Harry G. Goshgarian

Transporter protein and drug-conjugated gold nanoparticles capable of bypassing the blood-brain barrier

Drug delivery to the central nervous system (CNS) is challenging due to the inability of many drugs to cross the blood-brain barrier (BBB). Here, we show that wheat germ agglutinin horse radish peroxidase (WGA-HRP) chemically conjugated to gold nanoparticles (AuNPs) can be transported to the spinal cord and brainstem following intramuscular injection into the diaphragm of rats. We synthesized and determined the size and chemical composition of a three-part nanoconjugate consisting of WGA-HRP, AuNPs, and drugs for the treatment of diaphragm paralysis associated with high cervical spinal cord injury (SCI). Upon injection into the diaphragm muscle of rats, we show that the nanoconjugate is capable of delivering the drug at a much lower dose than the unconjugated drug injected systemically to effectively induce respiratory recovery in rats following SCI. This study not only demonstrates a promising strategy to deliver drugs to the CNS bypassing the BBB but also contributes a potential nanotherapy for the treatment of respiratory muscle paralysis resulted from cervical SCI.

Spontaneous Functional Recovery in a Paralyzed Hemidiaphragm Following Upper Cervical Spinal Cord Injury in Adult Rats

Previous studies have shown that latent respiratory pathways can be activated by as phyxia or systemic theophylline administration to restore function to a hemidiaphragm paralyzed by C2 spinal cord hemisection in adult female rats. Based on this premise, electrophysiologic recording techniques were employed in the present investigation to first determine qualitatively whether latent respiratory pathways are activated spon taneously following prolonged post hemisection periods (4-16 weeks) without any therapeutic intervention. Our second objective in a separate group of hemisected an imals was to quantitate any documented functional recovery under the following stan dardized recording conditions: bilateral vagotomy, paralysis with pancuronium bro mide, artificial ventilation, and constant PCO 2 (maintained at 25 mmHg).

Identification of the axon pathways which mediate functional recovery of a paralyzed hemidiaphragm following spinal cord hemisection in the adult rat

Despite extensive neurophysiological work carried out to characterize the crossed phrenic phenomenon, relatively little is known about the morphological substrate of this reflex which restores function to a hemidiaphragm paralyzed by spinal cord injury. In the present study WGA-HRP was injected into normal and functionally recovered hemidiaphragm muscle in rats during the crossed phrenic phenomenon. The retrograde transynaptic transport characteristics of WGA-HRP was utilized to delineate the source of the neurons which mediate the crossed phrenic phenomenon. The results indicated that the neurons which drive phrenic motoneurons in spinal hemisected rats during the crossed phrenic phenomenon are located bilaterally in the rostral ventral respiratory group (rVRG) of the medulla. No transneuronal labeling of propriospinal neurons was noted in either normal or spinal-hemisected rats. Thus, propriospinal neurons do not relay respiratory drive to phrenic motoneurons. The neurons of the rVRG project monosynaptically to phrenic motoneurons. The present results suggest that both crossed and uncrossed bulbospinal pathways from the rVRG collateralize to both the left and right phrenic nucleic and functional recovery of a hemidiaphragm paralyzed by ipsilateral spinal cord hemisection is mediated by supraspinal neurons from both sides of the brain stem. These results are important to our complete understanding of the mechanisms which govern motor recovery in mammals following spinal cord injury.

Effect of Spinal Cord Injury on the Respiratory System: Basic Research and Current Clinical Treatment Options

Abstract Summary: Spinal cord injury (SCI) often leads to an impairment of the respiratory system. The more rostral the level of injury, the more likely the injury will affect ventilation. In fact, respiratory insufficiency is the number one cause of mortality and morbidity after SCI. This review highlights the progress that has been made in basic and clinical research, while noting the gaps in our knowledge. Basic research has focused on a hemisection injury model to examine methods aimed at improving respiratory function after SCI, but contusion injury models have also been used. Increasing synaptic plasticity, strengthening spared axonal pathways, and the disinhibition of phrenic motor neurons all result in the activation of a latent respiratory motor pathway that restores function to a previously paralyzed hemidiaphragm in animal models. Human clinical studies have revealed that respiratory function is negatively impacted by SCI. Respiratory muscle training regimens may improve inspiratory function after SCI, but more thorough and carefully designed studies are needed to adequately address this issue. Phrenic nerve and diaphragm pacing are options available to wean patients from standard mechanical ventilation. The techniques aimed at improving respiratory function in humans with SCI have both pros and cons, but having more options available to the clinician allows for more individualized treatment, resulting in better patient care. Despite significant progress in both basic and clinical research, there is still a significant gap in our understanding of the effect of SCI on the respiratory system.

Dendritic organization of phrenic motoneurons in the adult rat

The dendritic organization of the phrenic nucleus as a whole was studied after injections of the B-subunit of cholera toxin conjugated to horseradish peroxidase were made into the diaphragm of adult rats. Transverse, sagittal, and horizontal sections through the phrenic nucleus (C3-C5) were incubated according to a modified tetramethylbenzidine HRP technique. The conjugated form of HRP used in this study has a special affinity for the GM1 ganglioside receptors on neuronal cell surfaces. As a result, extensive labeling of the terminal dendritic fields of a large number of phrenic motoneurons occurred simultaneously. The results showed that the majority of the dendrites of phrenic motoneurons were tightly organized rostrocaudally and confined to the boundaries of the column made up of the phrenic cell bodies. In addition, analysis of transverse and horizontal sections revealed dendritic bundles radiating at right angles to the long axis of the cell column in the following directions: dorsolateral into the dorsal half of the lateral funiculus, lateral into the lateral funiculus, ventromedial into the lateral half of the anterior funiculus, ventrolateral into the ventral half of the lateral funiculus, and dorsal into the intermediate gray matter. Some dendritic bundles were measured as far as 900 microns from phrenic cell bodies into the white matter. The horizontal sections also showed that there was a periodicity in the arrangement of the dendritic fascicles in that they were separated by distances ranging from 180 to 250 microns. From the analysis of phrenic dendritic distribution the present results suggest that the majority of synaptic input to phrenic motoneurons occurs within the column of the phrenic cell bodies. In addition, there is evidence to suggest that a synaptic input may also occur directly on distal phrenic dendrites in the lateral and ventral funiculi of the spinal cord white matter.

The ultrastructure and synaptic architecture of phrenic motor neurons in the spinal cord of the adult rat

SummaryAlthough light microscopic studies have analysed phrenic motor neurons in several different species, there has never been an ultrastructural investigation of identified phrenic motor neurons. In addition, electrophysiological studies have raised questions relating to the function of phrenic motor neurons which may be answered only by direct electron microscopic investigation. Thus, the present study was carried out to provide a detailed ultrastructural analysis of identified phrenic motor neurons. Phrenic motor neurons in the spinal cord of the rat were labelled by retrogradely transported horseradish peroxidase (HRP) after transecting the phrenic nerve in the neck and applying the enzyme directly to the central stump of the transected nerve. The results showed that the general ultrastructural characteristics of phrenic motor neurons were similar to those previously reported for other spinal motor neurons. However, phrenic primary dendrites appeared to be isolated from all other dendritic profiles in the neuropil. Primary dendrites were not fasciculated. Fasciculation occurred only among the more distal secondary and tertiary phrenic dendritic branches. Direct dendrodendritic or dendrosomatic apposition was rarely seen; gap junctions between directly apposing phrenic neuronal membranes were not observed. The membranes of adjacent phrenic neuronal profiles were most frequently separated by intervening sheaths of astroglial processes. Myelinated phrenic axons and a phrenic axon collateral were identified. The initial portion of the phrenic axon collateral was cone-shaped, lacked myelin, and thus resembled a miniature axon hillock. In one instance, a large accumulation of polyribosomes was observed within the hillock-like structure of a phrenic axon collateral. Eight morphological types of synaptic boutons, M, P, NFs, S, NFf, F, G and C were classified according to criteria used by previous investigators. Most of these endings (M, NFs, NFf, S and F) made synaptic contact with profiles of labelled phrenic somata and dendrites. F, NFf, and S boutons also terminated on phrenic axon hillocks. C and G boutons contacted exclusively phrenic somata and small calibre dendrites, respectively. P boutons established axo-axonic synaptic contacts with the M and NFs bouton. The morphological findings of the present study provide new data that may be related to phrenic synchronized output and presynaptic inhibition of primary afferents terminating on phrenic motor neurons.

Decussation of bulbospinal respiratory axons at the level of the phrenic nuclei in adult rats : a possible substrate for the crossed phrenic phenomenon

The axonal trajectories of inspiratory bulbospinal neurons were examined after deposition of the anterograde neuronal tracer phaseolus vulgaris leucoagglutinin (PHA-L) into the rostral ventral respiratory group in rats. At the level of the phrenic nucleus, PHA-L-labeled bulbospinal axons crossed the midline of the spinal cord in both the anterior gray and the anterior white commissure. These spinally decussating neurons provide a possible anatomical substrate for the respiratory reflex known as the crossed phrenic phenomenon.

Quantitative assessment of phrenic nerve functional recovery mediated by the crossed phrenic reflex at various time intervals after spinal cord injury

The present study was carried out to determine if augmentation of phrenic nerve activity during the crossed phrenic phenomenon temporally coincides with the morphological changes in the phrenic nucleus that we have observed in previous studies. This investigation consisted of two experiments in spinal cord hemisected young adult female Sprague-Dawley rats. Crossed phrenic activity was quantitatively assessed from the left phrenic nerve after bilateral vagotomy and sectioning of the right phrenic and accessory phrenic nerves. The first experiment involved serial recordings of crossed phrenic activity performed on each of 4 animals at hourly intervals ranging from 1 to 6 h after spinal cord hemisection. The second experiment consisted of single recordings from each of 24 animals at one of the following time intervals after hemisection: 1/2, 1, 2, 4, 12, and 24 h. Recording conditions were standardized at each recording session in both experiments by paralyzing the animals, regulating temperature and blood pressure, and controlling end tidal PCO2 with a volume ventilator. Crossed phrenic activity was induced by stopping the ventilator and quantitated by measuring the area under the integrated waveform of the largest respiratory burst. The results revealed a small, statistically insignificant increase in crossed phrenic activity at 1 h compared to the 30-min recordings. At 2 h there was a large, statistically significant increase in activity. Experiment one showed further increases from 3 to 6 h. The second experiment showed a smaller increase from 2 to 4 h and then maintained this level at 12 and 24 h.(ABSTRACT TRUNCATED AT 250 WORDS)

The role of cervical afferent nerve fiber inhibition of the crossed phrenic phenomenon

Abstract High cervical spinal cord hemisection produces a permanent paralysis of the ipsilateral hemidiaphragm. In many species, function is restored to this paretic hemidiaphragm if the contralateral hemidiaphragm is paralyzed by transecting the phrenic nerve. This response is termed the “crossed phrenic phenomenon.” The present study determines the long-term effects on diaphragmatic function after anesthetization or crushing the contralateral phrenic nerve, or after cutting its dorsal roots in rats subjected to a high cervical spinal cord hemisection. Dorsal root transection was the only procedure which resulted in a partial functional recovery of the hemidiaphragm paralyzed by the spinal cord hemisection without a loss of function in the contralateral hemidiaphragm. The results suggest that afferent nerve fibers in the contralateral phrenic nerve may normally inhibit the functional expression of the crossed phrenic pathway, although the precise mechanism for this inhibition is not yet known.

Ultrastructural Changes in the Rat Phrenic Nucleus Developing within 2 h after Cervical Spinal Cord Hemisection

The present study was conducted to describe the ultrastructural changes which occur in the young adult rat phrenic nucleus within 2 h after an ipsilateral C2 spinal cord hemisection. The main objective was to determine if there is a temporal relationship between specific ultrastructural changes in the phrenic nucleus and a significant augmentation of crossed phrenic nerve activity which occurs as early as 2 h after hemisection. Phrenic motoneurons were identified at electron microscopic levels by retrograde HRP labeling. Ultrastructural features in the phrenic nucleus of control and experimental rats were qualitatively analyzed and then quantitated. At 2 h posthemisection, there was a significant increase in the mean percentage of phrenic dendrodendritic appositions. In the control rats, 4.73 +/- 0.18% of phrenic dendrites were in apposition, and this percentage increased significantly to 8.58 +/- 0.54% at 2 h after injury. Furthermore, the mean lengths of asymmetrical and symmetrical synaptic active zones increased significantly at 2 h posthemisection from control lengths of 0.372 +/- 0.009 microns and 0.404 +/- 0.007 microns to 0.410 +/- 0.011 microns and 0.513 +/- 0.032 microns, respectively, in experimental rats. The phrenic nucleus is therefore capable of morphological plasticity as early as 2 h after spinal cord hemisection and this plasticity coincides temporally with the physiological augmentation of crossed phrenic nerve activity at 2 h. The data further suggest that these morphological changes may be part of the substrate for the unmasking of ineffective synapses during the crossed phrenic phenomenon.