Ovine Hemisection Model of Spinal Cord Injury.

INTRODUCTION: We are developing ovine models of spinal cord injury to test novel neuromodulation-based methods on spasticity. The hemisection has been reported in a number of large animal studies. Our aim is to duplicate a hemisection injury in the sheep. Our effort is explored here. Methods and Results: Three sheep underwent hemi-sectioning of the spinal cord. Quantitative gait analysis was completed both pre- and post-injury. While measurable differences in most of the 20 gait metrics were observed, relatively few were above the predicted thresholds based on error levels expected from the data. Variations in severity of injury across the three sheep were observed. Conclusions: The hemisection ovine model of spinal cord injury shows promise as a large-animal platform for developing new therapies for treating spinal cord injuries. While variability in injury severity was observed across animals, as has been observed with weight drop-based SCI models, the hemi-section approach has the advantages of procedural ease and reduced technical complexity.

The Hemisection Approach in Large Animal Models of Spinal Cord Injury: Overview of Methods and Applications.

Introduction: Translating basic science research into a safe and effective therapy for spinal cord injury (SCI) requires suitable large animal models for testing both implantable devices and biologic approaches to better approximate human anatomy and function. Hemisection lesions, routinely used for investigational purposes in small animals, are less frequently described in large animals that might be appropriate for translational studies. Size constraints of small animals (mice and rats) limits the predictability of the findings when scaled up. Our goal is to review the status of hemisection SCI in large animals across species and time to prepare for the testing of a novel intradural spinal cord stimulation device for control of spasticity in an ovine model. Methods and Results: We surveyed the literature on hemisection in quadrupeds and nonhuman primates, and catalogued the species, protocols and outcomes of the experimental work in this field. Feline, lapine, canine, simian, porcine, ovine and bovine models were the primary focal points. There is a consistent body of literature reporting use of the hemisection approach in large animals, but with differences in surgical technique depending on the goals and nature of the individual studies. While the injuries are not always consistent, the experimental variability is generally lower than that of the contusion-based approach. In general, as the body size of the animal increases, animal care requirements and the associated costs follow. In most cases, this is inversely correlated with the number of animals used in hemisection models. Conclusions: The hemisection approach to modeling SCI is straightforward compared with other methods such as the contusive impact and enables the transection of isolated ascending and descending tracts and segment specific cell bodies. This has certain advantages in models investigating post-injury axonal regrowth. However, this approach is not generally in line with the patho-physiologies encountered in SCI patients. Even so, the ability to achieve more control over the level of injury makes it a useful adjunct to contusive and ischemic approaches, and suggests a useful role in future translational studies.

Properties and mechanisms of drug release from matrix tablets containing poly(ethylene oxide) and poly(acrylic acid) as release retardants.

The interactions between poly(ethylene oxide) (PEO) and poly(acrylic acid) (PAA) in aqueous medium at pH 6.8 were investigated in the current study. We have also studied the effect of interpolymer interactions and various formulation variables, including the molecular weight of PEO, the ratio between PEO and PAA, the crystallinity of PEO, and the presence of an acidifying agent, on the release of theophylline from matrix tablets containing both PEO and PAA as release retardants. At pH 6.8, the synergy in solution viscosity between PEO and PAA as the result of ion-dipole interaction was observed in this study. The release of theophylline from the matrix tablets containing physical mixtures of PEO and PAA was found to be a function of dissolution medium pH because of the pH-dependent interactions between these two polymers. Because of the formation of water insoluble interpolymer complex between PEO and PAA in aqueous medium at pH below 4.0, the release of theophylline was independent of PEO molecular weight and was controlled by Fickian diffusion mechanism in 0.01N hydrochloric acid solution. In comparison, the drug release was a function of PEO molecular weight and followed the anomalous transport mechanism in phosphate buffer pH 6.8. The presence of PAA exerted opposite effects on the release of theophylline in phosphate buffer pH 6.8. In one aspect, theophylline release was accelerated because the erosion of PAA was much faster than that of PEO at pH6.8. On the opposite aspect, theophylline release was slowed down because of the formation of insoluble complex inside the gel layer as the result of the acidic microenvironment induced by PAA, and the increase in the viscosity of the gel layer as the result of the synergy between PEO and PAA. These two opposite effects offset each other. As a result, the release of theophylline remained statistically the same even when 75% PEO in the formulation was replaced with PAA. In phosphate buffer pH 6.8, the release of theophylline was independent of the crystalline form of PEO. The release profile remained identical whether PEO was present as a semicrystalline powder blend with PAA or an amorphous complex with PAA in the matrix tablets. It has also been observed that the presence of citric acid as an acidifying agent had negligible effect on the drug release rate.

Spermiogenesis and sperm ultrastructure in Troglocephalus rhinobatidis, Neoheterocotyle rhinobatidis and Merizocotyle australensis (Platyhelminthes, Monogenea, Monopisthocotylea, Monocotylidae).

The ultrastructural events of spermiogenesis and the ultrastructure of nature sperm of 3 species of monocotylid monogeneans are described. Two of these species, Troglocephalus rhinobatidis and Neoheterocotyle rhinobatidis are extremely similar in all the aspects studied, and their placement in separate subfamilies is questioned. Evidence is presented in both species for extensive distalward movement during spermiogenesis of an ornamented region associated with cortical microtubules, originally located in the zone of differentiation. Spermatids of Merizocotyle australis lack this ornamentation and the mature sperm also lacks cortical microtubules. Troglocephalus rhinobatidis exhibits the highest number (128) of spermatids in an isogenic group recorded for a flatworm to date. Sperm of all 3 species have 2 normal axonemes, shifted slightly relative to one another. Comparative data are presented on sperm and spermiogenesis of all monocotylids examined to date and the phylogenetic implications are discussed.

Spermatogenesis in Udonella (Platyhelminthes, Udonellidea) and the phylogenetic position of the genus.

Spermatids are joined (cytophores). Two centrioles lateral and/or proximal to the nucleus form axonemes, arranged parallel with each other, that extend into a short distal cytoplasmic process. The nucleus elongates, and mitochondria fuse to form a single elongate mitochondrion. The part of the spermatid containing the axonemes, nucleus and mitochondrion elongates to a thread-like spermatozoon, surrounded at its base (at the level near the basal bodies) by an arching membrane where the sperm detaches from the residual cytoplasm. At no stage are there free flagella (except for short free flagellar tips), an intercentriolar body or a median cytoplasmic process. A few peripheral microtubules and a compact dense body, not membrane bound and closely associated with the mitochondrion, were seen in spermatids. Spermiogenesis in Udonella is interpreted as derived from proximo-distal fusion by a shifting of the basal bodies deeper into the cytoplasm and a consequent loss of the median cytoplasmic process. Both the ultrastructure of the protonephridia and spermiogenesis indicate that Udonella does not represent the sister group of all other neodermatans, but is a secondarily modified "advanced" neodermatan. Lack of convincing synapomorphies indicates that the taxon Cercomeridea as a defined by Brooks (1989a, b) is invalid.

Ultrastructure of the buccal complex of Polylabroides australis (Monogenea, Polyopisthocotylea, Microcotylidae).

The two buccal suckers have a largely muscular septum each and, near the pharynx, dorsal swellings ("valve apparatus") extend into the lumen of the suckers. In the posterior parts of the suckers, the tegument forms a "rim" containing much electron-dense material. The walls of the suckers are mainly formed by muscle filaments, and there are several large vacuolated masses containing lipid (?) globules and vacuoles. The most anterior part of the pharynx contains a thick layer of electron-dense material which also lines the pharyngeal wall facing the lumen. Nine types of sensory receptors, all with electron-dense collars, were observed in the tegument near the suckers, in the mouth cavity and its extensions, and in the anterior part of the pharynx. Some are uniciliate, one is biciliate, one multiciliate, and two (including the pharyngeal receptors) are nonciliate. The greatest variety of receptors, including receptor complexes consisting of three receptor types, is found in a posterior ventral extension of the mouth cavity. It is suggested that this extension can be protruded into the suckers, facilitated by numerous muscle filaments in the tissue around it.--Septate suckers, mouth cavity and its extension, valve apparatus and pharynx are likely to form a functional whole, the "buccal complex", used for food intake.

Re-examination of spermatogenesis of Multicotyle purvisi (Platyhelminthes, Aspidogastrea).

Examination of spermatogenesis in Multicotyle purvisi revealed that the process follows the classical pattern previously described for many parasitic platyhelminths, especially other Aspidogastrea and Digenea. Free flagella (with an intercentriolar body between the basal bodies and with striated flagellar rootlets) and a median cytoplasmic process grow from a zone of differentiation. The flagella then fuse with the process in a proximo-distal direction and nucleus and mitochondrion grow out into the elongating spermatid. Spermatids are pinched off at the base, and rootlets and intercentriolar body remain behind in the residual cytoplasm. It is suggested that the aberrant processes described in an earlier report constitute a pathological condition or a response to stress. Changes in the intercentriolar body during spermiogenesis are documented and for the first time in a platyhelminth it is demonstrated that rootlets and intercentriolar bodies are discarded and left behind in the residual cytoplasm.

Ultrastructure of sperm and spermiogenesis in the monocotylid monogeneans Monocotyle helicophallus and Calicotyle australiensis (Platyhelminthes).

Spermiogenesis in Monocotyle helicophallus involves formation of free flagella which rotate to lie parallel before fusing. There is no median cytoplasmic process. A small number of microtubules (usually 2-4) is associated with the axonemes in the zone of differentiation. Nucleus and a fused mitochondrion migrate alongside the axonemes and one basal body moves distally. A coil of nucleus, originally in the cytophore, also moves out along the shaft. The mature spermatozoan has only a single axoneme for a short distance at each end, with two associated microtubules at the proximal end, as well as one axoneme, nucleus and mitochondrion throughout most of its length (the nucleus being enlarged and roughly coiled in one region), and a short region of two overlapping axonemes. We interpret our findings as two axonemes arranged almost end to end, one extending from the proximal end to some point in the mid-region, where the second axoneme begins and continues to the distal end of the sperm. Spermatozoa of Calicotyle australiensis develop from a zone of differentiation which has two basal bodies and a complete ring of cortical microtubules. Two initially free axonemes fuse with each other and there is no median cytoplasmic process. Spermatids form within parallel canals in the cytophore, by backward movement of the zone of differentiation. Prior to detachment, an electron-dense spiral end-piece forms around and proximal to the basal body region. Sixty-four spermatids are present in each isogenic group. With the study of spermiogenesis in more species of Monogenea Monopisthocotylea, it is apparent that the previously designated sperm patterns 2 and 3 are not distinct and should be combined and re-defined. Species previously designated as having sperm patterns 2 and 3 can all be accommodated by the description "two normal axonemes or one normal and one shortened, altered or displaced axoneme, and none, one or a few cortical microtubules remaining in a region of the sperm derived from the zone of differentiation in which a few or a complete ring of microtubules was present".

Ultrastructure of sperm and spermatogenesis of Lobatostoma manteri (Trematoda, Aspidogastrea).

Mature sperm has two axonemes of the 9 + '1' pattern incorporated in the sperm body, a row of peripheral microtubules interrupted along part of the sperm by the axonemes, some microtubules in the interior of the sperm and a long lateral extension (lobe) of the sperm body, an elongate nucleus and mitochondrion, and many dense rod-like structures. A supporting rod extends underneath a specialized region consisting of alternating thin and thick transverse rows of irregular dense patches, and with surface ridges around (all or) most of the surface of the sperm. Primary spermatocytes in the prophase of the first meiotic division have synaptonemal complex(es), and are rich in mitochondria. In early spermiogenesis, mitochondria are arranged around the surface of the nucleus, a dense layer appears at one pole of the nucleus, close to an apposed dense layer at the cell membrane in which a row of microtubules develops. The intercentriolar (= central) body develops close to the nucleus. The fully developed intercentriolar body has a regular striation and is located perpendicular and close to the surface of the nucleus. Two flagella extend into the space surrounding the outgoing median process, their basal bodies are located perpendicular to the intercentriolar body and their cross-striated rootlets extend along the surface of the rounded nucleus. At a later stage, rootlets and flagella become more parallel with the intercentriolar body, the nucleus and the fused mitochondria migrate into the median process, and the flagella become incorporated into the median process (= sperm body). The outgrowing spermatozoa are connected to the cytoplasm of the cytophore by dense arching membranes. Finally, rootlets of flagella are resorbed and the spermatozoa are pinched off close to the basal bodies. Two species (Lobatostoma and Multicotyle) of the same family differ strongly in the type of spermiogenesis, although their mature sperm is of the same basic type, i.e. spermiogenesis is not necessarily more useful for phylogenetic considerations than sperm structure.

Ultrastructure of the protonephridial system, of Anoplodiscus cirrusspiralis (Monogenea Monopisthocotylea).

The flame bulb is formed by a terminal cell and a proximal canal cell. The weir consists of interdigitating ribs all of which form one circle, i.e. alternating ribs do not have distinctly 'internal' or 'external' positions. Cytoplasmic cords are absent and all ribs, i.e. those continuous with the proximal canal cell and those continuous with the terminal cell, form external leptotriches. At least some external leptotriches have interconnected branches extending along the flame bulb. Internal leptotriches are not branched and arise from the basal perikaryon of the terminal cell. In the cytoplasmic cylinder at the tip of the flame bulb, structures resembling incomplete septate junctions were seen. However, neither the cytoplasmic cylinder nor the small protonephridial capillaries contain complete septate junctions as found in all other Monogenea Polyopisthocotylea, Monogenea Monopisthocotylea, Trematoda Aspidogastrea and Trematoda Digenea examined to date. In the lack of a septate junction, Anoplodiscus resembles Udonella, Amphilinidea, Gyrocotylidea and Eucestoda. However, the presence in this species of rudimentary septate junctions in the small capillaries and of complete junctions in larger ones indicates that complete junctions have been secondarily lost. Anoplodiscus resembles the Monogenea and Trematoda in the presence of lamellae in the larger protonephridial ducts. For the first time in a monogenean, the ultrastructure of the excretory bladder is described. A nucleated convoluted duct opens through a narrow connecting duct into the bladder, which in turn opens through a narrow connecting duct into the excretory pore lined by tegument. Convoluted duct, connecting ducts and bladder are lined by a lamellated epitheliu.(ABSTRACT TRUNCATED AT 250 WORDS)

Ultrastructure of the eyes of the larva of Neoheterocotyle rhinobatidis (Platyhelminthes, Monopisthocotylea), and phylogenetic implications.

The oncomiracidium of Neoheterocotyle rhinobatidis (Monogenea, Monopisthocotylea, Monocotylidae) has two pairs of eyes, each eye with a lens and pigment cup. The anterior eyes have a single rhadomere; the posterior ones, two rhadomeres. Lenses are part of the pigment cup cells, as indicated by cytoplasmic connections between them and the pigment cups, and they are of mitochondrial origin because mitochondrial cristae are present in the periphery of the lenses. This is the first time that mitochondrial lenses have been shown to exist in a neodermatan. Such lenses may be a synapomorphy of a large taxon comprising the Neodermata and its turbellarian sister groups, or they may have evolved convergently in several not closely related groups as a result of strong selection pressure to find a suitable habitat or host.

Symbioses in Amphipholis squamata (Echinodermata, Ophiuroidea, Amphiuridae): geographical variation of infestation and effect of symbionts on the host's light production.

Populations of the polychromatic and bioluminescent species Amphipholis squamata from eight locations were examined for internal and external symbionts. At three locations (two in the United Kingdom and one in Papua New Guinea), no symbionts were present, while four species were recovered from the remaining locations: Cancerilla tubulata and Parachordeumium amphiurae (copepods), Rhopalura ophiocomae (orthonectid) and an undescribed species of rhabdocoel turbellarian. No ophiuroid individual hosted more than one symbiont species, despite the presence of two or more within a population. Symbiont presence and prevalence varied with location, and with colour variety, but with no apparent pattern or trends. Light-production characteristics of the host were affected by the presence of all symbionts except C. tubulata. These effects, however, did not vary between colour varieties or between geographical locations, but were specific to the symbiont species: the presence of P. amphiurae resulted in enhanced intensity of light production, while that of R. ophiocomae and the turbellarian species resulted in reduced intensity. The kinetics of light production (time until maximum output) were altered only by the presence of the turbellarian. Changes in the light-production characteristics are discussed in relation to morphological, energetical and physiological effects of the symbioses.

Contributions to the phylogeny of Platyhelminthes based on partial sequencing of 18S ribosomal DNA.

Partial sequencing of the 18S ribosomal DNA gene of one nemertean and 13 free-living and parasitic Platyhelminthes (556 nucleotides), and of one nemertean and 20 Platyhelminthes (556 nucleotides) was used to test several hypotheses concerning the phylogenetic relationships of Platyhelminthes. The following conclusions were reached: the Neodermata is monophyletic; Trematoda (Aspidogastrea and Digenea) is monophyletic, although a sister group relationship of the Aspidogastrea and all other Neodermata cannot be definitely ruled out; the Cestoda comprising the Eucestoda, Amphilinidea and Gyrocotylidea is monophyletic; it is unresolved whether the Monogenea is paraphyletic; neither Gyrocotylidea and Monopisthocotylea nor Gyrocotylidea and Monogenea as a whole are sister groups; Anoplodiscus is a monopisthocotylean monogenean; none of Proseriata, Pterastericolidae/Umagillidae, Kalyptorhynchia, Rhabdocoela as a whole, Dalyelliida or the Temnocephalidae is the sister group of the Neodermata; there is some evidence that a larger taxon consisting of Proseriata, Tricladida and Rhabdocoela may be the sister group of the Neodermata but definitive evidence for a sister group relationship between the Neodermata and any turbellarian taxon is lacking; Rhabdocoela and Lecithoepitheliata are not closely related; it is unresolved whether the Rhabdocoela is monophyletic; Umagillidae, Pterastericolidae and Temnocephalidae belong to one monophylum; the Temnocephalidae are very close to the dalyelliids; Tricladida and Rhabdocoela are sister groups, the exact position of the Catenulida and Nemertini in relation to the Platyhelminthes has not been resolved, although Catenulida and Lecithoepitheliata may belong to one clade.

Supradiaphragmatic middle aorta syndrome--MR and angiographic imaging.

We describe the case of an asymptomatic 24-year-old man with hypertension who was investigated for aortic coarctation but found on MR scanning to have narrowing of the distal thoracic aorta. Stenosis of the thoracolumbar aorta--the Middle Aorta Syndrome--is rare and is usually found below the diaphragm. The MRI and angiographic findings are presented.

Inflammatory response and antigen localization following immunization with influenza virus ISCOMs.

The inflammatory response, antigen retention, and antigen localization was studied in mice after immunization with influenza virus glycoproteins presented in two physically defined forms--micelles and ISCOMS (immunostimulating complexes). Two hours after intraperitoneal injection, the proportion of polymorphonuclear leukocytes (PMNs) in peritoneal lavage cells increased from less than 1% to 82% in ISCOM-treated mice and from less than 1% to 41% of the total cell count in micelle-treated mice. For both treatment groups, the proportion of PMNs returned to around zero 24 h postimmunization. Total recovery of radioactive antigen was significantly greater (P less than 0.05) in ISCOM-treated than in micelle-treated mice at one, two, and eight days postinjection. At all times tested, animals given ISCOMs had significantly more radioactive antigen in their spleens than animals given micelles. By electron microscopy ISCOMs were found to attach externally to the plasma membrane or within phagosomes of macrophages in close association with the membranes.

Mechanism of the burst fracture in the thoracolumbar spine. The effect of loading rate.

STUDY DESIGN: Calf lumbar spine motion segments were randomly assigned to two groups. After insertion of a transducer capable of measuring transient occlusion of the spinal canal during impact, a low rate axial impact was applied in one group and a high rate load in the other. Post-injury computed tomography scans and peak canal occlusions were measured to determine the effect of rate of load application on occlusion of the spinal canal. OBJECTIVES: This study was designed to determine if for the same direction of impact and total energy delivered, occlusion of the spinal canal postvertebral fracture was related to the rate at which the impact was delivered (time from zero to peak load). SUMMARY OF BACKGROUND DATA: Several reports based on clinical observations have hypothesized that axial burst fractures, which displace bone fragments into the canal, occur because of internal pressurization and explosion of the vertebral body. The extent of bursting of the vertebra may depend on the rate of pressurization of the body, which could be related to the rate at which the load is applied. METHOD: Using calf lumbar spines, a transducer was placed within the spinal canal, after removal of the cord, to measure canal occlusion during impact. One group received axial compressive impacts at a mean loading rate of 400 msec (zero to peak load) using a materials-testing machine. The energy of failure was determined and used to select a drop weight and distance for the high loading rate tests, which would yield equivalent impact energy. The second group received impacts at a loading rate of of 20 msec. The post-injury radiographs and canal occlusion measurements were compared. RESULTS: The same mean energy of impact was used in the fractures for both groups. Post-injury radiographs of the low loading rate group showed compressive fractures with a mean canal occlusion of 6.84%, whereas the high loading rate group had burst fractures with mean canal encroachment of 47.6% (P = 0.0007). CONCLUSIONS: For the same energy and direction of impact, a high impact loading rate produces fractures with significant canal encroachment, whereas minimal encroachment is seen for fractures produced at a low loading rate.

The effect of post-injury spinal position on canal occlusion in a cervical spine burst fracture model.

STUDY DESIGN: The canal space of burst-fractured, human cervical spine specimens was monitored to determine the extent to which spinal position affected post-injury occlusion. OBJECTIVE: To test the null hypothesis that there is no difference in spinal canal occlusion as a function of spinal positioning for a burst-fractured cervical spine model. SUMMARY OF BACKGROUND DATA: Although previous studies have documented the effect of spinal positioning on canal geometry in intact cadaver spines, to the authors' knowledge, none has examined this relationship specifically in a burst fracture model. METHODS: Eight human cervical spine specimens (levels C1 to T3) were fractured by axial impact, and the resulting burst injuries were documented using post-injury radiographs and computed tomography scans. Canal occlusion was measured using a custom transducer in which water was circulated through a section of flexible tygon tubing that was passed through the spinal canal. Any impingement on the tubing produced a rise in fluid pressure that was monitored with a pressure transducer. Each spine was positioned in flexion, extension, lateral (and off-axis) bending, axial rotation, traction, and compression, while canal occlusion and angular position were monitored. Occlusion values for each position were compared with measurements taken with the spine in neutral position. RESULTS: Compared with neutral position, compression, extension, and extension combined with lateral bending significantly increased canal occlusion, whereas flexion decreased the extent of occlusion. In extension, the observed mechanism of occlusion was ligamentum flavum bulge caused by ligament laxity resulting from reduced vertebral body height. CONCLUSIONS: Increased compression of the spinal cord after injury may lead to more extensive neurologic loss. This study demonstrated that placing a burst-fractured cervical spine into either extension or compression significantly increased canal occlusion as compared with occlusion in a neutral position.