Neurosurgery

Laboratories

Research Projects

Brain trauma is a devastating condition that affects over 2 million people in the United States each year. However, the mechanisms of brain trauma have only begun to be explained. It has been determined that some unique features of the physical damage induced by brain trauma can trigger progressive degenerative damage. This startling finding is the basis of the Smith Neurotrauma Lab's research efforts.

Extreme Stretch Growth

Engineering Nerve Constructs for Clinical Application

B.J. Pfister1, J. Huang1, E.L. Zager1, A. Iwata1, D.F.Meaney2, A.S. Cohen3,4, D.H.Smith1

Departments of (1)Neurosurgery, (2)Bioengineering, and (3)Pediatrics, University of Pennsylvania and Division of Neurology; (4)Children's Hospital of Philadelphia

Elongated axons

In the United States, tens of thousands of peripheral nerve injuries occur each year, many resulting in the loss of bodily functions and even permanent disability. The gold standard of peripheral nerve repair traditionally relies on a surgical procedure that involves the removal and transplantation of an autograft, a separate, less important length of nerve from the patient.

In some patients the damaged nerve will regenerate, using the autograft as a guide, leading to restoration of lost bodily functions. However, some major peripheral nerve deficits that can typically be repaired are not due to the limited availability of nerves that the patient can spare (for example, an insufficient supply of donor nerves for the reconstruction of a major brachial plexus injury).

There are a number of disadvantages associated with this technique including the additional loss of neurological function associated with the harvesting other nerves for grafting (including scarring and painful neuroma formation), increased post-operative pain due to additional incisions, increased risk of infection, and increased operating room and anesthesia time. For this approach, the extent of functional recovery is largely dependent on the distance of regeneration required to bridge the damaged nerve. Typically only about 50 percent of all patients will recover some useful functions and full recovery is rare [1,2].

In the case of spinal cord injuries, there is presently no effective treatment that can restore lost function. In contrast to peripheral nerve injuries, spinal cord injuries occur in an environment that is non-permissive to regeneration. While active research is focusing on the obstacles to regeneration within the spinal cord, an effective repair strategy has yet to be uncovered [3,4].

Currently, repair of spinal cord and peripheral nerve injuries relies on the ability of axon fibers (a nerve cell process that conducts nervous signals) to regenerate across the damaged area to restore nervous system communication. Accordingly, primary repair strategies have aimed to enhance and guide axon outgrowth by bridging the damaged area with materials of biologic or synthetic origin. Several approaches have been vigorously studied including: biomaterials to act as physical guides, transplantation of various cell types to support axon growth, administration of drugs to counteract elements that inhibit axon growth, and agents that enhance axon growth [1,5]. For peripheral nerve damage, these approaches have only been successful for injuries spanning a short distance, much smaller than what autogenous grafting can repair 6. For spinal cord injury, some approaches have been able to enhance the outgrowth of a few axons, but fall far short of number that would be necessary to restore lost function [5].

Here a distinct approach to engineering an effective man-made nerve construct for nerve repair is described. This construct consists of numerous bundles of axons, which are embedded in a collagen gel and packaged in a biocompatible conduit. Sized to the length of the damaged nerve, this construct can be directly transplanted to provide a living and functional connection. Researchers hypothesize that nerve constructs spanned by axons may establish or promote functional pathways necessary for nervous system repair that have not been achieved by any other approach. In order for this to be feasible, axons must be grown in a short period of time to lengths that can bridge any size lesion and consist of enough axons to adequately restore function.

Supporting a long held hypothesis [7], research has shown that tracts containing up to a million axons can be mechanically elongated in the laboratory at rates and to lengths that greatly exceeds what an axon can grow on its own. This process is similar to one of two distinct forms of axonal growth that occur in succession during development. First, axons grow out from the neural cell body and find their way to their final destination. After the axon integrates with its target, the growth of an animal induces the continued growth of axons as a result of mechanical stretching [7,8]. Anecdotal evidence of this form of growth can be found throughout nature. For example, the blue whale can grow an estimated 4cm per day and the giraffe’s neck increases by about 2cm per day at peak growth. It has also been shown that sensory axons in the deer antler are forced to grow at a rate of over 1cm per day 9. This process is referred to as axon stretch-growth that likely represents the primary mechanism that drives the formation of long nerves in animals.

Research has found that axons from embryonic dorsal root ganglion neurons (DRG, a type of neuron that resides in the peripheral nervous system) can sustain amazingly high stretch growth rates of up to 10mm/day and potentially much faster. Furthermore, axon tracts consisting of 105 to 106 axons can be stretch grown to 1cm in length within 4 days and up to 10cm in length in only 28 days while remaining healthy in culture and maintaining a normal structure 10. By exploiting this stretch growth process, ideal axonal tissue for transplant can be created in a matter of days for even extensive lesions.

Nonetheless, for clinical application, an embryonic source tissue for human transplantation is controversial and largely unattainable. A more ideal source of neurons would be from adult sources such as organ donors or from the patients themselves. The research team therefore examined the stretch-growth potential of axons from adult rat DRG neurons and subsequently from adult human donors for use in transplantable nerve constructs. Human DRG neurons were harvested from 18 patients, 14 who had undergone a pain management surgery, and 4 who were organ donors. It was found that human and rat DRG neurons survive in culture for more than three months and their axons can be successfully stretch-grown to a length of at least 1cm. The incorporation of elongated axonal tissue into a nerve construct offers an unexplored and potentially important new direction in bridging nerve lesions. Combining living axons derived from adult humans with other promising nerve construct designs may lead to a clinically applicable strategy for the repair of spinal cord and other nerve injuries.

Supported by Sharpe Trust and NIH grants AG21527, NS38104, NS45975.
References:

  1. Evans, G.R.D. 2001 Anat Rec 263(4):396-404
  2. Lee, S.K. 2000 J Am Acad Orthop Surg 8(4):243-52
  3. Fawcett, J. 2002 Spinal Cord 40(12):615-23
  4. McKerracher, L. 2001 Neurobiol Dis 8(1):11-8
  5. Bunge, M.B. 2001 Neuroscientist 7(4):325-39
  6. Hall, S. 2001 J Hand Surg [Br] 26(2):129-36
  7. Weiss, P. 1941 Growth, Third Growth Symposium 5: 163-203
  8. Bray, D. 1984 Dev Biol 102(2):379-89
  9. Gray, C. et.al. 1992 Neuroscience 50(4): 953-63
  10. Pfister, B.J. et. al. 2004 J Neurosci (in press, Sept 8, 2004)
Peripheral Nerve Transplant

peripheral nerve transplant

Objective

Traditional methods for repairing peripheral nerve deficits include bridging the nerve gap with an autologous graft or a bioabsorbable tube. The autologous graft is considered the gold standard for repair of significant lesions within the PNS, but its challenges include the limited supply of donor nerves and donor site morbidity. Researchers sought to test a new concept in which living dorsal root ganglion neurons and mechanically stretch-grown axons are transplanted into a 12mm sciatic nerve gap in the rat.

Design

Thirty rats were divided into four groups

  • Transplant Group: Nine rats were transplanted with the living nerve construct.
  • Reverse Autologous Graft Group: Nine rats were repaired with a reverse autologous graft.
  • Transection/No Repair Group: Six rats were injured without repair.
  • Sham Group: Six rats were subjected to sham surgery in which the sciatic nerve was exposed, but not transected.

All rats were survived for four months.

Outcome Measures

Behavior: Rats were tested using an angle board paradigm in which they were required to stand without slipping on increasingly greater angles of incline.

Electrophysiology

The repaired sciatic nerve segment was recorded extracellularly immediately before sacrifice. Nerve conduction velocities were calculated as a measure of regeneration.

Spinal Cord Transplant

Illustration of the spinal cord transplant

There are an estimated 10,000 patients who suffer spinal cord injury (SCI) each year in the United States and approximately 250,000 chronic SCI patients. Accordingly, there are extensive research efforts to develop techniques that enhance axon growth in the injured spinal cord. A primary goal of these efforts is to promote axon growth across the lesion to integrate with viable tissue on either side and create functional relays.

There have been numerous notable attempts at promoting axon bridges across spinal cord lesions. Some of the previous techniques have been successful in promoting axon sprouting into or around spinal cord lesions in animal models. While promising, this sprouting typically includes only a small number of spinal axons growing a limited distance. It has been proposed that the improvements in functional recovery often found after SCI in these transplanted animals is primarily due to physical and biochemical support for the host tissue surrounding the lesion, rather than the formation of new intraspinal circuits across the lesion. Considering that human SCI lesions typically extend several centimeters, bridging these lesions with axons of sufficient number and length to form functional relays remains an enormous challenge.

Survival of transplanted tissues

In contrast to inducing axon growth by promoting sprouting of axons, researchers have recently developed technology that induces rapid axon growth through continuous mechanical elongation of integrated axon tracts in vitro (Smith et al., 2001; Pfister et al., 2004). This axon stretch-growth technology was recently adapted to create nerve constructs consisting of living and functional axon tracts that are capable of bridging even extensive SCI lesions (Figure: Illustration of the SCI transplant). Most recently, it was found that transplanted elongated cultures from GFP transgenic rats survived four weeks in the injured spinal cord (Figure: Survival of transplanted tissues). These results demonstrate the promise of the lab’s nerve constructs consisting of stretch-grown axons to bridge even extensive spinal cord lesions. The research team is now investigating the functional recovery after SCI in the transplanted animals.

Cultured Axonal Injury
Traumatic Brain Injury

Neurodegenerative Changes Following Traumatic Brain Injury

Traumatic brain injury (TBI) is one of the most devastating diseases due to its high percentage of mortality and disability and its claim of over 1.5 million victims each year in the United States alone. Recent studies have addressed that brain trauma leads to an increased risk of developing Alzheimer's disease (AD) and induces the acute formation of AD-like plaques containing amyloidal-β (Aβ). However, the novel mechanisms of neurodegenerative processes following brain trauma have yet to be identified.

To further explore the potential link between brain trauma and neurodegeneration, researchers used experimental brain injury models in both the pig (by inducing coronal plane rotational acceleration 110°) and in the rat ( lateral fluid percussion injury) to identify acute and prolong neurodegenerative changes after injury. Both animal models can produce clinically relevant neuropathological sequelae in the brain. They have also used human brain material from autopsies to study the connection between traumatic brain injury and AD, and to confirm similar findings gained from animal studies.

  1. Progressive axonal pathology after TBI

    Progressive axonal pathology after TBI

    Traumatic axonal injury (TAI) is an important pathological finding. After rapid mechanical deformation of the brain during trauma, axonal cytoskeleton was damaged and axoplasmic transport was impaired. These processes cause the accumulation of transported proteins, including toxic proteins and peptides that can lead to secondary disconnection. Immunohistochemistry demonstrated high density of axonal swellings and axonal bulbs labeled by NF and APP antibodies persisted up to many years post-injury.

  2. Axonal Aβ accumulation after TBI

    Axonal AΒ accumulation after TBI

    Following brain trauma, a marked accumulation of APP has been found in damaged axons. Furthermore, conversion of axonal APP to Aβ has been observed both clinically and in experimental models. Widespread axonal Aβ in the white matter and examples of axonal Aβ associated with Aβ plaques were confirmed by double immunostaining in brain-injured patients, the rat TBI model, and the swine model of DAI.

  3. Aβ production mediated by β-secretase and presenilin after TBI

    Aβ production mediated by β-secretase and presenilin after TBI

    The primary mechanism for production of Aβ peptides is thought to be via transmembrance cleavage of APP by β-and g-secretases in AD. However, for brain trauma, the rapid production and aggregation of Aβ may result from unique mechanisms related to specific post-injury processes. Emerging data suggests that intra-axonal proteolysis of APP to Aβ is a primary process proposed for Aβ formation in traumatic brain injury. A recent study has found that Aβ production was mediated by beta-site APP cleaving enzyme (BACE) and the catalytic component of g -secretase, presenilin-1 (PS-1), within the axon membrane compartment of peripheral nerves. Further, it was demonstrated that APP functions as a kinesin-1 membrane receptor, mediating fast axonal transport of BACE and PS-1. Researchers found that the co-accumulation of Aβ with APP, BACE or PS-1in damaged axons in both acute and long-term survival human and animal models after brain trauma. These data support the hypothesis that trauma impairs axonal transport and leads to massive accumulation of several key axonal proteins that normally do not encounter each other in such high concentrations. The metabolic mechanism of Aβ formation in TBI shares similarities with Aβ formation in AD.