Erika Cvetko

Frequency of M-Cadherin-stained Satellite Cells Declines in Human Muscles During Aging

To answer the question of whether the satellite cell pool in human muscle is reduced during aging, we detected satellite cells in 30-μm-thick transverse sections under the confocal microscope by binding of M-cadherin antibody. The basal lamina was detected with laminin. Nuclei were stained with bisbenzimide or propidium iodide. Satellite cells were counted by applying the disector method and unbiased sampling design. To determine if there are age-related differences in muscle fiber types, morphometric characteristics of muscle fibers were examined on thin sections stained for myofibrillar ATPase. Autopsy samples of vastus lateralis muscle from six young (28.7 ± 2.3 years) and six old (70.8 ± 1.3 years) persons who had suffered sudden death were analyzed. Numbers of satellite cells per fiber length (Nsc/Lfib) and number of satellite cells per total number of nuclei (satellite cell nuclei + myonuclei) (Nsc/Nnucl) were significantly lower in the old group (p<0.05). We demonstrate the importance of proper sampling and counting in estimation of sparsely distributed structures such as satellite cells. Our results support the hypothesis that the satellite cell fraction declines during aging.

A novel method for evaluation of capillarity in human skeletal muscles from confocal 3D images

A well developed capillary bed is essential for proper function of skeletal muscles. We present for the first time a triple immunofluorescent method suitable for staining capillaries and muscle fibre outlines in thick sections of human skeletal muscle, applying antibodies against collagen IV (in red) and F8 (in green) as well as Ulex europaeus lectin, visualized in green fluorescence. Further, we present possibilities for quantitative evaluation of the capillary network which implies the length of capillaries per unit volume of muscle tissue (Lcap/Vmuscle) and the length of capillaries supplying individual muscle fibres per unit fibre length (Lcap/Lfib), per surface area (Lcap/Sfib) and per volume (Lcap/Vfib) as well as the course of capillaries in the muscle. The latter can be described by the tortuosity, orientation and mean capillary length. To get reasonable results we met the following requirements: i) high quality thick tissue sections, from which 3D image data were acquired; ii) immunofluorescent methods suitable for confocal microscopy; iii) penetration of the fluorescent dyes throughout the tissue section; iv) proper 3D image analysis methods for performing reliable measurements and v) control over relevant tissue deformations. The developed methodology is illustrated by results obtained from autopsy or biopsy samples of three human muscles, i.e. vastus lateralis, multifidus and masseter muscle that exhibit differences in genetic background, innervation, tasks and functional activity.

Cementum thickness in multirooted human molars: A histometric study by light microscopy

Cementum distribution was studied on transversely sectioned roots of 33 three-rooted maxillary and 33 two-rooted mandibular permanent molars. The roots were sectioned at a level midway between furcation and apical region. Cementum thickness was measured at the midpoint of each of the four root surfaces and at a site exhibiting maximal cementum thickness. A reflected light microscope was used. Midpoint cementum thickness ranged between 5 and 800 microm in maxillary molars and between 5 and 700 microm in mandibular molars. Maximal cementum thickness ranged between 25 and 1140 microm in maxillary molars and between 20 and 700 microm in mandibular molars. The results indicate that cementum tends to accumulate along interradicular surfaces of multirooted molars. In addition, a tendency was noted for more cementum to occur orally than vestibularly in roots of mandibular molars as well as in buccal roots of maxillary molars. Furthermore, this study indicates that cementum accumulates in root concavities (developmental depressions), although the exact mechanism still awaits a clear explanation. Previous studies have suggested that tensile forces stimulate cementum apposition. It may be assumed, however, that masticatory function and continuing eruption of the teeth exert no discernible effect on cementum thickness in the examined region of the root. In conclusion, the observed cementum distribution could be attributed to at least two factors: one is buccal drift and the second may be the effect of root concavities. From a clinical viewpoint, the established pattern of cementum distribution in multirooted molars should be considered in endodontic and periodontal treatment.

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.

Bilateral anomalous high position of the mandibular foramen: a case report

The inferior alveolar nerve block is one of the most common techniques for delivering dental anesthesia. Its success depends on placing the needle tip in close proximity to the mandibular foramen (MF). In certain cases, however, this nerve block fails, even when performed by the most experienced clinician. Anatomical variability may be one source of local anesthetic failure and includes bone and nerve variations. A case is presented of a bilateral anomalous high position of the MF, identified from the panoramic radiograph. An adjusted anesthetic technique (the Vazirani-Akinosi technique) was used to achieve local anesthesia before extraction of a lower second molar following an unsuccessful conventional indirect technique with a higher entry point. A description of the embryological basis for the anomalous high positioned MF and its impact from a practical and clinical perspective, with particular reference to local anatomy, is included.

Electron-microscopic imaging of endothoracic fascia in the thoracic paravertebral space in rats.

Background and Objectives Anesthesia and analgesia with paravertebral block are reportedly variable. Existence of an endothoracic fascia has been proposed as one of the possible mechanisms leading to variability. We undertook an electron-microscopy imaging study to investigate the endothoracic fascia in the thoracic paravertebral space (TPS) in rats. Methods Male Wistar rats were studied in accordance with the principles of laboratory animal care. After the rats were euthanized in a CO2 chamber, the thoracic paravertebral tissues were removed en bloc and cut into consecutive transverse sections of approximately 3 mm. Stereomicroscopy and electron-microscopy assessments were performed by 2 independent observers. Results The endothoracic fascia was consistently identified in all specimens. The fascia was located between the parietal pleura and the innermost intercostal muscles or ribs. Its thickness ranged from 15 to 27 &mgr;m (mean, 20 ± 3 &mgr;m). The endothoracic fascia divided the TPS in 2 compartments: one, extrapleural and anterolateral (EPC); another, subendothoracic and posteromedial (SETC). The spinal nerves with their ganglia were found within SETC, whereas the sympathetic ganglia were consistently located within the EPC. Conclusions The endothoracic fascia in rats appears to divide the TPS into EPC and SETC. These anatomic characteristics may have implications in thoracic paravertebral blockade.

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

Abstract 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.

Inflammatory response after injection of aqueous gel into subarachnoid space in piglets.

Background Ultrasound-guided neuraxial anesthesia requires the application of ultrasound gel between the transducer and the skin to avoid signal drop-off. As the needle is inserted, the gel may be introduced intrathecally. The purpose of this study was to examine the evidence of an inflammatory response in the subarachnoid space after intrathecal gel introduction. Methods Twelve piglets were sedated with azaperone 0.5 mg/kg intramuscularly and anesthetized via face mask (2%–4% isoflurane in 50% air-oxygen mixture). After collection of cerebrospinal fluid by lumbar puncture with a 22-gauge needle (baseline), 0.2 mL of ultrasound gel mixed with 1 mL of saline was injected intrathecally into 9 piglets (gel group). In 3 piglets (control group), 1 mL of saline was administered. Behavioral and neurologic assessments were recorded on a 4-grade scale. Following the preinjection and postinjection cerebrospinal fluid collection, the piglets were killed, and samples of spinal cord with meninges were excised. Five cross sections (1 mm apart) were processed using immunohistochemistry. Results After anesthesia, all piglets displayed short-lived mild (grade 1) motor and behavioral deficits. Mean ± SD protein concentrations in the gel and baseline samples were 14.1 ± 3.0 and 1.3 ± 0.5 g/L, respectively (P = 0.001). No differences were found in protein concentration between baseline (1.8 ± 0.7 g/L) and control samples (2.8 ± 0.8 g/L) (P = 0.4). In the gel group, numerous immunopositive cells were found in the pia, arachnoid, and inner layer of dura. Conclusion Subarachnoid injection of ultrasound gel in piglets results in an inflammatory response within neuraxial space.

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.

A case of an unusual arrangement of numerous tributaries to the middle temporal vein and its fenestration

With the increasing use of free tissue grafts for extensive reconstruction of the head and neck, it is important to document and understand the types of vascular variations. We report an unusual arrangement of numerous interconnecting tributaries to the medial temporal vein (MTV) in a 73-year-old male cadaver. Another unusual feature was the division of the MTV into three parallel segments, which reunited into a single stem. The presence of an extensive venous plexus in the temporal region may be hazardous for surgical operations. Surgeons performing vascular or reconstructive surgery should be aware of this unexpected variation of temporal region venous drainage in the hope of preventing inadvertent injury.