Lentiviral vector delivery of short hairpin RNA to NG2 and neurotrophin-3 promotes locomotor recovery in injured rat spinal cord.

BACKGROUND AIMS: In this study we investigated the effect of neurotrophin-3 (NT-3) and knockdown of NG2, one of the main inhibitory chondroitin sulfate proteoglycans (CSPG), in the glial scar following spinal cord injury (SCI). METHODS: Short hairpin (sh) RNA were designed to target NG2 and were cloned into a lentiviral vector (LV). A LV was also constructed containing NT-3. LV expressing NT-3, shRNA to NG2 or combinations of both vectors were injected directly into contused rat spinal cords 1 week post-injury. Six weeks post-injection of LV, spinal cords were examined by histology for changes in scar size and by immunohistochemistry for changes in expression of CSPG, NT-3, astrocytes, neurons and microglia/macrophages. Motor function was assessed using the Basso, Beattie and Bresnahan (BBB) locomotor scale. RESULTS: Animals that received the combination treatment of LV shNG2 and LV NT-3 showed reduced scar size. These animals also showed an increase in levels of neurons and NG2, a decrease in levels of astrocytes and a significant functional recovery as assessed using the BBB locomotor scale at 2 weeks post-treatment. CONCLUSIONS: The improvement in locomotor recovery and decrease in scar size shows the potential of this gene therapy approach as a therapeutic treatment for SCI.

Lentiviral vector-mediated knockdown of the NG2 [corrected] proteoglycan or expression of neurotrophin-3 promotes neurite outgrowth in a cell culture model of the glial scar.

BACKGROUND: Following spinal cord injury, a highly inhibitory environment for axonal regeneration develops. One of the main sources of this inhibition is the glial scar that is formed after injury by reactive astrocytes. The inhibitory environment is mainly a result of chondroitin sulphate proteoglycans (CSPGs). NG2, [corrected] one of the main inhibitory CSPGs, is up-regulated following spinal cord injury. METHODS: Small interfering RNA (siRNA) was designed to target NG2 and this short hairpin RNA (shRNA) was cloned into a lentiviral vector (LV). The neurotrophic factor neurotrophin-3 (NT-3) promotes the growth and survival of developing neurites and has also been shown to aid regeneration. NT-3 was also cloned into a LV. In vitro assessment of these vectors using a coculture system of dorsal root ganglia (DRG) neurones and Neu7 astrocytes was carried out. The Neu7 cell line is a rat astrocyte cell line that overexpresses NG2, thereby mimicking the inhibitory environment following spinal cord injury. RESULTS AND DISCUSSION: These experiments show that both the knockdown of NG2 via shRNA and over-expression of NT-3 can significantly increase neurite growth, although a combination of both vectors did not confer any additional benefit over the vectors used individually. These LVs show promising potential for growth and survival of neurites in injured central nervous system tissue (CNS).

Opening and closing mechanisms of the leatherback sea turtle larynx: a crucial role for the tongue.

A combination of dissection and computed tomography scanning has provided significant novel insights into the structure and function of the Dermochelys coriacea larynx and its associated muscles. Several previously unknown features of the laryngeal aditus (glottis) are described and their functional significance in its opening and closure are considered. The tongue plays an essential part in producing and maintaining closure during dives and feeding bouts. Closure is brought about by compression of the glottis under the action of the two hyoglossus muscles. The tongue thus plays the role of the epiglottis of mammals, sealing the entrance to the larynx. As is already clear, opening is brought about by abduction of the arytenoid cartilages. In addition, there is a powerful mechanism for maintaining the larynx in close apposition to the hyoid plate during feeding and neck flexion, thereby enhancing the efficiency of feeding.

Thoracic paravertebral block using real-time ultrasound guidance.

BACKGROUND: We developed a technique for ultrasound-guided paravertebral block, which was subsequently applied in the clinical setting. METHODS: An initial cadaver study was used to develop a technique that was used in the clinical setting on patients undergoing breast cancer surgery. RESULTS: Paravertebral catheters were correctly placed in the cadaveric trial in 8 of 10 attempts. In the clinical study, all blocked patients (n = 9) had evidence of thoracic wall sensory block and analgesia postoperatively. CONCLUSIONS: Determined by anatomical dissection, we have described the ultrasound features of the thoracic paravertebral space and performed clinically successful ultrasound-guided paravertebral block.

Fat head: an analysis of head and neck insulation in the leatherback turtle (Dermochelys coriacea).

Adult leatherback turtles are gigantothermic/endothermic when foraging in cool temperate waters, maintaining a core body temperature within the main body cavity of ca. 25 degrees C despite encountering surface temperatures of ca. 15 degrees C and temperatures as low as 0.4 degrees C during dives. Leatherbacks also eat very large quantities of cold, gelatinous prey (medusae and pyrosomas). We hypothesised that the head and neck of the leatherback would have structural features to minimise cephalic heat loss and limit cooling of the head and neck during food ingestion. By gross dissection and analytical computed tomography (validated by ground truthing dissection) of an embalmed specimen we confirmed this prediction. 21% of the head and neck was occupied by adipose tissue. This occurred as intracranial blubber, encapsulating the salt glands, medial portions of the eyeballs, plus the neurocranium and brain. The dorsal and lateral surfaces of the neck featured thick blubber pads whereas the carotid arteries and jugular veins were deeply buried in the neck and protected laterally by blubber. The oesophagus was surrounded by a thick sheath of adipose tissue whereas the oropharyngeal cavity had an adipose layer between it and the bony proportion of the palate, providing further ventral insulation for salt glands and neurocranium.

Ontogenetic changes in tracheal structure facilitate deep dives and cold water foraging in adult leatherback sea turtles.

Adult leatherbacks are large animals (300-500 kg), overlapping in size with marine pinniped and cetacean species. Unlike marine mammals, they start their aquatic life as 40-50 g hatchlings, so undergo a 10,000-fold increase in body mass during independent existence. Hatchlings are limited to the tropics and near-surface water. Adults, obligate predators on gelatinous plankton, encounter cold water at depth (<1280 m) or high latitude and are gigantotherms that maintain elevated core body temperatures in cold water. This study shows that there are great ontogenetic changes in tracheal structure related to diving and exposure to cold. Hatchling leatherbacks have a conventional reptilian tracheal structure with circular cartilaginous rings interspersed with extensive connective tissue. The adult trachea is an almost continuous ellipsoidal cartilaginous tube composed of interlocking plates, and will collapse easily in the upper part of the water column during dives, thus avoiding pressure-related structural and physiological problems. It is lined with an extensive, dense erectile vascular plexus that will warm and humidify cold inspired air and possibly retain heat on expiration. A sub-luminal lymphatic plexus is also present. Mammals and birds have independently evolved nasal turbinates to fulfil such a respiratory thermocontrol function; for them, turbinates are regarded as diagnostic of endothermy. This is the first demonstration of a turbinate equivalent in a living reptile.

Hypertonic Saline Attenuates the Pro-metastatic Effects of LPS by Reducing Tumor Cell Migration, Proliferation and MMP-9 Expression.

BACKGROUND: Lipopolysaccharide (LPS) promotes tumor metastases. The aim of this study was to determine the ability of a hypertonic environment to attenuate the pro metastatic properties of LPS both in vitro, and in vivo. METHODS: LPS stimulated, and unstimulated, 4T1 tumor cells were cultured in either an isotonic or hypertonic environment. The effect on invasion, migration, pro-matellomatrixproteinase 9 (proMMP-9) expression, proliferation, and microscopic cell structure was assessed. Lung metastases were induced in C57 mice with systemic hypertonicity in unstimulated and stimulated mice. The metastatic burden was assessed by estimation of lung/body weight ratio, pleural nodules and clonogenic assay. RESULTS: In vitro, a hypertonic environment reduced proMMP-9 expression (0.012 versus 1.16, P < 0.001) invasion (0.06 versus 0.119, P = 0.005), tumor cell proliferation (0.035 versus 0.041, P = 0.001), while inducing structural changes to tumor cells reducing overall cell volume. In vivo, the induction of transient systemic hypertonicity reduced metastatic burden as demonstrated by reduced lung nodules (4 versus 8, P = 0.004) and colonies on clonogenic assay (12 versus 43, P = 0.04). CONCLUSION: The in vitro exposure of tumor cells to a hypertonic environment reduces tumor cell migration and proliferation. Transient systemic hypertonicity can reduce the metastatic burden following intra-operative exposure to LPS in vivo.

Training tomorrow's anatomists today: a partnership approach.

Anatomy is recognized to play a central role in the education and training of clinicians, healthcare professionals, and scientists. However, in recent years, the perceived decline in popularity of anatomy has led to a deficiency in the numbers of new anatomy educators. The tide is now turning with anatomy once again taking its rightful place in a wide of variety of disciplines, and therefore it is imperative that a new generation of anatomists is in place to meet this need. In response, the Anatomical Society of Great Britain and Ireland has made the training of the next set of anatomists, one of its strategic priorities, and in collaboration with the American Association of Anatomists has developed a dedicated Training Program. The overall aim of the Program is to provide trainees with the necessary knowledge, understanding, aptitudes, and attitudes in appropriate detail, sufficient to enable them to teach and examine Anatomy with full competence at the undergraduate and postgraduate level. The Program offers opportunities to consolidate knowledge and deepen understanding of anatomy, improve skills in teaching and communication with students, and be competent in preparing teaching materials and assessment modalities. The Program uses a distance-learning approach with an incorporated Residential School and is particularly aimed at those undertaking a career in the biosciences. Early indications suggest that initiatives such as the development of this Training Program will help deliver the next generation of anatomists and ensure that anatomy continues to play a fundamental role in the education of clinicians, healthcare professionals, and scientists.

Bilateral abducent and facial nerve palsies following fourth ventricle shunting: two case reports.

CASE REPORTS: Treatment of isolated fourth ventricle syndrome is difficult and there is no widely agreed method. Fourth ventriculo-peritoneal shunting is the most commonly utilized procedure for the management of this syndrome. Complications from shunting are common and are usually related to malfunction, infection, dislocation and overdrainage. We present two unusual cases in which both patients developed bilateral abducens and facial nerve palsies following shunting of an isolated fourth ventricle. Magnetic resonance imaging (MRI) in both cases revealed collapse of the fourth ventricles with downward displacement of the brain stem. In the first case the trans-tentorial pressure difference was equilibrated with the aid of a "Y" connector between the supratentorial and infratentorial shunts, with full recovery of the neurological deficits; in the second case this approach failed and following a complicated neurosurgical course successful endoscopic aqueductal stenting was performed. DISCUSSION: Pathogenesis of cranial nerve palsies following fourth ventricle shunting and the rationale of treatment are discussed and the literature is reviewed.

Axons and glial interfaces: ultrastructural studies.

At most vertebrate nerve transitional zones (TZs) there is a glial barrier which is pierced by axons passing between the CNS and PNS. Myelinated axons traverse this in individual tunnels. The same is true of larger non-myelinated axons. This holds widely among the vertebrates, for example, the large motor axons of the sea-lamprey Petromyzon (which also possess TZ specializations not found in mammals). Smaller non-myelinated axons traverse the TZ glial tunnels as fascicles and so the barriers are correspondingly less comprehensive for them. Accordingly, in nerves composed of non-myelinated axons, such as the vomeronasal or the olfactory, a TZ barrier stretching across the nerve is effectivelyabsent. The chordateAmphioxus differsfrom the vertebrates in lacking a TZ barrier throughout. Invertebrates also lack glial barriers at the TZs between ganglia and interconnecting nerve trunks. The glial barrier at the dorsal spinal root TZ (DRTZ) has considerable value for analysing protocols aimed at achieving CNS regeneration, because it provides a useful model of the gliotic reaction at sites of CNS injury. Also, it is especially amenable to morphometric analysis, and so enables objective quantification of different protocols. Being adjacent to the subarachnoid space, it is accessible for experimental intervention. The DRTZ was used to investigate the value of neurotrophin 3 (NT3) in promoting axon regeneration across the TZ barrier and into the CNS following dorsal root crush. It promoted extensive regeneration and vigorous non-myelinated axonal ensheathment. On average, around 40% of regenerating axons grew across the interface, compared with virtually none in its absence. These may have traversed the interface through loci occupied by axons prior to degeneration. Many regenerating axons became myelinated, both centrally and peripherally.

The developing cervical spinal ventral commissure of the rat: a highly controlled axon-glial system.

The floor plate of the neural tube is of major importance in determining axonal behaviour, such that, having crossed, decussating axons do not cross back again. The ventral commissure (VC) of the spinal cord forms immediately ventral to the floor plate shortly after neural tube closure. It is the principal location in which decussating axons cross the midline. It is probably also of major importance in neural tube development, but has received relatively little attention. This study analyses the growth and development of the rat VC and also axon-glial relationships within it throughout the crucial prenatal period of extensive transmedian axon growth, when key biochemical interactions between the two tissues are taking place. The morphometric, stereological and immunohistochemical methods used show that the axonal and glial populations remain in a finely balanced equilibrium throughout a period of almost a hundred-fold growth of both elements. At all stages axons are highly segregated into small bundles of constant size by glial processes, to which they are closely apposed. Thus, glial-axon contact is remarkably precocious, uniquely intimate and persists throughout VC development. This suggests that the relationship between the two tissues is highly controlled through interactions between them. The VC is likely to be the physical basis of a second set of glial-axonal interactions, namely, those which are well known to influence axon crossing behaviour. In mediating these, the extensive axon-glial contact is an ideal arrangement for molecular transfer between them, and is probably the substrate for altering axon responsiveness and ensuring reliable transmedian decussation. The VC is therefore a segregating matrix temporally and spatially specialised for a range of key developmental axon-glial interactions.

Neurotrophin-3-mediated regeneration and recovery of proprioception following dorsal rhizotomy.

Injured dorsal root axons fail to regenerate into the adult spinal cord, leading to permanent sensory loss. We investigated the ability of intrathecal neurotrophin-3 (NT3) to promote axonal regeneration across the dorsal root entry zone (DREZ) and functional recovery in adult rats. Quantitative electron microscopy showed robust penetration of CNS tissue by regenerating sensory axons treated with NT3 at 1 and 2 weeks postrhizotomy. Light and electron microscopical anterograde tracing experiments showed that these axons reentered appropriate and ectopic laminae of the dorsal horn, where they formed vesicle-filled synaptic buttons. Cord dorsum potential recordings confirmed that these were functional. In behavioral studies, NT3-treated (but not untreated or vehicle-treated) rats regained proprioception. Recovery depended on NT3-mediated sensory regeneration: preventing regeneration by root excision prevented recovery. NT3 treatment allows sensory axons to overcome inhibition present at the DREZ and may thus serve to promote functional recovery following dorsal root avulsions in humans.