Gizelda T. Casella

Improved method for harvesting human Schwann cells from mature peripheral nerve and expansion in vitro.

The use of cellular prostheses containing large populations of Schwann cells (SC) has been proposed as a future therapeutic approach in the repair of neural tissue. We have sought to define an efficient protocol for the harvest and expansion of human SC from mature human peripheral nerve. We evaluated SC proliferation occurring within fresh explants and studied the relationship between certain parameters (cell yield, purity, and rate of SC proliferation) and the conditions of maintenance of nerve explants prior to dissociation. In addition, we studied SC proliferation after dissociation in a variety of conditions. We observed that SC within explants divide at a low rate during the first 3 weeks following explantation; this proliferation falls to near zero during the fourth week. The cell yield, SC purity, and proliferation rate following dissociation were all increased when nerve explants were exposed to heregulin/forskolin for 2 weeks prior to dissociation. Electron microscopic analysis showed that heregulin/forskolin exerted trophic effects on SC within explants. Following dissociation, SC growth in heregulin/forskolin‐containing medium was more rapid on laminin or collagen than on poly‐L‐lysine. These results provide new insights into human SC biology and suggest several procedural improvements for harvesting and expanding these cells. The new method we describe shortens our previous procedure by 4–6 weeks and provides a 30–50‐fold increase in the number of SC obtained relative to the earlier procedure.

New Vascular Tissue Rapidly Replaces Neural Parenchyma and Vessels Destroyed by a Contusion Injury to the Rat Spinal Cord

Blood vessels identified by laminin staining were studied in uninjured spinal cord and at 2, 4, 7, and 14 days following a moderate contusion (weight drop) injury. At 2 days after injury most blood vessels had been destroyed in the lesion epicenter; neurons and astrocytes were also absent, and few ED1+ cells were seen infiltrating the lesion center. By 4 days, laminin associated with vessel staining was increased and ED1+ cells appeared to be more numerous in the lesion. By 7 days after injury, the new vessels formed a continuous cordon oriented longitudinally through the lesion center. ED1+ cells were abundant at this time point and were found in the same area as the newly formed vessels. Astrocyte migration from the margins of the lesion into the new cordon was apparent. By 14 days, a decrease in the number of vessels in the lesion center was observed; in contrast, astrocytes were more prominent in those areas. In addition to providing a blood supply to the lesion site, protecting the demise of the newly formed vascular bridge might provide an early scaffold to hasten axonal regeneration across the injury site.

Clinical Outcomes Using Modest Intravascular Hypothermia After Acute Cervical Spinal Cord Injury

BACKGROUNDAlthough a number of neuroprotective strategies have been tested after spinal cord injury (SCI), no treatments have been established as a standard of care. OBJECTIVEWe report the clinical outcomes at 1-year median follow-up, using endovascular hypothermia after SCI and a detailed analysis of the complications. METHODSWe performed a retrospective analysis of American Spinal Injury Association and International Medical Society of Paraplegia Impairment Scale (AIS) scores and complications in 14 patients with SCI presenting with a complete cervical SCI (AIS A). All patients were treated with 48 hours of modest (33°C) intravascular hypothermia. The comparison group was composed of 14 age- and injury-matched subjects treated at the same institution. RESULTSSix of the 14 cooled patients (42.8%) were incomplete at final follow-up (50.2 [9.7] weeks). Three patients improved to AIS B, 2 patients improved to AIS C, and 1 patient improved to AIS D. Complications were predominantly respiratory and infectious in nature. However, in the control group, a similar number of complications was observed. Adverse events such as coagulopathy, deep venous thrombosis, and pulmonary embolism were not seen in the patients undergoing hypothermia. CONCLUSIONThis study is the first phase 1 clinical trial on the safety and outcome with the use of endovascular hypothermia in the treatment of acute cervical SCI. In this small cohort of patients with SCI, complication rates were similar to those of normothermic patients with an associated AIS A conversion rate of 42.8%.

Clinical application of modest hypothermia after spinal cord injury.

There is widespread interest in the use of hypothermia in the treatment of CNS injury. While there is considerable experience in the use of cooling for a variety of brain pathologies, limited data exist after spinal cord injury. In the past few years, technological advances in the induction and maintenance of cooling have been achieved and can potentially allow for a more accurate evaluation of this form of treatment. We report a series of 14 patients with an average age of 39.4 years (range, 16-62 years) with acute, complete (AIS A) cervical spinal cord injuries who underwent a protocol using an intravascular cooling catheter to achieve modest (33 degrees C) systemic hypothermia. There was an excellent correlation between intravascular and intrathecal cerebrospinal fluid temperature. The average time between injury and induction of hypothermia was 9.17 +/- 2.24 h (mean +/- SEM); the time to target temperature was 2.72 +/- 0.42 h; the duration of cooling at target temperature was 47.6 +/- 3.1 h; the average total length of time of cooling was 93.6 +/- 4 h. There was a positive correlation between temperature and heart rate. Most documented adverse events were respiratory in nature. We were able to effectively deliver systemic cooling using the cooling catheters with minimal variation in body temperature. The study represents the largest, modern series of hypothermia treatment of acute spinal cord injury with intravascular cooling techniques and provides needed baseline data for outcome studies to include larger multi-center, randomized trials.

Endothelial cell loss is not a major cause of neuronal and glial cell death following contusion injury of the spinal cord

Contusion of the spinal cord causes an immediate local loss of neurons and disruption of vasculature; additional loss continues thereafter. To explore the possibility of a causal link between delayed endothelial cell (EC) death and secondary neural cell loss, we evaluated neural and endothelial cell survival, and measured inflammatory cell infiltration, at times up to 48 h after contusion injury to the adult rat thoracic spinal cord. Female Fischer rats (200 g), subjected to moderate (10 g x 12.5 mm) weight drop injuries by the MASCIS (NYU) impactor, were analyzed at 15 min and at 1, 8, 24 and 48 h. ECs, neurons, astrocytes, oligodendrocytes, neutrophils and activated macrophages/microglia were counted in transverse sections. At the injury site, 90% of all neurons died within 48 h of injury; no medium-large diameter neurons survived beyond 48 h. EC death occurred with kinetics similar to glial cell death. Because, in the injury site, most cell death occurred before 8 h, it preceded inflammatory cell infiltration. Three millimeters rostral and caudal to the injury epicenter neuronal numbers were stable for 8 h, and a sharp decrease in neuronal numbers beginning at 8 h strongly correlated with the onset of inflammatory cell infiltration. Glial and blood vessel numbers remained relatively stable in these areas up to 48 h. These results suggest that the loss of ECs during the first 48 h after a contusion injury is not a major cause of neuronal and glial cell death and, in tissue adjacent to the epicenter, inflammatory cell infiltration leads to neuronal loss.

A Prospective, Randomized Trial Comparing Expansile Cervical Laminoplasty and Cervical Laminectomy and Fusion for Multilevel Cervical Myelopathy

BACKGROUND: Controversy exists as to the best posterior operative procedure to treat multilevel compressive cervical spondylotic myelopathy. OBJECTIVE: To determine clinical, radiological, and patient satisfaction outcomes between expansile cervical laminoplasty (ECL) and cervical laminectomy and fusion (CLF). METHODS: We performed a prospective, randomized study of ECL vs CLF in patients suffering from cervical spondylotic myelopathy. End points included the Short Form-36, Neck Disability Index, Visual Analog Scale, modified Japanese Orthopedic Association score, Nurick score, and radiographic measures. RESULTS: A survey of academic North American spine surgeons (n = 30) demonstrated that CLF is the most commonly used (70%) posterior procedure to treat multilevel spondylotic cervical myelopathy. A total of 16 patients were randomized: 7 to CLF and 9 to ECL. Both groups showed improvements in their Nurick grade and Japanese Orthopedic Association score postoperatively, but only the improvement in the Nurick grade for the ECL group was statistically significant (P < .05). The cervical range of motion between C2 and C7 was reduced by 75% in the CLF group and by only 20% in the ECL group in a comparison of preoperative and postoperative range of motion. The overall increase in canal area was significantly (P < .001) greater in the CLF group, but there was a suggestion that the adjacent level was more narrowed in the CLF group in as little as 1 year postoperatively. CONCLUSION: In many respects, ECL compares favorably to CLF. Although the patient numbers were small, there were significant improvements in pain measures in the ECL group while still maintaining range of motion. Restoration of spinal canal area was superior in the CLF group.

Density dependent regulation of human Schwann cell proliferation.

Cessation of division is prerequisite for Schwann cell differentiation but regulation of this critical function is poorly understood. Heregulin/forskolin‐induced growth of human Schwann cells (HSCs) in vitro was found to be strongly regulated by cell density and thus could model some aspects of negative growth‐regulation in vivo. To better understand this phenomenon, the production of an autocrine growth‐inhibitor and the role of contact‐inhibition were investigated. The possible involvement of two membrane proteins, contactinhibin (CI) and peripheral myelin protein 22 (PMP22) in regulating growth was studied. Thymidine‐labeling of HSCs on collagen‐coated dishes was inhibited at cell densities less than one tenth of the density at maximal growth‐inhibition. Medium from high density cultures did not inhibit the thymidine‐labeling of HSCs at low density, a result that argues against the production of a soluble inhibitor. The expression of CI and PMP22 in nerve and HSCs, and the effect of a function‐blocking antibody to CI on HSC growth, were determined. CI was detected in fresh nerve by western blotting, and could easily be detected by immunocytochemistry in cultured HSCs by five days and for several weeks thereafter. Twenty‐four hour treatment with anti‐CI antibody did not increase the thymidine‐labeling of HSCs at any density but a significant increase in HSC number was observed in cultures treated with anti‐CI for 20 days. This increase was not related to decreased cell death. PMP22, unlike other myelin proteins, was not down‐regulated after nerve dissociation and by seven days nearly all HSCs were PMP22 positive. These results provide evidence for a contact‐mediated mechanism of growth‐regulation in HSCs and suggest that CI is involved in this mechanism. GLIA 30:165–177, 2000.

The immunophilin ligand FK506, but not the P38 kinase inhibitor SB203580, improves function of adult rat muscle reinnervated from transplants of embryonic neurons.

Injury to the adult CNS often involves death of motoneurons, resulting in the paralysis and progressive atrophy of muscle. There is no effective therapy to replace motoneurons in the CNS. Our strategy to replace neurons and to rescue denervated muscles is to transplant dissociated embryonic day 14-15 (E14-15) ventral spinal cord cells into the distal stump of a peripheral nerve near the denervated muscles. Here, we test whether long-term delivery of two pharmacological inhibitors to denervated muscle, FK506 or SB203580, enhances reinnervation of muscle from embryonic cells transplanted in the tibial nerve of adult Fischer rats. FK506, SB203580 (2.5 mg/kg) or saline was delivered under the fascia of the medial gastrocnemius muscle for 4 weeks, beginning when muscles were denervated by section of the sciatic nerve. After 1 week of nerve degeneration, one million E14-15 ventral spinal cord cells were transplanted into the distal tibial nerve stump of each rat in the three treatment groups. Ten weeks later, all cell transplants had neuron-specific nuclear protein (NeuN) positive neurons. Neuron survival and axon regeneration were similar across treatments. An average (+/-S.E.) of 210+/-66, 100+/-36 and 176+/-58 myelinated axons grew distally from the cell transplants of rats with muscles treated with FK506, SB203580 or saline, respectively. Regenerating axons in muscles of all three treatments groups were detected with antibodies against phosphorylated neurofilaments and synaptophysin, and motor end plates were labeled with alpha-bungarotoxin. Muscles of rats that received transplants of media only had no axon growth, indicating that the muscles were denervated. The mean muscle fiber areas of rats that received cell transplants and had long-term delivery of FK506, SB203580 or saline to muscles were significantly larger than those of denervated muscle fibers. Thus, cell transplantation reduced muscle atrophy. Transplantation of embryonic cells also resulted in functional muscle reinnervation. Electromyographic activity and force were evoked from >90% of the muscles of rats with cell transplants, but not from denervated muscles. FK506-treated muscles were significantly more fatigue resistant than naive control muscles. FK506-treated muscles also had significantly stronger motor units than those in SB203580 or saline-treated muscles. These data suggest that a pathway regulated by FK506 improves the function of muscles reinnervated by embryonic neurons placed in peripheral nerve.

Motoneuron Replacement for Reinnervation of Skeletal Muscle in Adult Rats

Abstract Reinnervation is needed to rescue muscle when motoneurons die in disease or injury. Embryonic ventral spinal cord cells transplanted into peripheral nerve reinnervate muscle and reduce atrophy, but low motoneuron survival may limit motor unit formation. We tested whether transplantation of a purified population of embryonic motoneurons into peripheral nerve (mean ± SE, 146,458 ± 4,011 motoneurons) resulted in more motor units and reinnervation than transplantation of a mixed population of ventral spinal cord cells (72,075 ± 12,329 motoneurons). Ten weeks after either kind of transplant, similar numbers of neurons expressed choline acetyl transferase and/or Islet-1. Motoneuron numbers always exceeded the reinnervated motor unit count. Most motor end plate were simple plaques. Reinnervation significantly reduced muscle fiber atrophy. These data show that the number of transplanted motoneurons and motoneuron survival do not limit muscle reinnervation. Incomplete differentiation of motoneurons in nerve and lack of muscle activity may result in immature neuromuscular junctions that limit reinnervation and function.

Neurotrophic factors improve muscle reinnervation from embryonic neurons.

Motoneurons die in diseases like amyotrophic lateral sclerosis and after spinal cord trauma, inducing muscle denervation. We tested whether transplantation of embryonic cells with neurotrophic factors into peripheral nerve of adult rats improves muscle reinnervation and motor unit function more than cells alone. One week after sciatic nerve section, embryonic ventral spinal cord cells were transplanted into the tibial nerve with or without glial cell line‐derived neurotrophic factor, hepatocyte growth factor, and insulin‐like growth factor‐1. These cells represented the only neuron source for muscle reinnervation. Ten weeks after transplantation, all medial gastrocnemius muscles contracted in response to electrical stimulation of cell transplants with factors. Only 80% of muscles responded with cells alone. Factors and cells resulted in survival of more motoneurons and reinnervation of more muscle fibers for a given axon (motor unit) number. Greater reinnervation from embryonic cells may enhance muscle excitation by patterned electrical stimulation. Muscle Nerve 42: 788–797, 2010