Andrea Martinuzzi

De novo neuromuscular junction formation on human muscle fibres cultured in monolayer and innervated by foetal rat spinal cord: ultrastructural and ultrastructural--cytochemical studies.

SummaryUltrastructural features of neuromuscular junction formation and transverse tubule development were studied utilizing a newly developed model in which human muscle fibres cultured in monolayer are innervated by foetal rat spinal cord with dorsal root ganglia attached. At early innervation (7–10 days), when distinct ‘boutons’ are contacting muscle fibres, the contacts of nerve terminals with the muscle fibres are, ultrastructurally, superficial and unorganized, and there is no basal lamina-like material between nerve terminals and muscle fibres. A bouton consists, ultrastructurally, of a cluster of small nerve terminals contacting the muscle fibre. At 2–3 weeks of innervation, shallow ‘beds’ are formed on the muscle fibre just beneath nerve terminals, and occasionally there are irregular and miniscule fragments of basal lamina-like material in the cleft. There is no Schwann cell apposing the nerve terminal at this stage of innervation. After 4–5 weeks of innervation there is more definite basal lamina material in the cleft and suggestive postsynaptic plasmalemmal densities and invaginations. However, there is no Schwann cell apposing the nerve terminal at this stage. At 6–8 weeks of innervation, deep postsynaptic folds are present, a Schwann cell apposes the nerve terminal, and basal lamina surrounds the entire muscle fibre. At all four stages of innervation examined, ultrastructural cytochemistry of alpha-bungarotoxin binding reveals that nicotinic ACh receptors are located exclusively at the neuromuscular junctions. After 1–2 weeks of innervation, very few lanthanum-positive transverse tubules are observed and only in close proximity to the surface membrane. After 3 weeks of innervation, more lanthanum-positive tubules are present, and they are located deeper within the muscle fibre. Five weeks after innervation, somewhat more elaborated tubules (but no lateral sacs) appear, and honeycomb structures are often present. After 6–7 weeks of innervation the tubular system is very elaborate and lateral sacs are present. Hence, this study describes consecutive stages of the formation of neuromuscular junctions and transverse tubules in innervated cultured human muscle, and provides an important basis to which similar studies related to the diseased human muscle can be compared.

Primary obstruction of the fourth ventricle outlets: neuroendoscopic approach and anatomic description.

OBJECTIVEPrimary obstruction of the foramina of Magendie and Luschka is an uncommon and still unclear cause of noncommunicating hydrocephalus. The aim of this work is the description, for the first time, of the inner aspect of these velar obstructions of the fourth ventricle outlets and the demonstration of the efficacy of neuroendoscopic treatment. METHODSOf 240 hydrocephalic patients treated in our institution with endoscopic third ventriculostomy, a subgroup of 10 cases with closure of the fourth ventricular outlets without associated Chiari malformation and syringomyelia was selected. In all of these cases, a transaqueductal endoscopic navigation of the fourth ventricle was performed, and the obstructed outlets were inspected. All of the clinical data and, in particular, the videotape records of endoscopic operations, as well as the cine-magnetic resonance imaging scans, were reviewed to evaluate their patency status. RESULTSVarious degrees of stenosis were found endoscopically: restriction of the Magendie contour with thick and opaque membrane, transparent spider web-like membrane, and dense membrane with fissures acting as valves. Endoscopic third ventriculostomy was effective in almost all patients, although we noticed an unforeseen high incidence of closure of the stoma. The restored normal cerebrospinal fluid flux after ventriculocisternostomy and magendieplasty was demonstrated by comparative study of cerebrospinal fluid flow measurements by cine-magnetic resonance imaging. CONCLUSIONThis report demonstrates the effectiveness of neuroendoscopic third ventriculostomy as well as magendiestomy in cases of tetraventricular hydrocephalus attributable to primary obstruction of the outlets of the fourth ventricle and, for the first time, presents direct images of various types of outlet obstructive pathology.

Asynchronous regulation of muscle specific isozymes of creatine kinase, glycogen phosphorylase, lactic dehydrogenase and phosphoglycerate mutase in innervated and non-innervated cultured human muscle.

Expression of muscle specific isozymes (MSIs) of creatine kinase (CK, EC 2.7.3.2), glycogen phosphorylase (GP, EC 2.4.1.1), lactate dehydrogenase (LDH, EC 1.1.1.27) and phosphoglycerate mutase (PGAM, EC 2.7.5.3) was studied both in cultured human muscle fibers which had been innervated (InnCHMFs) for 20-83 days, and in their non-innervated (non-InnCHMFs) sister control. In non-InnCHMFs, the MSI of PGAM was never detected, and there was no change in the expression of the MSI of CK during the entire period examined; the expression of MSIs of LDH and GP showed linear increase during the entire period of growth. The expression of MSIs of all 4 enzymes was significantly enhanced in InnCHMFs as compared to non-innervated control. The expression of MSIs of GP and PGAM, and to a lesser degree of LDH increased significantly in correlation with the duration of innervation; the MSI of CK increased linearly only up to 54 days of innervation and plateaued afterward. This study demonstrates: (1) innervation of cultured human muscle fibers by fetal rat spinal cord exerts a time-related maturational influence on their cellular isoenzymatic pattern; (2) to achieve induction and characteristic time-related expression of various MSIs, the requirements for neuronal influences seem to differ.

Endoscopic Anatomy of the Cerebral Aqueduct

OBJECTIVE What is known about the cerebral aqueduct is derived mainly from the legacy of classic histology and from the most recent advanced neuroimaging technologies. In fact, although this important structure is frequently glimpsed by neurosurgeons, only limited anatomic contributions have been added by microsurgery to its direct in vivo description. A review of our surgical experience in navigating the fourth ventricle prompted us to revisit the classical anatomic descriptions of the aqueduct and compare them using the novel perspective of neuroendoscopy. METHODS We reviewed video recordings of 65 transaqueductal explorations of the fourth ventricle using flexible endoscopes, which were performed in our center to treat various pathological conditions. Forty-one patients were selected as being more informative for anatomic description. They include 21 patients with communicating normal pressure hydrocephalus, 6 patients with intraventricular hemorrhage, 5 patients with membranous obstruction of the foramen of Magendie, 5 patients with trapped fourth ventricle as evidenced after aqueductoplasty, 3 patients with colloid cysts, and 1 patient with craniopharyngioma with apparently normal aqueduct, which was navigated to aspirate small fragments of colloid and tiny clots. RESULTS Patients with normal-sized third ventricles confirmed the typical triangular shape of the aqueductal adytum, whereas all pathological aqueducts invariably had an oval contour. The posterior commissure, a faint trace of the median sulcus, and the rubral eminences were the structures invariably noticed. Five segments of the aqueduct were always identifiable: the adytum, first constriction, ampulla, second constriction, and posterior part or egressus. CONCLUSION Neuroendoscopy provides a novel perspective into the inner aqueductal wall and supplies an incomparable view of the intracanalicular anatomic structures.

Developmental expression of the muscle-specific isozyme of phosphoglycerate mutase in human muscle cultured in monolayer and innervated by fetal rat spinal cord

The electrophoretic pattern of phosphoglycerate mutase of adult innervated normal human muscle is composed predominantly of the muscle-specific isozyme, whereas the electrophoretic pattern of aneurally cultured human muscle is composed only of the brain-specific isozyme. We studied the transition of the isozymes (phosphogluterate mutase) in human muscle cultured in monolayer and innervated for 20 to 83 days by rat embryo spinal cord explants. In this culture system, regions of innervated muscle fibers in close proximity to the ventral part of the spinal cord explant continuously contracted and the contractions were reversibly blocked by 1 mM d-tubocurarine. In those innervated cultured human muscle fibers, the total activity of phosphoglycerate mutase was increased and the muscle-specific isozyme was expressed. The amount of muscle-specific isozyme directly correlated with the duration of innervation. This study demonstrated that expression of the gene for the muscle-specific isozyme of phosphoglycerate mutase in human muscle cultured in monolayer is influenced by de novo innervation.

Aquaporin 1 expression in cystic hemangioblastomas

Hemangioblastomas of the central nervous system (CNS) become clinically manifest through the development of huge associated cysts. The mechanism underlying these fluid collections is as yet largely unexplained. Aquaporins (AQPs) are cell proteins responsible for transmembrane water transport that have been extensively studied in the last 10 years. We analyzed AQP1 water channel expression by an immunostaining technique in ten specimens of operated cerebellar cystic hemangioblastomas, for which complete clinical and follow-up records were available. Tumour and associated cyst volumes were determined by neuroimaging and then compared with immunohistochemical scores. Stromal cancer cells showed surprisingly high AQP1 expression, and huge cyst volume development showed correlation with higher immunostaining scores. Heavy AQP1 expression in cystic hemangioblastomas could shed new light on the mechanisms of satellite cyst development.

Effects of electrical stimulation and tetrodotoxin paralysis on expression of muscle-specific isozymes of four enzymes in aneurally cultured embryonic rat muscle

We studied the effect of electrical stimulation and a sodium channel blocker (tetrodotoxin) on the expression of muscle-specific isozymes of creatine kinase, glycogen phosphorylase, phosphoglycerate mutase, and lactate dehydrogenase in aneurally cultured embryonic rat muscle. Muscle contractile activity slightly accelerated the accumulation of muscle-specific isozyme of creatine kinase in early cultures (4 days of experiment), but no increase in the expression of muscle-specific isozymes of any enzyme was present in older cultures (11 days of experiment). We conclude that muscle contractile activity is not a main regulator of isozyme maturation in this system.

Paralysis of innervated cultured human muscle fibers affects enzymes differentially

Abstract: Increased accumulation of muscle‐specific isozyme (MSI) of creatine kinase (CK), lactate dehydrogenase (LDH), glycogen phosphorylase (GP), and phosphoglycerate mutase (PGAM) occurs with development and indicates muscle fiber maturation. The expression of MSIs of those four enzymes is greatly enhanced in innervated‐contracting as compared to noninnervated and noncontracting cultured human muscle fibers. We have now studied the effect of contractile activity on developmental accumulation of MSIs in innervated‐contracting, innervated‐paralyzed (2 μM tetrodotoxin for 30 days), and noninnervated‐noncontracting cultured human muscle fibers. Muscle acetylcholinesterase (AChE) and total enzyme activities were also studied under the same conditions. We observed a different dependency on contractile activity between total enzymatic activities of CK., LDH, and AChE, which were substantially reduced after paralysis, and GP and PGAM, which were unchanged. The expression of MSIs of CK, GP, PGAM, and LDH was always significantly increased in innervated as compared to noninnervated fibers. While the expression of MSIs of GP and PGAM was the same in contracting‐innervated and paralyzed‐innervated muscle fibers, the expression of MSIs of CK and LDH in paralyzed‐innervated muscle fibers was very slightly decreased as compared to their contracting‐innervated controls. Our studies demonstrate that in human muscle: (1) total enzymatic activities and the expression of MSIs of GP and PGAM are regulated by neuronal effect(s); (2) total enzymatic activities of CK, LDH, and AChE depend mainly on muscle contractile activity; and (3) MSIs of CK and LDH are regulated predominantly by neuronal factors and to a much lesser degree by muscle contractile activity.