Neurological and histological outcomes after subarachnoid injection of a liposomal bupivacaine suspension in pigs: a pilot study.

BACKGROUND: An injectable liposomal bupivacaine suspension (EXPAREL™) is approved by the US Food and Drug Administration for analgesia by tissue infiltration and interscalene brachial plexus, but not for use in the neuraxial space. This pilot study describes neurological and histological outcomes of escalating doses of this extended-release formulation of bupivacaine after subarachnoid administration. METHODS: Twenty-five pigs (Sus scrofa domesticus) weighing 36.2 (4.4) kg were randomly assigned to one of five groups to receive a subarachnoid injection of sodium chloride 0.9%, 3 ml (negative control), preservative-free bupivacaine hydrochloride 0.5%, 3 ml (positive control), or one of three doses of liposomal bupivacaine suspension 1.33%: 1.5, 3, or 5 ml. After recovering from general anaesthesia, neurological outcomes were assessed by blinded observers. Three weeks later, the animals were sacrificed for histological evaluations of neurotoxicity. RESULTS: Animals that received sodium chloride 0.9%, bupivacaine hydrochloride, or liposomal bupivacaine 1.5 ml recovered within 2, 5, or 4 h, respectively. Animals that received liposomal bupivacaine 3 or 5 ml exhibited signs of neuraxial block (decreased nociception and proprioception) up to 32 h after injection. No histological evidence of neurotoxicity was found in any of the groups. CONCLUSIONS: Subarachnoid administration of liposomal bupivacaine in pigs exhibited a dose-response effect, and resulted in longer duration of neuraxial block than bupivacaine hydrochloride without histological evidence of neurotoxicity. Our study contributes preliminary data to inform further toxicological assessments and regulatory approval before subarachnoid administration in humans.

Concomitant fenestration and phlebectasia of the right internal jugular vein: a unique finding in anatomical dissection.

The internal jugular vein (IJV) is an important vascular structure for oncologists and radiologists and is also a frequently used central venous route. The varia-tions in the pattern of its course, and knowledge of its variations, are important. During the anatomical dissection of a 70-year-old male cadaver, fenestration involving a short segment (2.5 cm) of the right IJV in the superior part of the carotid triangle and a large dilatation (phlebectasia) of the IJV, involving its nonfenestrated segment were found - a case that has not previously been reported. The aetiology and clinical implications of the concurrent anomalies are described. Clinicians and surgeons performing neck vascular or reconstru-ctive surgery should be made aware of both IJV variations in order to prevent inadvertent injury and avoid invasive investigations and inappropriate treatment.

Neurotoxicity of perineural vs intraneural-extrafascicular injection of liposomal bupivacaine in the porcine model of sciatic nerve block.

Liposomal bupivacaine is a prolonged-release local anaesthetic, the neurotoxicity of which has not yet been determined. We used quantitative histomorphometric and immunohistochemical analyses to evaluate the neurotoxic effect of liposomal bupivacaine after perineural and intraneural (extrafascicular) injection of the sciatic nerve in pigs. In this double-blind prospective randomised trial, 4 ml liposomal bupivacaine 1.3% was injected either perineurally (n = 5) or intraneurally extrafascicularly (n = 5). Intraneural-extrafascicular injection of saline (n = 5) was used as a control. After emergence from anaesthesia, neurological examinations were conducted over two weeks. After harvesting the sciatic nerves, no changes in nerve fibre density or myelin width indicative of nerve injury were observed in any of the groups. Intraneural injections resulted in longer sensory blockade than perineural (p < 0.003) without persistent motor or sensory deficit. Sciatic nerve block with liposomal bupivacaine in pigs did not result in histological evidence of nerve injury.

The utility of three-dimensional optical projection tomography in nerve injection injury imaging.

The examination of nerve microarchitecture has hitherto been limited solely to two-dimensional imaging techniques. The objective of this study was to evaluate the ability of optical projection tomography to discern the nerve microarchitecture and injection injury in three dimensions. Five piglets were studied, whose median and lingual nerves were unilaterally injected post mortem with preset volumes of local anaesthetic, excised and subsequently made transparent with benzyl alcohol benzyl benzoate. Images were captured in three dimensions. The same contralateral nerves were used as controls. Using optical projection tomography, we observed differences between the internal organisation of the median and the lingual nerves, which potentially explain the variations in their susceptibility to injury. This was demonstrated in three dimensions as a disruption to the fascicles in the lingual nerve, and their displacement in the median nerve. This new technology offers potential for studying nerve microarchitecture topography and its tolerance to injection injury.

Unilateral absence of foramen spinosum with bilateral ophthalmic origin of the middle meningeal artery: case report and review of the literature.

Bilateral ophthalmic origin of the middle meningeal artery with an unilateral absence of foramen spinosum has not yet been described. We report on a skull with endocranial meningeal grooves indicating bilateral ophthalmic origin of the middle meningeal artery, however, its branches were normal both in their position and distribution. In addition, a rare venous sinus variation was present unilaterally - a sinus of Hyrtl. Imaging identification of the anomalous origin of the middle meningeal artery is important while planning surgical and endovascular interventions in the middle cranial fossa and the orbit.

Wearing of complete dentures reduces slow fibre and enhances hybrid fibre fraction in masseter muscle.

Edentulous conditions and use of complete dentures alter the function of jaw muscles, which is presumably reflected in the myosin heavy chain (MyHC) isoform composition. This study is the first dealing with MyHC isoforms expression in edentulous persons with the aim to clarify to which extent the decreased functional load following teeth loss contributes to the changed muscle phenotype during ageing. We analysed MyHC expression in old masseter muscle at decreased and full functional load by comparing age-matched edentulous and dentate subjects. Edentulous subjects had upper and lower complete dentures. Dentate subjects had at least 24 natural teeth in continuous dental arches with two molars present in each quadrant and normal intermaxillary relationship. The adaptive response to the reduced masticatory load was lower numerical and area proportion of MyHC-1 expressing fibres and higher numerical proportion of hybrid fibres in edentulous compared with dentate subjects with no significant difference in the proportion of MyHC-neo-expressing fibres between both groups. We conclude that the observed differences in the proportion of fibre types between denture wearers and dentate subjects cannot be ascribed to degenerative changes intrinsic to the ageing muscle, but to functional differences in muscle activity and to morphological alterations of stomatognathic system accompanying the complete teeth loss.

Variant course of the submental vein.

Superficial head and neck vessels are increasingly used in surgical procedures and are especially important in facial transplantation surgeries. We report a variant course of the submental vein observed during a dissection of a 79-year-old-male embalmed cadaver: a left submental vein running caudally across the intermediate tendon of the digastric muscle, separating from the course of the submental artery and emptying directly into the common facial vein in the carotid triangle. Such course may complicate submental island flap harvesting, as well as the graft manipulations and reattachment, since the vein could be damaged during the conventional procedure. This report therefore extends current knowledge of the variations of the vascular anatomy of the head and neck.

Platysma muscle additionally innervated by a variant anterior branch of the great auricular nerve.

Damage to the great auricular nerve, with consequent clinical deficits, is a common surgical complication in facial aesthetic and in head and neck procedures such as parotidectomy, neck dissection, rhytidectomy and platysma flap operations. Hence, a thorough knowledge of nerve anatomy, particularly its potential variations, is critical in reducing the associated operative morbidity. Accordingly, we report an unusual variation of the anterior branch of the great auricular nerve noted in an 81-year-old female cadaver. The nerve was observed to course into the submandibular region anterior and superficial to the internal jugular vein, communicating with the cervical branch of the facial nerve, while independently innervating the platysma muscle. Although several anatomical variations of the branches of the cervical plexus have been documented, our report describes unique innervation of the platysma muscle by the great auricular nerve, which provides a new insight on the motor component of the nerve.

Biomechanical comparison of the three techniques for arthroscopic suprapectoral biceps tenodesis: implant-free intraosseous tendon fixation with Cobra Guide, interference screw and suture anchor.

PURPOSE: A new arthroscopic technique with Cobra Guide (CG) was developed to enable fast, controlled and strong intraosseous biceps tenodesis while avoiding an implant. The purpose of this study was to compare the newly developed suture-only biceps tenodesis technique [arthroscopic suprapectoral intraosseous implant-free biceps tenodesis (ASIIBT) with the new CG] to classical interference screws (IS) and suture anchors (SA) in terms of construct resistance to failure. MATERIALS AND METHODS: Fifty-eight human cadaveric shoulders were randomized into three treatment groups. Twenty shoulders received an IS, 19 SA and 19 ASIIBT. A biceps tenodesis was performed according to the techniques listed above. Cyclic loading tests on a dynamic loading testing device were used to measure and compare the resistance to failure pullout between the three groups. Hartley's Fmax test and Tukey's Honest Significant Difference method were used for statistical analysis. RESULTS: The construct with the greatest resistance was ASIIBT. Its resistance was statistically higher compared to the IS technique (p = 0.001). Resistance compared to the SA technique was not statistically significant (p = 0.123), although in seven cases ASIIBT resisted more than 50 cycles at 200 N, while the SA technique reached 50 cycles at 200 N in just two cases. During cyclic loading, each specimen failed at the tenodesis site. CONCLUSIONS: ASIIBT showed higher failure loads compared with IS and SA. Better construct performance of ASIIBT is due to greater absorption of distension forces which may improve final tenodesis healing. Also, the absence of an implant lowers additional costs and the chances for postoperative complications may be decreased significantly.

Software for muscle fibre type classification and analysis.

Fibre type determination requires a large series of differently stained muscle sections. The manual identification of individual fibres through the series is tedious and time consuming. This paper presents a software that enables (i) adjusting the position of individual fibres through a series of differently stained sections (image registration) and identification of individual fibres through the series as well as (ii) muscle fibre classification and (iii) quantitative analysis. The data output of the system is the following: numerical and areal proportions of fibre types, fibre type size and optical density (grey level) of the final reaction product in every fibre. The muscle fibre type can be determined stepwise, based on one set of stained sections while further, newly stained sections can be added to the already defined muscle fibre profile. Several advantages of the presented software application in skeletal muscle research are presented. The system is semiquantitative, flexible, and user friendly.

Cross-over type of supernumerary digastric muscle.

A cross-over type asymmetric anomaly of the anterior belly of the right digastricmuscle was observed during a cadaveric dissection of the submental region.Three irregularly-shaped supernumerary muscle bundles were found between theanterior bellies of the digastric muscles. Although the anomalies of the digastricmuscles are often observed, this complicated pattern has not been previouslyreported. Our findings and previous reports illustrate the morphogenetic complexityof the anterior belly of the digastric muscle, and their potential importancein confounding clinical evaluation or complicating surgical procedures in thesubmental region.

Effect of ageing on the myosin heavy chain composition of the human sternocleidomastoid muscle.

The myosin heavy chain (MyHC) composition of ageing limb muscles is transformed into a slower phenotype and expresses fast-twitch fibre type atrophy, presumably due to age-related motor unit remodelling and a change in the patterns of physical activity. It is not known if ageing affects the sternocleidomastoid muscle (SCM) in a similar way. The goal of the study was to analyze the MyHC composition and the size of muscle fibres in the ageing SCM by immunohistochemical methods and quantitative analysis and stereology using our own software for morphometry. We hypothesize that with ageing the MyHC composition of SCM transforms similarly as in ageing limb muscles, but the size of the muscle fibres is less effected as in limb muscles. The study was performed on the autopsy samples of the SCM in 12 older males. The results were compared with those published in our previous study on 15 young adult males. An ageing SCM transforms into a slower MyHC profile: the percentage of slow-twitch fibres is enhanced (numerical proportion 44.6 vs. 31.5%, P<0.05; area proportion 57.2 vs. 38.4%, P<0.05). The share of hybrid 2a/2x fibres is diminished (numerical proportion 14.1 vs. 26.8%, P<0.05), the area proportion of all fast-twitch fibres expressing MyHC-2a and 2x is smaller (50.6 vs. 63.5%, P<0.05), and the area proportion of fibres expressing the fastest myosin isoform MyHC-2x is smaller too (19.0 vs. 34.5%, P<0.05). The slower phenotype with the preferential reduction of the fibres expressing the fastest MyHC-2x provide circumstantial evidence for: (i) more fast-twitch than slow-twitch motor units being lost; and (ii) reinnervation by the surviving motor units. There appears to be no significant influence on muscle fibre size, which is congruent with relatively unchanged SCM activity during life.

Myosin heavy chain composition of the human sternocleidomastoid muscle.

The sternocleidomastoid (SCM) muscle is one of the neck muscles responsible for head posture and control of head movement. It functions in rotation, inclination, protraction, extension and flexion of the head, whilst chewing and in exerting increased respiratory efforts. This study is the first one describing the myosin heavy chain (MyHC) isoform composition of the SCM muscle of presumably healthy young males for the purpose of better understanding the contractile properties of the muscle as well as to help in evaluation of pathologically altered structure of the muscle. Autopsy samples were processed immunohistochemically to reveal the MyHC isoform composition. The muscle fibres expressed MyHC-1 (31.5%), -2a (29.7%) and -2x (4.3%) or co-expressed MyHC-2a with MyHC-2x (26.8%), MyHC-1 with MyHC-2a (4.1%) and/or MyHC-1, -2a with -2x (1.1%). In addition to the MyHC isoforms, characteristic of adult limb muscles, a very low percentage of muscle fibres (0.2-2.7%) expressed MyHC-neo, which is normally not found in adult limb muscles. Only two samples exhibited MyHC-neo at a rather higher percentage (6.3% and 7.5%) of muscle fibres. The high share of hybrid fibres and the presence of MyHC-neo in the SCM muscle differ from that of adult limb muscles where hybrid fibres are rare and the expression of immature MyHC isoforms occurs only in pathological or experimental conditions. Since the SCM muscle shares the same embryogenic potential as limb muscles, its distinct MyHC expression appears to be associated with twin innervation and with the intrinsic specialisation to perform multiple functions.

Software for muscle fibre type classification and analysis.

Fibre type determination requires a large series of differently stained muscle sections. The manual identification of individual fibres through the series is tedious and time consuming. This paper presents a software that enables (i) adjusting the position of individual fibres through a series of differently stained sections (image registration) and identification of individual fibres through the series as well as (ii) muscle fibre classification and (iii) quantitative analysis. The data output of the system is the following: numerical and areal proportions of fibre types, fibre type size and optical density (grey level) of the final reaction product in every fibre. The muscle fibre type can be determined stepwise, based on one set of stained sections while further, newly stained sections can be added to the already defined muscle fibre profile. Several advantages of the presented software application in skeletal muscle research are presented. The system is semiquantitative, flexible, and user friendly.

Effect of chronic uncontrollable stress on myosin heavy chain expression in rat masseter muscle.

This study has evaluated the influence of chronic uncontrollable painless stress on the expression of myosin heavy chain (MyHC) isoforms in deep and superficial parts of the rat masseter muscle. Muscle cross sections of three month old Wistar rats were processed for immunohistochemical staining. In control muscle the three adult fast MyHC isoforms (-2a, -2x/d, -2b), were nonhomogenously distributed: in the smaller anterior part of the muscle only fast MyHC isoforms were expressed. The major superficial and posterior part of the muscle fibres also contained MyHC-1 (10%) and only 7% expressed of MyHC-2b. Psychological stress significantly modified the expression of particular MyHC isoforms in both parts of the muscle. In the anterior part of the experimental masseter the proportion of MyHC-2b expressing fibres decreased and 10% of the muscle fibres expressed MyHC-1. The superficial and posterior part of the experimental muscle contained significantly more muscle fibres expressing MyHC-2b isoforms. Regarding whole cross-sectional area transition towards faster phenotype was evident due to a higher percentage of either pure MyHC-2b containing fibres or hybrid MyHC-2x/d and -2b expressing fibres. The results of this study demonstrate that psychological stress is accompanied by a modification of muscle phenotype which is region specific. Opposite changes in deep and superficial part of the muscle could be ascribed to differences in genetic programme, function or susceptibility to hormones in both parts of masseter.

Fiber types in the mouse levator auris longus muscle: a convenient preparation to study muscle and nerve plasticity.

The histochemical composition of the levator auris longus (LAL) muscle has been investigated in adult NMRi mice. Histochemical reaction for myofibrillar adenosine triphosphatase (ATPase) after preincubation in alkaline and acidic media, nicotine amideadenine-dinucleotide dehidrogenase (NADH-dehydrogenase), and alpha-glycerophosphate dehydrogenase were performed on cryosections of LAL muscle. Expression of myosin heavy chain (MyHC) isoforms was detected with the immunoperoxidase method applying monoclonal antibodies against MyHC isoforms -1, -2a, -2x/d, and -2b, as well as by sodium dodecylsulfate (SDS) glycerol gel electrophoresis. The muscle was proven to be a pure fast-twitch muscle. The most numerous fibers in LAL muscles contained MyHC-2b and some MyHC-2a. Histochemically, pure IIA fibers with oxidative metabolism and pure IIB fibers with glycolytic metabolism were detected. In contrast to the majority of mature control muscles, numerous hybrid fibers coexpressing MyHC-2x/d with MyHC-2a or MyHC-2b were present. Both hybrids were oxidative-glycolytic; additionally, some hybrids containing MyHC-2a were oxidative. In one out of six muscles, traces of MyHC-1 were detected both with immunoperoxidase staining and with SDS glycerol gel electrophoresis. Rare fibers that exceptionally expressed small amounts of MyHC-1 always coexpressed MyHC-2a, which is an additional proof that pure type I fibers do not exist in LAL. Due to these histochemical characteristics and to its previously described morphological features, the use of the LAL muscle as a model for various studies, particularly muscle and nerve interactions, is emphasized.

Viability of fibroblasts in cell culture after treatment with different chemical retraction agents.

Prior to fixed prosthodontic impression procedures, temporary horizontal retraction of the free gingival tissue should be accomplished apically to the preparation finishing line. The mechanical-chemical method using cotton retraction cords of various sizes impregnated with various retraction chemicals is the most commonly employed retraction technique. Most retraction agents have pH values from 0.8 to 0.3, and are therefore hazardous to the cut dentine and periodontal tissues. Sympathomimetic vasoconstrictors introduced recently have a pH of 5.6, and are free of systemic side-effects. The present study using the dye exclusion test, colony forming ability test and colorimetric assay was undertaken to evaluate cytotoxic effects of four chemical retraction agents on cultured V-79 fibroblasts, and the dependence of cytotoxicity on the agent concentration and time of exposure. Original concentrations of retraction agents produced stronger cytotoxic effects than dilutions of 1:1 and 1:10. The most aggressive agent, 25% aluminium chloride, took only 1 min to damage all cell cultures. The proportion of cells damaged after 10 min of exposure to tetrahydrozoline was 60%, which was significantly less compared with other chemicals tested. With the colony forming ability test using retraction agents diluted to 1:10 the greatest number of colonies emerged in samples treated with tetrahydrozoline (statistical significance: P < 0.01). The colorimetric assay showed equal cytotoxic effects for 25% aluminium sulphate and tetrahydrozoline. The colorimetric test used in the study has proved an ergonomic, accurate and reliable test for cytotoxicity determination.

Gingival tissue inflammatory response following treatment with chemical retraction agents in Beagle dogs.

A normal finding of a clinically healthy gingiva is minimal inflammatory infiltration of subepithelial connective tissue which increases after exposure of gingiva to retraction agents. The study investigates and compares the inflammatory potential of four retraction agents on the gingival connective tissue. The gingiva of Beaagle dogs was exposed for 10 minutes to chemical agents. Byopsies were taken one hour, 24 hours and 7 days after treatment. Morphometric analysis of treated and control gingiva was performed on parasagittal tissue sections to compare the gingival tissue inflammatory potential of four retraction chemicals. Retraction agents produced no significant effect on connective tissue under the junctional epithelium. On the other hand the connective tissue under sulcular epithelium exhibits extensive inflammatory infiltration. The most aggressive retraction agent being Racestyptine and the least Visine, which therefore could be recommended for clinical practice.

Alpha 2-macroglobulin in gingival fluid: correlation with alveolar bone loss in periodontal disease.

The concentration of total, free and bound (complexed forms) of alpha 2-macroglobulin was measured by crossimmunoelectrophoresis in patients with different degrees of periodontal disease as indicated by the gingival index (GI) and the proportion of alveolar bone loss (ABL). The concentration of total alpha 2-M (bound and free forms) is lower in gingival fluid taken from sites with more inflamed gingivae. Its concentration decreases with an increase in the pocket depth and the alveolar bone loss at the sites of fluid collection. The concentration of alpha 2-M bound form, i.e., the presence of alpha 2-M: protease complexes, in gingival fluid is low or absent at the sites with more pronounced bone loss (above 15%). Our results support the hypothesis that unbalanced protease activity damages the periodontal tissue. Not only the proteolytic but also inhibitory activities are altered and correspond to the severity of the disease.

Alpha 2-macroglobulin in the sera of patients with periodontal disease.

The concentration of alpha 2-macroglobulin (alpha 2-M) in the sera of 86 blood donors was measured by laser nephelometry. Plaque index (PII), gingival index (GI) and probing depth were recorded around all existing teeth. All measured clinical parameters correlated positively with the concentration of alpha 2-M in the sera of the examined persons, but statistically significant positive correlation was found only in with the following: average gingival index and alpha 2-M concentration in the sera of females and average probing depth and alpha 2-M concentration in the sera of all examined females and males. This study supports the hypothesis that in long-standing chronic periodontal disease, the increased levels of proteinases might trigger the synthesis of alpha 2-macroglobulin in the host. The role of alpha 2-M as a proteinase scavenger and immunosuppressive agent is discussed briefly.