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Intramuscular Injections Along the Motor End Plates: A Minimally Invasive Approach to Shuttle Tracers Directly into Motor Neurons

Diseases affecting the integrity of spinal cord motor neurons are amongst the most debilitating neurological conditions. Over the last decades, the ...

University of New South Wales

The purpose of this study was to examine the effects of the epithelium on processes involved in stromal wound healing. Lamellar epithelial-stromal flaps were produced in rabbit corneas with a microkeratome. Peripheral corneal epithelial tissue, central corneal epithelial tissue, or no epithelial tissue (control) was introduced beneath the flap. Corneas were removed at time points from 4 hr to 1 month after surgery. Tissue sections were analyzed with immunocytochemistry for Keratin 3 (K3) to detect epithelial antigen, terminal deoxyribonucleotidyl transferase-mediated dUTP-digoxigenin nick-end labelling (TUNEL) assay to detect apoptosis, immunocytochemistry for Ki67 to detect cell proliferation, and immunocytochemistry for alpha-smooth muscle actin (SMA) to detect myofibroblasts. K3 was detected at the level of the interface from 4 hr to 1 month after surgery in corneas in which epithelial tissue was introduced, but not control corneas, with the exception of one that developed epithelial in growth. Keratocyte apoptosis was significantly higher at 4 hr after flap formation in both groups in which corneal epithelial tissue was introduced beneath the flap compared with controls. Keratocyte proliferation was significantly greater at 72 hr in corneas in which epithelial tissue was introduced beneath the flap compared to the controls. Corneas in which epithelial tissue was introduced into the interface, but not control corneas, had stromal cells expressing alpha-SMA in the stroma anterior and posterior to the interface at 1 week and 1 month after surgery. This was also noted in the control cornea in which there was epithelial ingrowth. Signals derived from the corneal epithelium promote keratocyte apoptosis. Keratocyte proliferation is higher in corneas that have lamellar surgery when epithelial tissue is introduced into the interface. Epithelium-derived signals also participate in the generation and/or maintenance of myofibroblasts in the corneal stroma.

Effect of ectopic epithelial tissue within the stroma on keratocyte apoptosis, mitosis, and myofibroblast transformation.

Development of gene transfer methods that can precisely deliver therapeutic genes to the localized or targeted tissue(s) would be highly beneficial in developing new gene therapy approaches and may also extend animal models for studying in vivo gene function and regulation at molecular levels in the selected tissues. We investigated lipid- and AAV-mediated gene transfer in rabbit cornea using a lamellar flap-technique. The goals of this study were to (1) analyze methods for in situ gene transfer into keratocytes, (2) identify efficient and suitable vectors for gene transfer into keratocytes, and (3) characterize times of first detectable expression, localization and duration of transgene expression in keratocytes with different vectors. A lamellar flap was produced in the rabbit cornea with a microkeratome. Recombinant adeno-associated viral vector (rAAV) expressing either beta-galactosidase (rAAV-beta-gal) or chloramphenicol acetyltransferase (rAAV-CAT) reporter genes, or plasmid-cationic lipid complexes expressing CAT (pMP6-CAT) or beta-galactosidase (pTR-beta-gal) were applied beneath the lamellar flap for two minutes. The flap was repositioned and eyelids sutured overnight. Corneas were removed at 4hr, 12hr, 36hr, 3 days, 7 days, or 10 days after application and either fixed in 2% formaldehyde, cryosectioned and stained for beta-galactosidase activity or homogenized and measured for CAT levels by ELISA. Corneas infected with rAAV-beta-gal vector showed positive beta-gal staining in the center and periphery of the flap interface in whole corneas and corneal beds at 3, 7, and 10 days, but not at earlier time points. Corneas treated with pTR-beta-gal plasmid vector showed positive beta-gal expression at the interface at 4, 12 and 36hr, but not at 3 or 7 days. The posterior surface of the lamellar interface where the vector was applied showed more expression than the overlying anterior surface with both plasmid and viral vectors. The level of gene expression was less with plasmid vector than viral vector monitored using beta-gal staining. CAT-ELISA confirmed expression of the CAT reporter gene with either the plasmid or rAAV vector. These results demonstrate that foreign genes can be introduced into keratocytes with plasmid or viral vectors using a lamellar flap to gain access to the stroma. The expression profile of the reporter genes depended on the vector. Transfection of keratocytes with plasmid vectors produced rapid expression of the reporter genes, but for a short duration. Reporter gene expression following transduction by rAAV vector was delayed several days, but was at higher levels and for a longer duration. This is the first report to demonstrate selective gene transfer into keratocytes and would be highly useful in studying function and regulation of genes in vivo and may eventually furnish a tool for the treatment of corneal dystrophies.

Gene transfer into rabbit keratocytes using AAV and lipid-mediated plasmid DNA vectors with a lamellar flap for stromal access.

The aim of this study was to semi-quantitatively analyze stromal cell apoptosis, stromal cell proliferation, and myofibroblast generation over time points from 4hr to 3 months in rabbit eyes having photorefractive keratectomy (PRK) or laser in situ keratomeliusis (LASIK). Stromal cell necrosis and inflammatory cell infiltration were also studied. PRK for low myopia (-4.5diopters [D]), PRK for high myopia (-9.0D), and LASIK for high myopia (-9.0D) were performed in rabbit eyes, and corneas were obtained for examination at 4, 24, and 72hr, 1 and 4 weeks, and 3 months after surgery. A total of 144 rabbits were included in the study. Stromal cell apoptosis, proliferation, and myofibroblast generation were evaluated semi-quantitatively by TUNEL assay, immunocytochemical analysis of Ki67, and immunocytochemical analysis of alpha-smooth muscle actin, respectively. Stromal cell necrosis and characteristics of other cell types in the stroma were evaluated by electron microscopy. Keratocyte apoptosis and the subsequent proliferation and generation of myofibroblasts were qualitatively and quantitatively different in PRK for high myopia compared to either PRK for low myopia or LASIK for high myopia. Stromal cell necrosis becomes a significant form of cell death by 24hr after injury and may involve corneal fibroblasts, myofibroblasts, and inflammatory cells. Large numbers of polymorphonuclear cells and monocytes invade the cornea by 24hr after surgery and persist for over 1 week. The qualitative and quantitative differences in the cellular wound healing response after PRK for high and low myopia and LASIK for high myopia are likely determinants of the clinical differences in refractive outcome and some of the complications, such as regression and haze, seen after these procedures.

Apoptosis, necrosis, proliferation, and myofibroblast generation in the stroma following LASIK and PRK.

Apoptosis in the cornea in response to epithelial injury: significance to wound healing and dry eye.

Corneal injury. A relatively pure model of stromal-epithelial interactions in wound healing.

Lack of an optimal in vitro model of human corneal epithelial (HCE) cells is a major limitation in studying normal functions and gene regulations in HCE. Moreover, availability of a multi-layered HCE culture can reduce the usage of animals in the toxicity testing of consumer products. We have developed tetracycline-responsive human papilloma virus (HPV) 16-E6/E7 transduced HCE clones showing tight regulation of proliferation and normal differentiation. Expression of HPV16-E6/E7 mRNA and HPV16-E7 and keratin K3 proteins was examined by RNase protection assay and western blotting, respectively, in presence and absence (+/-) of Dox in identified clones. Localization of cornea-specific keratin k3 in +/- of Dox was evaluated by immunocytochemistry. The response of growth factors such as hepatocyte growth factor (HGF) and epidermal growth factor to the cellular proliferation in +/- of Dox in the newly identified clones was measured by cell counting. Cellular morphology, formation of multi-layered cultures at air-liquid interface and ultrastructural features were evaluated by light and transmission electron microscopy. The physical barrier established by the newly developed clones was determined by the transepithelial permeability to sodium fluorescein and transepithelial electrical resistance assays in the airlifted-stratified cultures.

Development of genetically engineered tet HPV16-E6/E7 transduced human corneal epithelial clones having tight regulation of proliferation and normal differentiation.

Playing a sport or a musical instrument is presumed to improve motor ability. One would therefore predict that children who play a sport or music are better at motor imagery tasks, which rely on an intact cortical proprioceptive representation and precise motor planning, than children who do not. The authors tested this prediction.

Are children who play a sport or a musical instrument better at motor imagery than children who do not?

The sense of body ownership can be easily disrupted during illusions and the most common illusion is the rubber hand illusion. An idea that is rapidly gaining popularity in clinical pain medicine is that body ownership illusions can be used to modify pathological pain sensations and induce analgesia. However, this idea has not been empirically evaluated. Two separate research laboratories undertook independent randomized repeated measures experiments, both designed to detect an effect of the rubber hand illusion on experimentally induced hand pain. In Experiment 1, 16 healthy volunteers rated the pain evoked by noxious heat stimuli (5 s duration; interstimulus interval 25 s) of set temperatures (47°, 48° and 49°C) during the rubber hand illusion or during a control condition. There was a main effect of stimulus temperature on pain ratings, but no main effect of condition (p = 0.32), nor a condition x temperature interaction (p = 0.31). In Experiment 2, 20 healthy volunteers underwent quantitative sensory testing to determine heat and cold pain thresholds during the rubber hand illusion or during a control condition. Secondary analyses involved heat and cold detection thresholds and paradoxical heat sensations. Again, there was no main effect of condition on heat pain threshold (p = 0.17), nor on cold pain threshold (p = 0.65), nor on any of the secondary measures (p<0.56 for all). We conclude that the rubber hand illusion does not induce analgesia.

No pain relief with the rubber hand illusion.

Lower motor neuron dysfunction is one of the most debilitating motor conditions. In this regard, transgenic mouse models of various lower motor neuron dysfunctions provide insight into the mechanisms underlying these pathologies and can also aid the development of new therapies. Viral-mediated gene therapy can take advantage of the muscle-motor neuron topographical relationship to shuttle therapeutic genes into specific populations of motor neurons in these mouse models. In this context, motor end plates (MEPs) are highly specialized regions on the skeletal musculature that offer direct access to the pre-synaptic nerve terminals, henceforth to the spinal cord motor neurons. The aim of this study was two-folded. First, it was to characterize the exact position of the MEP regions for several muscles of the mouse forelimb using acetylcholinesterase histochemistry. This MEP-muscle map was then used to guide a series of intramuscular injections of Fluoro-Gold (FG) in order to characterize the distribution of the innervating motor neurons. This analysis revealed that the MEPs are typically organized in an orthogonal fashion across the muscle fibers and extends throughout the full width of each muscle. Furthermore, targeting the full length of the MEP regions gave rise labeled motor neurons that are organized into columns spanning through more spinal cord segments than previously reported. The present analysis suggests that targeting the full width of the muscles' MEP regions with FG increases the somatic availability of the tracer. This process ensures a greater uptake of the tracer by the pre-synaptic nerve terminals, hence maximizing the labeling in spinal cord motor neurons. This investigation should have positive implications for future studies involving the somatic delivery of therapeutic genes into motor neurons for the treatment of various motor dysfunctions.

Targeting the full length of the motor end plate regions in the mouse forelimb increases the uptake of fluoro-gold into corresponding spinal cord motor neurons.

Rahul Mohan

Translational Neuroscience Facility,
School of Medical Sciences,
Translational Neuroscience Facility, School of Medical Sciences

University of New South Wales

Intramuscular Injections Along the Motor End Plates: A Minimally Invasive Approach to Shuttle Tracers Directly into Motor Neurons

Rahul Mohan

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University of New South Wales

Intramuscular Injections Along the Motor End Plates: A Minimally Invasive Approach to Shuttle Tracers Directly into Motor Neurons

Translational Neuroscience Facility,
School of Medical Sciences,
Translational Neuroscience Facility, School of Medical Sciences