Here, we analyzed the impact of paternal care, i.e. the father's emotional contribution towards his offspring, on the synaptic development of the anterior cingulate cortex. Our light and electron microscopic comparison of biparentally raised control animals and animals which were raised in single-mother families revealed no significant differences in spine densities on the apical dendrites of layer II/III pyramidal neurons and of asymmetric and symmetric spine synapses. However, significantly reduced densities (-33%) of symmetric shaft synapses were found in layer II of the fatherless animals compared to controls. This finding indicates an imbalance between excitatory and inhibitory synapses in the anterior cingulate cortex of father-deprived animals. Our results query the general assumption that a father has less impact on the synaptic maturation of his offspring's brain than the mother.

Light microscopical study of nitric oxide synthase I-positive neurons, including fibres in the vestibular nuclear complex of the cat.

Nitric oxide is a gaseous neurotransmitter that is synthesized by the enzyme nitric oxide synthase I (NOS I). At present, little is known of NOS I-positive neurons in the vestibular nuclear complex of the cat (VNCc). The aim of the present study was to examine the morphology, distribution patterns and interconnections of NOS I-positive neurons, including fibres in the VNCc. Five adult cats were used as experimental animals. All cats were anaesthetized and perfused transcardially. Brains were removed, postfixed, cut on a freezing microtome and stained in three different ways. Every third section was treated with the Nissl method, other sections were stained either histochemically for NADPH diaphorase or immunohistochemically for NOS I. The atlas of Berman (1928) was used for orientation in the morphometric study. NOS I-positive neurons and fibres were found in all parts of VNCc: medial vestibular nucleus (MVN); lateral vestibular nucleus (LVN); superior vestibular nucleus (SVN); inferior vestibular nucleus (IVN); X, Y, Z groups and Cajal's nucleus. The NOS I-positive neurons were classified according to their size (small, medium-sized, large neurons type I and type II) and their shape (oval, fusiform, triangular, pear-shaped, multipolar and irregular). In every nucleus, a specific neuronal population was observed. In SVN, a large number of interconnections between NOS I-positive neurons were identified. In MVN, chain-like rolls of small neurons were found. Tiny interconnections between MVN and mesencephalic reticular formation were present. Our data provide information on the morphology, distribution patterns and interconnections of NOS I-positive neurons in the VNCc and can be extrapolated to other mammals.

Parvalbumin-like immunostaining in the cat inferior colliculus. Light and electron microscopic investigation.

The presence of the calcium-binding protein parvalbumin (PV) was studied in neuronal elements of the cat's inferior colliculus (IC) by means of light and electron microscopic immunocytochemistry. Immunostaining of PV was detected in all three main parts of the IC. Several subtypes of large neurons that differed in size and shape were immunostained, comprising approx. 15% of the total number of PV-containing neurons. Approx. half of the labeled neurons were medium sized. Two types of small neurons were found to be PV synthesizing, and comprised approx. 35% of the total PV-containing population. Ultrastructurally, many dendrites were heavily immunolabeled, and the reaction product was present in dendritic spines as well. Several types of synaptic boutons contained reaction product, and terminated on both labeled and unlabeled postsynaptic targets forming asymmetric and symmetric synapses. Approx. 70% of all PV-immunolabeled terminals contained round synaptic vesicles and formed asymmetric synapses. The majority of these boutons were of the "large round" type and corresponded to the terminals of cochlear nuclei. A lower number were of the "small round" type, and were probably corticotectal terminals. The remaining 30% of PV-containing terminals contained pleomorphic or elongated vesicles and formed symmetric synapses. These terminals corresponded with "P" and "F1" bouton types. Part of these boutons appeared to arise from nuclei of the lateral lemniscus and the superior olive, and a certain percentage likely represented endings of inhibitory interneurons.

Postnatal development of neurons expressing NADPH-diaphorase and parvalbumin in the parietal cortex of male and female rats.

Expression of the enzyme nicotinamide adenine dinucleotide phosphate-diaphorase (NADPH-d) and the calcium-binding protein parvalbumin was studied in the parietal cortex of male and female rats during postnatal development at 20, 60 and 90 days of age. First, localization of the activity of NADPH-d was combined with the immunohistochemical localization of parvalbumin to facilitate recognition of morphological details and distribution patterns of these two types of cortical neurons. Double staining of neurons for parvalbumin and NADPH-d was never found. Second, it was found that NADPH-d is a simple and proper marker for quantitative studies. Morphometric analysis revealed sexual dimorphism in the density of NADPH-d-positive neurons in 20 days-old prepubertal rats. Females showed higher amounts of NADPH-d-positive neurons than males. No sex-dependent differences were detected in 60 days-old pubertal and 90 days-old postpubertal rats. The present results suggest that sex differences in the number of NADPH-d-positive neurons in the rat parietal cortex may be related to epigenetic effects of gonadal hormones in the early prepubertal period of postnatal development.