Mohamed Lehar

Pigment epithelium-derived factor (PEDF) protects motor neurons from chronic glutamate-mediated neurodegeneration.

Although pigment epithelium-derived factor (PEDF) is a neurotrophic factor that may aid the development, differentiation, and survival of adjacent neural retinae, the wider distribution of PEDF mRNA in the central nervous system suggested to us that this factor could have pleiotropic neurotrophic and neuroprotective effects on nonretinal neurons. We examined the distribution of PEDF mRNA and its transcript in the spinal cord. By immunohistochemistry and western blot analysis using an antihuman PEDF antiserum of known specificity, we found that PEDF protein is present in spinal cord, cerebrospinal fluid, and skeletal muscle and that its mRNA appears concentrated in motor neurons of the human spinal cord. These observations indicate that PEDF could have potential autocrine and paracrine effects on motor neurons, as well as being target-derived. We analyzed the pharmacologic utility of PEDF in a postnatal organotypic culture model of motor neuron degeneration and proved it is highly neuroprotective. The effect was biologically important, significantly sparing the spinal cords gross organotypic morphological appearance and preserving motor neuron choline acetyltransferase (ChAT). PEDF alone did not increase ChAT, indicating that the observed effect is neuroprotective, not merely an upregulation of motor neuron ChAT. Further, PEDF preserved motor neuron number, proving a survival effect. We hypothesize that PEDF may play important roles in the survival and maintenance of spinal motor neurons in their neuroprotection against acquired insults in postnatal life. It should be developed further as a therapeutic strategy for motor neuron diseases such as amyotrophic lateral sclerosis (ALS).

Sensory ataxic neuropathy as the presenting feature of a novel mitochondrial disease

Four unrelated patients presented with a severe sensory ataxic neuropathy in association with dysarthria and chronic progressive external ophthalmoplegia. Electrophysiologic and pathologic studies showed severe axonal loss disproportionately affecting sensory nerves. Molecular genetic analysis revealed multiple mitochondrial DNA deletions in muscle and peripheral nerve. Sensory ataxic neuropathy may be the predominant and presenting manifestation of a mitochondrial disorder, and a mitochondrial etiology should be included in its differential diagnosis. The triad of sensory ataxic neuropathy, dysarthria, and opthalmoparesis (SANDO) may represent a novel mitochondrial disease associated with multiple mitochondrial DNA deletions.

Effect of platelet rich plasma and fibrin sealant on facial nerve regeneration in a rat model

Objective: To investigate the effects of platelet rich plasma (PRP) and fibrin sealant (FS) on facial nerve regeneration.

Synaptic alterations at inner hair cells precede spiral ganglion cell loss in aging C57BL/6J mice

Hearing deficits have often been associated with loss of or damage to receptor hair cells and/or degeneration of spiral ganglion cells. There are, however, some physiological abnormalities that are not reliably attributed to loss of these cells. The afferent synapse between radial fibers of spiral ganglion neurons and inner hair cells (IHCs) emerges as another site that could be involved in transmission abnormalities. We tested the hypothesis that the structure of these afferent terminals would differ between young animals and older animals with significant hearing loss. Afferent endings and their synapses were examined by transmission electron microscopy at approximately 45% distance from the basal end of the cochlea in 2-3 month-old and 8-12 month-old C57BL/6J mice. The number of terminals in older animals was reduced by half compared to younger animals. In contrast, there was no difference in the density of SGCs between the age groups. Older animals featured enlarged terminals and mitochondria and enlarged postsynaptic densities and presynaptic bodies. These morphological changes may be a combination of pathologic, adaptive and compensatory responses to sensory dysfunction. Improved knowledge of these processes is necessary to understand the role of afferent connectivity in dysfunction of the aging cochlea.

Ophthalmic Involvement in Myo-neuro-gastrointestinal Encephalopathy Syndrome

We studied the clinical, histopathologic, neuroradiologic, biochemical, and genetic profile of a patient with the myo-neuro-gastrointestinal encephalopathy syndrome, a recently described multisystem mitochondriopathy characterized by blepharoptosis and ophthalmoparesis. The patient had severe intestinal pseudo-obstruction and a mixed demyelinating and axonal neuropathy. Abnormal collections of mitochondria in nerve and muscle as well as diffuse white matter disease were present. Cytochrome oxidase activity in muscle mitochondria was reduced. No mitochondrial DNA deletions were detected.

Myosin Heavy Chain Composition in Normal and Atrophic Equine Laryngeal Muscle

The myosin heavy chain (MHC) composition of a given muscle determines the contractile properties and, therefore, the fiber type distribution of the muscle. MHC isoform expression in the laryngeal muscle is modulated by neural input and function, and it represents the cellular level changes that occur with denervation and reinnervation of skeletal muscle. The objective of this study was to evaluate the pattern of MHC isoform expression in laryngeal muscle harvested from normal cadavers and cadavers with naturally occurring left laryngeal hemiplegia secondary to recurrent laryngeal neuropathy. Left and right thyroarytenoideus (TA) and cricoarytenoideus dorsalis (CAD) were obtained from 7 horses affected with left-sided intrinsic laryngeal muscle atrophy and from 2 normal horses. Frozen sections were evaluated histologically for degree of atrophy and fiber type composition. MHC isoform expression was determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of muscle protein. Histologic atrophy was seen in all atrophic muscles and some right-sided muscles of 3 affected horses, as well as the left TA of 1 normal horse. Fiber type grouping or loss of type I muscle fibers was observed in the left-sided laryngeal muscles in all but 1 affected horse, as well as in the right muscles of 2 affected horses, and the left TA of 1 normal horse. SDS-PAGE showed 2 bands corresponding to the type I and type IIB myosin isoforms in the CAD and TA of the 2 normal horses. Affected horses demonstrated a trend toward increased expression of the type IIB isoform and decreased expression of the type I isoform in atrophic muscles. This study confirmed the presence of histologic abnormalities in grossly normal equine laryngeal muscle, and it demonstrated an increased expression of type IIB MHC with a concurrent decreased expression of type I MHC in affected muscles. Evaluation of muscle fiber changes at the cellular level under denervated and reinnervated conditions may aid in assessing future strategies for reinnervation or regeneration of atrophic laryngeal muscle.

Efficient quantification of afferent cochlear ultrastructure using design-based stereology

The afferent synapse between the auditory nerve fiber and the inner hair cell (IHC) represents a critical junction for hearing. Elucidation of the structure at this site will help establish the substrate for normal sound encoding as well as pathologic processes associated with hearing dysfunction. Previous applications of unbiased (design-based) stereological principles have expanded our knowledge of neuro-morphological changes evident with the light microscope. Applying these principles at the level of the synapse is a promising morphometric approach for the efficient sampling of large reference spaces with electron microscopy. This study tests the accuracy of using ultra-thin sections at a fixed interval, known as disector pairs, to quantify afferent innervation density. We analyzed the total numbers of afferent terminals, synaptic thickenings, and synaptic bodies associated with each IHC in the C57BL/6J mouse cochlea, and confirmed the accuracy of the stereological approach in comparison to three-dimensional reconstructions of serial alternate sections. The higher sampling efficiency of the disector pair method rapidly increases precision while also reducing the largest source of variability, inter-animal differences. We conclude that ultrastructural quantification of afferent innervation can be accomplished in the cochlea using efficient design-based stereology.

High efficiency gene delivery into laryngeal muscle with bidirectional electroporation

Objective The impact of polarity change on the efficiency of in vivo electroporative (EP) gene transfection was assessed in rat laryngeal muscle. Study Design and Setting High (HV) and low field voltage (LV) were combined with polarity changes to determine transfection in 5 different conditions: 1) without EP (EP[-]), 2) HV+LV (HL), 3) HV+LV followed by HV+LV with no change in polarity (HLHL unidirectional), 4) HV+LV followed by HV+LV with opposite polarity (HLHL bidirectional), 5) HV+LV followed by LV with opposite polarity (HLL bidirectional). Results HLL bidirectional sequence showed the best result with less interindividual variability and extended expression period. With the exception of repeated high voltage sequences, EP parameters were not likely to induce cell injury or inflammation. Conclusion HLL bidirectional electroporative gene delivery produces high transfection rates with limited tissue trauma. Significance Bidirectional EP provides a safe and highly efficient method for therapeutic gene delivery into skeletal muscle.

Laryngeal muscle surface receptors identified using random phage library

Objectives: The ultimate goal of this study is to improve the efficiency of gene transfer in mammalian muscle by developing targeted adenoviral vectors. Altering the tropism of viral vectors to recognize tissue specific antigens is one method to achieve this goal. This approach requires identification of cell‐surface receptors and the insertion of target peptide sequences into the adenoviral fiber protein. In this study, phage biopanning was performed on cultured rat skeletal and laryngeal muscle to identify cell‐surface receptors.

Proteomic analysis of rat laryngeal muscle following denervation and reinnervation

Laryngeal muscle atrophy induced by nerve injury is a major factor contributing to the disabling symptoms associated with laryngeal paralysis. Alterations of global proteins in rat laryngeal muscle following denervation were, therefore, studied using proteomic techniques. Twenty-eight adult Sprague-Dawley rats were divided into normal control and denervated groups. The thyroarytenoid (TA) muscle was excised 60 days after right recurrent laryngeal nerve was resected. Protein separation and identification were preformed using 2-DE and MALDI-MS with database search. Forty-four proteins were found to have significant alteration in expression level after denervation. The majority of these proteins (57%), most of them associated with energy metabolism, cellular proliferation and differentiation, signal transduction and stress reaction, were decreased levels of expression in denervated TA muscle. The remaining 43% of the proteins, most of them involved with protein degradation, immunoreactivity, injury repair, contraction, and microtubular formation, were found to have increased levels of expression. The protein modification sites by phosphorylation were detected in 22% of the identified proteins that presented multiple-spot patterns on 2-D gel. Significant changes in protein expression in denervated laryngeal muscle may provide potential therapeutic strategies for the treatment of laryngeal paralysis.