Henrich Cheng

Spinal cord repair in adult paraplegic rats: partial restoration of hind limb function.

Complete spinal cord gaps in adult rats were bridged with multiple intercostal nerve grafts that redirected specific pathways from white to gray matter. The grafted area was stabilized with fibrin glue containing acidic fibroblast growth factor and by compressive wiring of posterior spinal processes. Hind limb function improved progressively during the first 6 months, as assessed by two scoring systems. The corticospinal tract regenerated through the grafted area to the lumbar enlargement, as did several bulbospinal pathways. These data suggest a possible repair strategy for spinal cord injury.

Conversion of human umbilical cord mesenchymal stem cells in Wharton's jelly to dopaminergic neurons in vitro: potential therapeutic application for Parkinsonism.

Human mesenchymal stem cells isolated from Whartons jelly of the umbilical cord were induced to transform into dopaminergic neurons in vitro through stepwise culturing in neuron‐conditioned medium, sonic hedgehog, and FGF8. The success rate was 12.7%, as characterized by positive staining for tyrosine hydroxylase (TH), the rate‐limiting catecholaminergic synthesizing enzyme, and dopamine being released into the culture medium. Transplantation of such cells into the striatum of rats previously made Parkinsonian by unilateral striatal lesioning with the dopaminergic neurotoxin 6‐hydroxydopamine partially corrected the lesion‐induced amphetamine‐evoked rotation. Viability of the transplanted cells at least 4 months after transplantation was identified by positive TH staining and migration of 1.4 mm both rostrally and caudally. These results suggest that human umbilical mesenchymal stem cells have the potential for treatment of Parkinsons disease.

In vitro differentiation of size-sieved stem cells into electrically active neural cells.

Size‐sieved stem (SS) cells isolated from human bone marrow and propagated in vitro are a population of cells with consistent marker typing, and can form bone, fat, and cartilage. In this experiment, we demonstrated that SS cells could be induced to differentiate into neural cells under experimental cell culture conditions. Five hours after exposure to antioxidant agents (β‐mercaptoethanol ± retinoic acid) in serum‐free conditions, SS cells expressed the protein for nestin, neuron‐specific enolase (NSE), neuron‐specific nuclear protein (NeuN), and neuron‐specific tubulin‐1 (TuJ‐1), and the mRNA for NSE and Tau. Immunofluorescence showed that almost all the cells (>98%) expressed NeuN and TuJ‐1. After 5 days of β‐mercaptoethanol treatment, the SS cells expressed neurofilament high protein but not mitogen‐activated protein‐2, glial filament acidic protein, and galactocerebroside. For such long‐term‐treated cells, voltage‐sensitive ionic current could be detected by electrophysiological recording, and the intracellular calcium ion, Ca2+, concentration can be elevated by high potassium (K+) buffer and glutamate. These findings suggest that SS cells may be an alternative source of undifferentiated cells for cell therapy and gene therapy in neural dysfunction.

Gait analysis of adult paraplegic rats after spinal cord repair

This study presents a novel detailed method of analysis of rat gait and uses this method to demonstrate recovery of forward locomotion patterns in adult rats made paraplegic by surgical spinal cord transection and subjected to a novel strategy for spinal cord repair. Six normal rats were compared to five animals in which the cord was transected at T8-T9, and a 5-mm segment of the spinal cord removed, and to seven animals in which, following spinal cord transection and removal of a spinal cord segment, multiple intercostal peripheral nerve bridges were implanted, rerouting pathways from white to gray matter in both directions. The implanted area was filled with fibrin glue containing acidic fibroblast growth factor. Details of the repair strategy have been published (H. Cheng, Y. Cao, and L. Olson, 1996, Science 273: 510-513). Gait analysis was carried out 3 and 4 months after surgery and once in the normal animals. Animals were allowed to walk across a runway with a transparent floor. Each test consisted of five trials, and each trial was videorecorded from underneath. Using frame-by-frame playback, individual footprints were then recorded regarding location and order of limb use, as well as step quality (degree of weight bearing, etc.). These data allowed measuring runway transit time, five different measures of step numbers, all possible temporal patterns of limb use, stride length, and base of support. Transected controls remained paralyzed in the hindlimbs with only occasional reflex hindlimb movements without weight bearing. Animals subjected to the full repair procedure were significantly faster than the controls, used their hindlimbs for 25-30% of the movements, and regained several of the specific limb recruitment patterns used by normal rats. Taken together, the gait analysis data demonstrate remarkable recovery of coordinated gait in the repaired animals, which was significantly better than controls for all relevant parameters, while at the same time clearly inferior to normal rats for most of the examined parameters. We conclude that normal rats use a multitude of interchangeable step sequence patterns, and that our spinal cord repair strategy leads to recovery of some of these patterns following complete spinal cord transection. These data suggest functionally relevant neuronal communication across the lesion.

Neuroprotection of glial cell line-derived neurotrophic factor in damaged spinal cords following contusive injury

Glial cell line‐derived neurotrophic factor (GDNF) acts as a potent survival factor for many neuronal populations, including spinal motoneurons, indicating the therapeutic promise of GDNF for neurological disorders. Injury to spinal cord (SCI) triggers processes destructive to ascending sensory and descending motor conduction and extends tissue loss, thereby leading to permanent behavioral dysfunction. In this study, we attempted to examine whether GDNF protects neurons from SCI and subsequently lessens locomotor deficit in SCI rats. We utilized the NYU weight‐drop device developed at New York University to induce spinal cord contusion at the T9–10 spinal segment. After SCI, GDNF was administrated into the cord 1–2 mm rostral and caudal to the epicenter. Animals receiving GDNF treatment showed significant improvement over phosphate‐buffered saline (PBS)‐treated controls on the Basso Beattie Bresnahan (BBB) locomotor rating scale (P < 0.01‐0.001). GDNF treatment increased the remaining neuronal fibers with calcitonin gene‐related peptide, neurofilament, and growth‐associated protein 43 immunoreactivity in injured spinal tissues compared with PBS‐treated controls. Moreover, treatment with GDNF caused approximately 50% cell survival in the contused spinal cord tissues. Examination of signal transduction triggered by GDNF indicated that GDNF injection transiently induced activation of the mitogen‐activated protein (MAP) kinase pathway in the spinal cord. Additionally, an up‐regulation of anti‐apoptotic Bcl‐2 levels in the contusive center of the damaged spinal cord was observed 24 hr post‐GDNF injection. Together our results show that GDNF exerts behavioral and anatomic neuroprotection following SCI. Additionally, GDNF‐activated MAP kinase and Bcl‐2 signaling may contribute to neuronal survival after spinal cord contusion.

High-Resolution MRI of Intact and Transected Rat Spinal Cord

Spinal cord transection at midthoracic level leads to an immediate loss of hindlimb motor function as well as to a progressive degeneration of descending and ascending spinal cord pathways. Thoracic spinal cord in unlesioned control rats and in rats 2 to 6 months after complete midthoracic transection were imaged in vivo using an ultrahigh-field (4.7 T) magnetic resonance spectrometer. High-resolution spin-echo and inversion-recovery pulse sequences were employed. In addition, the apparent diffusion coefficients (ADCs) in longitudinal and transverse directions of the spinal cord were determined. Anatomical MRI findings were confirmed in histological spinal cord tissue preparations. In healthy spinal cord, gray and white matter were easily discerned in proton density-weighted images. An infield resolution of max. 76 micrometers per pixel was achieved. In animals with chronic spinal cord transection changes in gray-white matter structure and contrast were observed toward the cut end. The spinal cord stumps showed a tapering off. This coincided with changes in the longitudinal/transverse ADC ratio. Fluid-filled cysts were found in most cases at the distal end of the rostral stump. The gap between the stumps contained richly vascularized scar tissue. Additional pathologic changes included intramedullary microcysts, vertebral dislocations, and in one animal compression of the spinal cord. In conclusion, MRI was found to be a useful method for in vivo investigation of anatomical and physiological changes following spinal cord transection and to estimate the degree of neural degeneration. In addition, MRI allows the description of the accurate extension of fluid spaces (e.g., cysts) and of water diffusion characteristics which cannot be achieved by other means in vivo.

Gene transfer of glial cell line-derived neurotrophic factor promotes functional recovery following spinal cord contusion

Neuronal cell death and the failure of axonal regeneration cause a permanent functional deficit following spinal cord injury (SCI). Administration of recombinant glial cell line-derived neurotrophic factor (GDNF) has previously been reported to rescue neurons following severe SCI, resulting in improved hindlimb locomotion in rats. In this study, thus, GDNF gene therapy using an adenoviral vector (rAd-GDNF) was examined in rats following SCI induced by dropping the NYU weight-drop impactor from a height of 25 mm onto spinal segment T9-T10. To evaluate the efficacy of intraspinal injection of recombinant adenovirus into the injured spinal cord, we observed green fluorescent protein (GFP) gene transfer in the contused spinal cord. GFP was effectively expressed in the injured spinal cord, and the most prominently transduced cells were astrocytes. The expression of GDNF was detected only in rats receiving rAd-GDNF, not the controls, and remained detectable around the injured site for at least 8 days. Open-field locomotion analysis revealed that rats receiving rAd-GDNF exhibited improved locomotor function and hindlimb weight support compared to the control groups. Immunohistochemical examination for the neuronal marker, calcitonin gene-related peptide (CGRP), showed an increase in CGRP+ neuronal fibers in the injured spinal cord in rats receiving rAd-GDNF treatment. Collectively, the results suggest that adenoviral gene transfer of GDNF can preserve neuronal fibers and promote hindlimb locomotor recovery from spinal cord contusion. This research should provide information for developing a clinical strategy for GDNF gene therapy.

Characterization of a Fibrin Glue–GDNF Slow-Release Preparation

One novel method to deliver trophic factor locally in the CNS is to mix it into fibrin glue. In the present studies, [125I]-labeled GDNF-containing fibrin glue balls were used to determine binding and spread of the trophic factor. First, the binding of different concentrations of [125I]-labeled GDNF in fibrin glue was determined in vitro. Within the six concentrations used (from 200 nM to 0.004 nM, 0 M as control), there was a strong linear correlation between the [125I]-GDNF concentration and the recovered radioactivity (r = 0.992). The mean bound radioactivity in 16 samples with 4 nM [125I]-GDNF was 71262 +/- 2710 CPM, and accounted for 89.8% of the mean initial count of free [125I]-GDNF (79369 +/- 3499 CPM). Second, [125I]-GDNF-containing glue balls were implanted into the anterior chamber of adult rats. The implanted fibrin glue balls decreased in size with time, but could still be identified on the irises 2 wk after implantation. Radioactivity was concentrated at the implantation sites in the early stages with a distribution in the surrounding iris tissue, which became separated into focal radioactive spots at the third week. Counts of radioactivity were significantly higher in the [125I]-GDNF glue ball-implanted irises than controls until 14 days after implantation. A study of the [125I] decay over time using least-squares linear regression demonstrated first-order kinetics (r = -0.98, p <0.02) with k = 0.0091 and T 1/2 = 76 h. Finally, [125I]-GDNF-containing glue balls were implanted in the spinal cord of adult rats. Radioactivity was concentrated at the implantation sites in the early stages and was later distributed more widely in the surrounding thoracic cord. The [125I]-GDNF-containing glue degraded over time and became a porous meshwork with decreasing radioactivity at the later time points. Radioactivity in the spinal cords subjected to implantation of [125I]-GDNF-containing glue balls was higher than in controls for 14 days. Study of the [125I] decay by time with least-squares linear regression demonstrated first-order kinetics (r = -0.97, p = 0.001) with T 1/2 = 75.6 h. We conclude that the trophic factor GDNF becomes bound in the fibrin glue matrix from which it is gradually released. Our results suggest that fibrin glue is an effective substrate for keeping a trophic factor localized in situ for a finite period, protected from the circulation, surrounding aqueous humor or CSF.

The effect of glial cell line-derived neurotrophic factor in fibrin glue on developing dopamine neurons

Glial cell line-derived neurotrophic factor (GDNF), a member of the transforming growth factor-β superfamily, promotes the survival, morphological differentiation, and high-affinity dopamine (DA) uptake of cultured nigral DA neurons. In order to test potential methodology for peptide delivery in vivo, GDNF-containing fibrin glue balls (8 μg/ball) were incorporated with pieces of fetal ventral mesencephalon (E15) and transplanted into the anterior chambers of sympathetically denervated adult rats. Five weeks after grafting, the numbers of TH-positive neurons and the nerve fiber density were significantly higher in the ventral mesencephalic grafts treated with GDNF-containing glue balls than in those treated with vehicle. In addition, the laminin and GFAP immunoreactivities were similar between the two groups. These data support the concept that GDNF is a potent trophic factor for DA neurons in vivo and suggest that fibrin glue may provide a unique and safe means to permit prolonged delivery of trophic molecules to CNS tissues.

Salvage surgery for recurrent nasopharyngeal carcinoma.

Objective To evaluate the efficacy of salvage surgery in the treatment of recurrent nasopharyngeal carcinoma (NPC) at the primary site.