Vadims Poukens

Tuberous sclerosis in a 20-week gestation fetus: immunohistochemical study.

Abstract We report an autopsy case of tuberous sclerosis complex (TSC) in a 20-week gestational age female fetus. The brain showed lesions suggestive of early cortical tubers and subependymal hamartomatous nodules. The large cells within these nodular clusters were variably immunoreactive for glial fibrillary acidic protein (GFAP) and vimentin and negative for synaptophysin and neurofilament. Subependymal radial glia expressed both vimentin and GFAP, but subpial radial glia either did not express these markers (in contrast to an age-matched control) or were absent. Tuberin expression was noted in heterotopic neurons in the white matter and brain cells consistent with Cajal Retzius cells in the neocortical molecular layer, very weakly in superficial cortical neurons, neurons in the basal ganglia, Purkinje cells and external granular cells of cerebellum, cranial nerve nuclei neurons, occasional germinal matrix cells, ependymal cells, choroid plexus epithelium, and pituitary gland neuroendocrine cells; it was not seen within the cells of subependymal nodules. The pattern of tuberin immunoreactivity was similar to that which we have observed in older TSC patients. Proliferating cell labeling indexes were comparable in the germinal matrix of the TSC patient and an age-matched control. Abnormal subpial radial glia may be responsible for some of the neuronal migration abnormalities that appear to result in neocortical tubers.

Extraocular connective tissue architecture

Extraocular muscle pulleys, now well known to be kinematically significant extraocular structures, have been noted in passing and described in fragments several times over the past two centuries. They were late to be fully appreciated because biomechanical modeling of the orbit was not available to derive their kinematic consequences, and because pulleys are distributed condensations of collagen, elastin and smooth muscle (SM) that are not sharply delineated. Might other mechanically significant distributed extraocular structures still be awaiting description?An imaging approach is useful for describing distributed structures, but does not seem suitable for assessing mechanical properties. However, an image that distinguished types and densities of constituent tissues could give strong hints about mechanical properties. Thus, we have developed methods for producing three dimensional (3D) images of extraocular tissues based on thin histochemically processed slices, which distinguish collagen, elastin, striated muscle and SM. Overall tissue distortions caused by embedding for sectioning, and individual-slice distortions caused by thin sectioning and subsequent histologic processing were corrected by ordered image warping with intrinsic fiducials. We describe an extraocular structure, partly included in Lockwoods ligament, which contains dense elastin and SM bands, and which might refine horizontal eye alignment as a function of vertical gaze, and torsion in down-gaze. This active structure might therefore be a factor in strabismus and a target of therapeutic intervention.

Surgical implications of the rectus extraocular muscle pulleys

PURPOSE Magnetic resonance imaging (MRI) shows that the paths of rectus extraocular muscle bellies remain fixed in the orbit during large ocular rotations, and across large surgical transpositions of their insertions. This stability of muscle paths is due to their passage through pulleys which are coupled to the orbit and located in a coronal plane anterior to the muscle bellies near the equator of the globe. Autopsy studies have shown the pulleys to be fibroelastic sleeves consisting of dense bands of collagen and elastin, suspended from the orbit and adjacent extraocular muscle sleeves by bands of similar composition. Immunohistochemical studies have revealed substantial smooth muscle in the pulley suspensions and in posterior Tenons fascia. The pulleys function as mechanical origins of the rectus extraocular muscles in the sense of determining extraocular muscle pulling directusons. This study was conducted to determine the theoretical effects of the pulleys on the outcome of rectus transposition surgery. METHODS The functional and anatomical evidence for the existence of the rectus extraocular muscle pulleys was reviewed. In two patients, binocular alignment data were collected using the Hess screen test before and after vertical rectus transposition surgeries for lateral rectus paralysis. Paths of the rectus extraocular muscles were determined using high resolution MRI. The OrbitTM 1.5 extraocular biosimulation program was employed to compute theoretical binocular alignment and muscle paths, under alternative conditions including or omitting the pulleys. RESULTS Pulleys are required to account for observed paths of rectus extraocular muscles following transposition surgery. In the absence of pulleys, transposition of the superior and inferior rectus muscles to the lateral rectus insertion for abducens paralysis would result in bizarre ocular misalignments not observed clinically. CONCLUSIONS The human orbit contains specialized musculofibroelastic tissues in and just posterior to Tenons fascia, which serve as pulleys, determining actions of rectus extraocular muscles. These pulleys are located in a roughly coronal plane just posterior to the equator of the globe. Unimpaired pulley function is essential to effective muscle transposition surgery.

Intramuscular Innervation of Primate Extraocular Muscles: Unique Compartmentalization in Horizontal Recti

PURPOSE It has been proposed that the lateral rectus (LR), like many skeletal and craniofacial muscles, comprises multiple neuromuscular compartments subserving different physiological functions. To explore the anatomic potential of compartmentalization in all four rectus extraocular muscles (EOMs), evidence was sought of possible regional selectivity in intramuscular innervation of all rectus EOMs. METHODS Whole orbits of two humans and one macaque monkey were serially sectioned at 10 μm thickness and stained with Massons trichrome. Three-dimensional reconstruction was performed of the intramuscular courses of motor nerves from the deep orbit to the anterior extents of their arborizations within all four rectus EOMs in each orbit. RESULTS Findings concorded in monkey and human orbits. Externally to the global surface of the lateral (LR) and medial rectus (MR) EOMs, motor nerve trunks bifurcated into approximately equal-sized branches before entering the global layer and observing a segregation of subsequent arborization into superior zones that exhibited minimal overlap along the length of the LR and only modest overlap for MR. In contrast, intramuscular branches of the superior and the nasal portion of the inferior rectus were highly mixed. CONCLUSIONS Consistent segregation of intramuscular motor nerve arborization suggests functionally distinct superior and inferior zones within the horizontal rectus EOMs in both humans and monkeys. Reduced or absent compartmentalization in vertical rectus EOMs supports a potential functional role for differential innervation in horizontal rectus zones that could mediate previously unrecognized vertical oculorotary actions.

Compartmentalized Innervation of Primate Lateral Rectus Muscle

PURPOSE Skeletal and craniofacial muscles are frequently composed of multiple neuromuscular compartments that serve different physiological functions. Evidence of possible regional selectivity in LR intramuscular innervation was sought in a study of the anatomic potential of lateral rectus (LR) muscle compartmentalization. METHODS Whole orbits of two humans and five macaque monkeys were serially sectioned at 10-microm thickness and stained with Masson trichrome. The abducens nerve (CN6) was traced anteriorly from the deep orbit as it branched to enter the LR and arborized among extraocular muscle (EOM) fibers. Three-dimensional reconstruction was performed in human and monkey orbits. RESULTS Findings were in concordance in the monkey and human orbits. External to the LR global surface, CN6 bifurcated into approximately equal-sized trunks before entering the global layer. Subsequent arborization showed a systematic topography, entering a well-defined inferior zone 0.4 to 2.5 mm more posteriorly than branches entering the largely nonoverlapping superior zone. Zonal innervation remained segregated anteriorly and laterally within the LR. CONCLUSIONS Consistent segregation of intramuscular CN6 arborization in humans and monkeys suggests functionally distinct superior and inferior zones for the LR. Since the LR is shaped as a broad vertical strap, segregated control of the two zones could activate them separately, potentially mediating previously unappreciated but substantial torsional and vertical oculorotary LR actions.

Effects of Intracranial Trochlear Neurectomy on the Structure of the Primate Superior Oblique Muscle

PURPOSE. Although cyclovertical strabismus in humans is frequently attributed to superior oblique (SO) palsy, anatomic effects of SO denervation have not been studied. Magnetic resonance imaging (MRI) and orbital histology was used to study the effects of acute trochlear (CN4) denervation on the monkey SO. METHODS. Five juvenile macaque monkeys were perfused with formalin for 5 weeks: 15 months after unilateral or bilateral 10-mm intracranial trochlear neurectomy. Denervated and fellow orbits were imaged by MRI, embedded whole in paraffin, serially sectioned at 10-mum thickness, and stained with Masson trichrome. Whole muscle and individual fiber cross sections were quantified in SO muscles throughout the orbit and traced larger fibers in one specimen where they were present. RESULTS. MRI demonstrated marked reduction in midorbital cross section in denervated SO muscles, with anterior shift of SO mass preserving overall volume. Muscle fibers exhibited variable atrophy along their lengths. Denervated orbital layer (OL) fiber cross sections were slightly but significantly reduced from control at most anteroposterior locations, but this reduction was much more profound in global layer (GL) fibers. Intraorbital and intramuscular CN4 were uniformly fibrotic. In one animal, there were scattered clusters of markedly hypertrophic GL fibers that exhibited only sparse myomyous junctions only anteriorly. CONCLUSIONS. CN4 denervation produces predominantly SO GL atrophy with relative OL sparing. Overall midorbital SO atrophy was evident by MRI as early as 5 weeks after denervation, as denervated SO volume shifted anteriorly. Occasional GL fiber hypertrophy suggests that at least some SO fibers extend essentially the full muscle length after trochlear neurectomy.

Characterization of Ocular Tissues Using Microindentation and Hertzian Viscoelastic Models

PURPOSE The authors applied a novel microindentation technique to characterize biomechanical properties of small ocular and orbital tissue specimens using the hertzian viscoelastic formulation, which defines material viscoelasticity in terms of the contact pressure required to maintain deformation by a harder body. METHODS They used a hard spherical indenter having 100 nm displacement and 100 μg force precision to impose small deformations on fresh bovine sclera, iris, crystalline lens, kidney fat, orbital pulley tissue, and orbital fatty tissue; normal human orbital fat, eyelid fat, and dermal fat; and orbital fat associated with thyroid eye disease. For each tissue, stress relaxation testing was performed using a range of ramp displacements. Results for single displacements were used to build quantitative hertzian models that were, in turn, compared with behavior for other displacements. Findings in orbital tissues were correlated with quantitative histology. RESULTS Viscoelastic properties of small specimens of orbital and ocular tissues were reliably characterized over a wide range of rates and displacements by microindentation using the hertzian formulation. Bovine and human orbital fatty tissues exhibited highly similar elastic and viscous behaviors, but all other orbital tissues exhibited a wide range of biomechanical properties. Stiffness of fatty tissues tissue depended strongly on the connective tissue content. CONCLUSIONS Relaxation testing by microindentation is a powerful method for characterization of time-dependent behaviors of a wide range of ocular and orbital tissues using small specimens, and provides data suitable to define finite element models of a wide range of tissue interactions.

Compartmental Innervation of the Superior Oblique Muscle in Mammals

PURPOSE Intramuscular innervation of mammalian horizontal rectus extraocular muscles (EOMs) is compartmental. We sought evidence of similar compartmental innervation of the superior oblique (SO) muscle. METHODS Three fresh bovine orbits and one human orbit were dissected to trace continuity of SO muscle and tendon fibers to the scleral insertions. Whole orbits were also obtained from four humans (two adults, a 17-month-old child, and a 33-week stillborn fetus), two rhesus monkeys, one rabbit, and one cow. Orbits were formalin fixed, embedded whole in paraffin, serially sectioned in the coronal plane at 10-μm thickness, and stained with Masson trichrome. Extraocular muscle fibers and branches of the trochlear nerve (CN4) were traced in serial sections and reconstructed in three dimensions. RESULTS In the human, the lateral SO belly is in continuity with tendon fibers inserting more posteriorly on the sclera for infraducting mechanical advantage, while the medial belly is continuous with anteriorly inserting fibers having mechanical advantage for incycloduction. Fibers in the monkey superior SO insert more posteriorly on the sclera to favor infraduction, while the inferior portion inserts more anteriorly to favor incycloduction. In all species, CN4 bifurcates prior to penetrating the SO belly. Each branch innervates a nonoverlapping compartment of EOM fibers, consisting of medial and lateral compartments in humans and monkeys, and superior and inferior compartments in cows and rabbits. CONCLUSIONS The SO muscle of humans and other mammals is compartmentally innervated in a manner that could permit separate CN4 branches to selectively influence vertical versus torsional action.

Nonclassical innervation patterns in mammalian extraocular muscles.

Purpose: The abducens (CN6) and oculomotor (CN3) nerves (nn) enter target extraocular muscles (EOMs) via their global surfaces; the trochlear (CN4) nerve enters the superior oblique (SO) muscle on its orbital surface. Motor nn are classically described as entering the EOMs in their middle thirds. We investigated EOM innervation that does not follow the classic pattern. Methods: Intact, whole orbits of two humans and one each monkey, cow, and rabbit were paraffin embedded, serially sectioned in coronal plane, and prepared with Masson’s trichrome and by choline acetyltransferase (ChAT) immunohistochemistry. Nerves innervating EOMs were traced from the orbital apex toward the scleral insertion, and some were reconstructed in three dimensions. Results: Classical motor nn positive for ChAT entered rectus and SO EOMs and coursed anteriorly, without usually exhibiting recurrent branches. In every orbit, nonclassical (NC) nn entered each EOM well posterior to classical motor nn. These NC nn entered and arborized in the posterior EOMs, mainly within the orbital layer (OL), but often traveled into the global layer or entered an adjacent EOM. Other NC nn originated in the orbital apex and entered each EOM through its orbital surface, ultimately anastomosing with classical motor nn. Mixed sensory and motor nn interconnected EOM spindles. Conclusions: EOMs exhibit a previously undescribed pattern of NC innervation originating in the proximal orbit that partially joins branches of the classical motor nn. This NC innervation appears preferential for the OL, and may have mixed supplemental motor and/or proprioceptive functions, perhaps depending upon species. The origin of the NC innervation is currently unknown.