Technical aspects of spinal cord injections for cell transplantation. Clinical and translational considerations

Brain Research Bulletin 84 (2011) 267–279

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Brain Research Bulletin journal homepage: www.elsevier.com/locate/brainresbull

Review

Technical aspects of spinal cord injections for cell transplantation. Clinical and translational considerations James Guest a,b,∗ , Francisco Benavides a , Kyle Padgett a,b , Eric Mendez b , Diego Tovar a a b

The Miami Project To Cure Paralysis, University of Miami, Miami, FL, USA Neurological Surgery, University of Miami, Miami, FL, USA

a r t i c l e

i n f o

Article history: Received 18 July 2010 Received in revised form 20 September 2010 Accepted 8 November 2010 Available online 16 November 2010 Keywords: Spinal cord injury Transplantation Cells Injection Pressure

a b s t r a c t Spinal cord injections may be used to transplant cellular suspensions for the experimental treatment of spinal cord injury. These injections cause some additional injury due to needle penetration, spinal cord motion during injection, creation of intraparenchymal pressure gradients and hydrodynamic dissection, instillation of a deforming cell mass and possible cord ischemia. It is important to understand these variables to maximize the safety of injections and avoid injury to spared structures. Surprisingly little knowledge exists regarding these variables. Further complicating spinal cord injections is the fact that intraparenchymal events are not evident during injections. As cell injections for spinal cord injury enter extensive clinical testing it is important to both optimize the procedures, and reduce the probability of technical failures. In this review current knowledge and key areas for knowledge advance are identified. These include a need for a more thorough understanding of how the spinal cord is affected by needle entry and dwell, needle-cord relative motion, instillation of highly concentrated cellular volumes, compliance of intact and damaged spinal cord tissue, radial tensile stresses and hydrodynamic forces created by injection, and the rates of pressure gradient dissipation in damaged and intact tissue. We propose that if the variables associated with injury can be identified, injection injury may be reduced and we illustrate the use of ultrasound to monitor injection in a spinal cord model. We also suggest that injectate backout or extrusion be reinterpreted as a clear indicator of excessive intraparenchymal pressure. The strengths and weaknesses of alternatives to direct intraparenchymal injection are also discussed. © 2010 Published by Elsevier Inc.

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Background . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Theoretical concepts related to spinal cord injections . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.1. Physical characteristics of the spinal cord . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.2. Impact of spinal cord motion on injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.3. Tissue compliance, injury volumes, and injections . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.4. Fluid clearance . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.5. Hydrodynamic injury . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.6. Volumes occupied or created by cell injections . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.7. Ischemia . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.8. Cell stability and survival . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.9. Neuroprotection during injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.10. Methods to measure injection injury . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Technical issues pertinent to cell injections . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.1. Devices for cell injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.2. Syringes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.3. Needle design . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

∗ Corresponding author at: Department of Neurological Surgery, University of Miami, Lois Pope LIFE Center, 1095 NW 14th Terrace (D4-6), Miami, FL 33136, USA. Tel.: +1 305 325 7059; fax: +1 305 243 5588. E-mail address: [email protected] (J. Guest). 0361-9230/$ – see front matter © 2010 Published by Elsevier Inc. doi:10.1016/j.brainresbull.2010.11.007

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3.4. Dosage . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.5. Localization of the injury epicenter . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.6. Lesion characteristics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.7. Maintenance of a dry injection area . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.8. Flow of the cellular suspension during injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.9. Injection technique and reduction of extrusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.10. Dural and wound closure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.11. Spinal cord conduction monitoring during injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.12. Open incisional cell delivery . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.13. Post injection imaging . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Alternatives to laminectomy based spinal cord injections . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.1. Delivery of cells into the CSF via lumbar puncture (LP) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.2. Intravenous delivery . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.3. Transvascular delivery . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Minimally invasive spinal cord injections . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Alternative methods of pressure injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Discussion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Competing interests . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

1. Background Spinal cord injuries typically have an injury epicenter where there is evolving tissue necrosis and cavity formation, axotomy, axonal demyelination, glial activation and scarring, as well as parallel endogenous repair processes [6] including neoangiogenesis and axonal sprouting. A longstanding therapeutic concept has been to place cells or tissues into the lesion site to augment repair and to reverse or attenuate the injury consequences. Earlier clinical studies used direct implantation of tissue fragments [89,54] into the spinal cord but maturing regulatory requirements requiring a high level of therapeutic specification and reproducibility, and the scaleup for multi-center clinical trials have led to an increased focus on delivery of purified cellular suspensions. Spinal cord cellular injections aim to place therapeutic cells to a desired target in the spinal cord parenchyma or injury region for experimental treatment of spinal cord injury (SCI) [33,34] or other diseases such as multiple sclerosis or amyotrophic lateral sclerosis (ALS). Clinical trials of intraparenchymal cell delivery in acute [43] and chronic SCI [55] have been conducted in the past several years with further trials imminent. While it may seem quite simple to inject into the spinal cord, cellular injections can be very damaging, and the creation of minimally injurious injections is both an art and science. Central nervous system (CNS) injections are used extensively in neuroscience, and injections of tiny volumes of drugs and solutes can be made with minimal tissue injury [67], but therapeutic spinal cord injections pose substantial additional challenges due to preexisting tissue injury, large injection volumes, high cell concentrations, and high delivery rates. To appreciate the problems associated with therapeutic injections it is important to have insights into spinal cord structure and tissue properties, motion, blood supply, injury responses, and the properties of injection devices. We propose that if the variables associated with injury can be clearly understood, injection injury may be reduced. Our observations arise from testing we have conducted in several animal models including rodents, primates and pigs in the course of planning for translation of autologous Schwann cell transplantation for SCI. Several current spinal cord cell injection strategies aim to inject cells to enhance the repair of preserved axons that may be demyelinated or lack trophic support in the injury epicenter. For this reason, some proposed clinical trials will inject pro-myelinating cells such as oligodendrocyte precursors [3], or Schwann cells [65] directly into the spinal cord at or near the injury epicenter.

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Whether cells should be injected into naïve versus fully damaged tissue is an a important consideration that may be dictated by the biology of the injected cells, e.g. their ability to migrate, survive, whether they divide, their mechanism of action, and the extent and shape of the injury. Preclinical studies aimed at treating neurodegenerative diseases such as amyotrophic lateral sclerosis have utilized small injections of neural stem cells into naïve cord [73] and human testing of this paradigm has been initiated (http://www.neurology.emory.edu/ALS/Stem%20Cell.html). Human SCI studies, however will need to use larger cell doses, either as multiple or single large injections. If this were not the case, the anticipated risks associated with cell injections could be reduced. A central problem with current clinical injection approaches is that the access to the spinal cord is a single event associated with a major surgery. If a series of smaller injections could be made over a longer time span, risks of injection-related cord injury per intervention might be reduced and overall efficacy increased. Injection injury to the spinal cord presumably occurs due to known mechanisms similar to those associated with traumatic injury e.g. compression, laceration, excitotoxicity, apoptosis, ischemia, etc. (reviewed in Hood and Guest [38]). Because recovery from SCI may depend on the preservation of a small number of preserved axons and neurons in the injury region, it is essential that these not be further depleted by procedures aimed at repair. Furthermore, added injuries at critical time points after the index injury might influence the extent of the evolving major sequelae of SCI such as pain, spasticity, and autonomic dysfunction in an unpredictable manner. Although very delicate small volume injections may be made over protracted periods in laboratory experiments (Fig. 1), clinical injections will need to be made within the realistic parameters of the human operating room in which surgical and anesthetic time, as well as complication risks associated with surgical duration are limiting parameters. 2. Theoretical concepts related to spinal cord injections 2.1. Physical characteristics of the spinal cord The spinal cord may be modeled as a cylinder of heterogeneous anisotropic elastic compact tissue [9], with a fibrous surface lining, suspended in fluid, tethered by small ligaments, and having intermittent motion and tissue waves related to cardiac pulsation

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Fig. 1. Microinjections of autologous iron oxide nanoparticle containing primate Schwann cells made in the primate cervical spinal cord using conventional stereotaxic techniques. All injections were made using a dilution of 10,000 cells per ␮l in DMEM-F12. Cells were delivered at 0.5 ␮l/min. The spinal cord was perfused 40 days after injection. (A) A series of vertical cellular trails were created in the primate cervical spinal cord. The site of each trail is marked on the dura by a suture. (B) A 5 ␮l injection of autologous primate cells in the medullary pyramid. Cells are identified by Prussian blue reaction. Note absence of cavitation and visible injection tract. (C) Typical stereotaxic micromanipulator with Hamilton microsyringe attached. (D–F) The trails which had iron oxide nanoparticle-loaded Schwann cells are visible on ex vivo MRI (4.7 T). Each trail consists of a 10 ␮l injection delivered over a 20 min period, with a 0.25 mm needle extraction dorsally each minute to create the linear trail. In G. an axial section shows the cellular trail within grey matter with minimal evident tissue disruption. Lack of migration is characteristic of implanted Schwann cells.

and respiration [7]. In the normal uninjured spinal cord, there are no anatomic dead spaces or expendable surgical tissue planes. This means that any entry to spinal cord from the surface causes some injury to axons, glia, or neurons. Normal spinal cord tissue is highly susceptible to edema and ischemia. Subarachnoid vessels are susceptible to vasospasm during exposure [3] and manipulation, and the vascular supply is tenuous in some regions such as the midthoracic water-shed area. Thus, the spinal cord is a very unforgiving structure unsuited to manipulation. The previously damaged spinal cord is also vulnerable to tissue injury but may offer some safer corridors for injection approach through degenerated tissue. The injection susceptibility of previously injured as compared to uninjured spinal cord tissue has not been described. In the acute and subacute period after a severe contusive or compression injury there is cord swelling, inflammation, and tissue softening with areas of necrosis that will eventually become cavitated. At chronic time points the epicenter of most damaged spinal cords with some preserved parenchyma has complete grey mater loss with a margin of pia and fibroblastic scar and an underlying thin rim of preserved gliotic white matter [17,26].

2.2. Impact of spinal cord motion on injection If the spinal cord moves around a rigid injection needle, injury will occur; therefore reduction of spinal cord motion is desirable for injections. Most spinal cord injections are performed under anesthesia with the subject in the prone position such that the thorax and abdomen rest against a firm surface. As a result, during breathing, the vertebral column rises and falls vertically, inducing considerable spinal cord motion, especially in the thoracic region. The human spinal cord also undergoes pulsatile longitudinal motion induced by cardiac pulsations [42,21,14], and in concert with CSF flow the spinal cord also undergoes small changes in area. Spinal cord motion consists of the direction and magnitude of total displacement and the velocity of motion [62]. Because this motion causes difficulties in acquiring magnetic resonance images, it has been extensively studied in this regard. In the intact spinal canal, CSF flow is associated with cycles of cranial and caudal cord motion [36,53]. Spinal cord and spine motion are not uniformly coupled and thus spine mounted injection devices [73] do not fully compensate for spinal cord motion. Thus, if the cord moves repet-

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itively relative to a fixed needle, a cavity as opposed to a narrow injury tract may be created due to the combined rostral-caudal and dorsal-ventral motions. The term “pistoning” is sometimes applied to this phenomenon. This injury may be proportional to the extent of motion and the duration of needle dwell within the tissue, although we speculate that it is non-linear, meaning that there is probably a time point at which additional motion adds no injury. Needle injury may also be related to trauma caused by pial penetration and the rate at which the needle penetrates the spinal cord. The human spinal cord is tethered by the denticulate ligaments between the pia mater and dura mater [19,86]. Generally, nerve roots do not come under tension during spinal cord physiological motion and thus are non-constraining. Motion may be constrained by aspects of surgical preparation such as dural tack-up sutures because denticulate ligaments attached to dura may be tensioned. Also due to subarachnoid scarring, spinal cord motion may be reduced at the injury site. Experimental surgical techniques to reduce the impact of respiratory motion during spinal cord injections have included suspension of the thorax via spinal process clamps so that the abdomen and rib cage move away from the spine during inhalation, or an elastic suspension sling [88]. Another technique is to use a period of apnea or very low tidal volume ventilation during injection. Prior to apnea the animal is pre-oxygenated so that the oxygen saturation is 100% and the blood contains extra diffused oxygen allowing several minutes of apnea tolerance prior to a drop in saturation. One consideration is that as PCO2 rises spinal cord blood flow may increase in regions where autoregulation is maintained [76], but several minutes of apnea can be tolerated in large experimental animals without apparent harmful effects (Levene, Solano, Guest, unpublished data). 2.3. Tissue compliance, injury volumes, and injections If injections are performed into spinal cord cavities or areas of fluid necrosis, the tissue compliance (C = V/P) may be high and allow larger volumes to be introduced without creating large tissue pressure gradients providing the initially occupying fluid or debris can be displaced out of the spinal cord. Thus it should be useful to match injection volumes to lesion volumes anticipating that excessive volumes may create damaging pressure gradients. Surprisingly, there are no publications regarding the volumes of human spinal cord injuries [58] although numerous studies have calculated injury volumes after SCI in animal models [50]. The lack of human studies with lesion volume calculations may reflect the fact that post SCI surgical decompression MRIs are not usually obtained unless there is a clinical indication during the subacute injury period. The approximate volume of chronic human spinal cord injuries may be readily calculated from clinical MRIs but calculation of acute injury volumes is more complex due to the difficulty in discerning “dead” tissue from viable. It is challenging to interpret the significance of volume in spinal tissue but a single nanoliter occupies a sphere with a radius of 124 ␮m, which is a meaningful space within spinal cord tissue. The entire estimated volume of the human T8 spinal cord segment is 690 mm3 , or 690 ␮l [47]. If we use a theoretical 3.5 mm radius × 18 mm length as a model cylinder of the T8 spinal cord segment and allow for 1 mm of preserved tissue rim, (2.5 mm radius × 18 mm length) we produce a lesion volume of 350 ␮l. Many strategies are envisioned to involve filling the injury volume with cells with a view to causing tissue formation to fill the lesion region. 2.4. Fluid clearance Cellular suspensions consist of combined fluid and cell mass components. Fluid clearance is a potential mechanism to increase

compliance during injections. Studies in the brain have demonstrated that some fluid clearance may occur by bulk fluid flow and diffusion along the extracellular spaces and absorption to the CSF or blood [72], especially through white matter. When focal edema is established, the extracellular spaces open increasing this flow. However, these extracellular channels consist of membrane interstices and ground substance may be traumatically dilated by acutely created excessive pressures. The rates of brain fluid clearance have usually been measured in hours and not minutes, however one study showed that with very slow pressure injections of solutes a diffusion rate of 44 nl/5 min was associated with excellent tissue preservation [63]. At that rate it would take 114 min to deliver 1 ␮l. For brain microinjections in rodents, a rate more than 1 ␮l/min has been associated with tissue damage and even higher rates are associated with a several fold increase in the effective injection size [67] and associated tissue damage. A very viscous injectate of highly concentrated cells contains only a relatively small amount of solute fluid that could be dissipated and may behave more like a deforming gel inducing tissue expansion, cavitation and dissection. It is unknown how the presence of a traumatically altered blood–spinal cord barrier in the context of SCI may affect fluid clearance following injection. 2.5. Hydrodynamic injury The key forces created within the spinal cord tissue by injection may be described in accordance with the tissue’s properties such as compliance, elastic modulus [49,87], stress and strain, and vulnerability to radial tensile stress- “tearing stress” in response to pressure gradients. A key question is- at what pressure level or gradient does traumatic dissection of the tissue occur? A pressure gradient is necessary for extrusion of a cell suspension from the needle into the tissue. Although a syringe and attached components may behave as a hydraulic system with equal pressures at all points, this is not true of the point where the injectate exits the syringe, where a lower pressure must exist. As injection proceeds the increasing volume within the tissue will eventually raise the intraparenchymal pressure due to decreasing compliance, creating a tissue pressure gradient within the injectate and surrounding parenchyma (Fig. 2). As a large volume is delivered, the spinal cord may distend visibly with resistance mainly provided by the pia mater. This gradient will naturally dissipate along the paths of least resistance and if intraparenchymal pressure exceeds the tensile and shearing limits of the parenchyma, spinal cord tissues will undergo hydrodynamic dissection due to fluid pressures. This means that the fluid pressure will dissect the tissue, opening up space, until the intraparenchymal pressure is again lowered (Fig. 2). We currently believe this is a major source of injection-related injury associated with large injection volumes (Fig. 3). The fact that the spinal cord surfaces are only exposed to barometric pressure during open surgery may permit a slighter greater cord distension (increased compliance) because the resistive CSF/venous pressure is lacking. We speculate that acutely or subacutely damaged spinal cord tissue may be more vulnerable to such fluid dissection due to regions of necrosis and inflammation. Aside from visible swelling or injectate backout another indicator of the occurrence of excessive injection pressures is extrusion of cells within the central canal, which may be seen histologically [28]. Additional evidence for the hydrodynamic dissection concept comes from autopsy studies. In some severe human spinal cord injuries instantaneous displacement of tissue occurs rostral and caudal to the compressive lesion, a phenomenon known as “toothpasting”, that is clearly related to an abrupt local pressure increase (J Guest, R Bunge, unpublished data). Regarding the magnitude of forces involved, under mechanical elongation testing it has been shown that strain failure of normal guinea pig grey matter and white matter occurs at 45 and 65kPa

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Fig. 2. Illustration of concept of hydrodynamic dissection pressure gradient-total injected volume curves. Left panel. As injection proceeds with delivery of an increasing volume, intraparenchymal pressure slowly rises linearly, the injectate is illustrated as a sphere which exerts a pressure. Middle panel. As tissue resistance limits are strained, the pressure rises in a non-linear fashion and ss the injectate volume increases in size, distension of the local spinal cord occurs. Right panel. At the “breaking pressure” there is failure of the local tissue resistance and the injectate dissects into tissue along the planes of least resistance leading to an abrupt reduction in tissue pressure. During this stage, as injection continues, dissection also continues and tissue pressures remain elevated above baseline.

respectively [40]. To assist understanding the significance of these numbers, they are approximately 50% of atmospheric pressure (101.3 kPa) [1 psi = 6.89 kPa = 51.7 mm Hg]. Average human blood pressure is 2.3/1.5 psi (16/10.3 kPa), which is more than sufficient to dissect a space occupying hematoma in the brain if a blood vessel fails. Therefore, relatively small pressures may be sufficient to cause hydrodynamic dissection in the spinal cord. Given that many microsyringes are engineered to withstand 1000 psi or more before failure, a potential to generate very large tissue pressures during injection exists. 2.6. Volumes occupied or created by cell injections Cells delivered as suspensions by injection disperse into the areas of least resistance. Some cell types subsequently show substantial migration while others do not, Schwann cells or bone marrow stromal cells show a tendency to spontaneously form linear bundles parallel to white matter tracts [37]. There is little control over the eventual structure induced by the cells three dimensionally. A cellular injection technique that is under explored is the progressive extrusion of cells to form cellular trails using a needle withdrawal extrusion technique [57,16]. For cells with limited migration this may be an excellent technique to create linear cell trails to facilitate regeneration (Fig. 1). An apparently low risk injection paradigm is to inject cells into a preexisting cavity surrounded by a shell of preserved parenchyma. Here the main risks are to preserved subpial axons where dorsal needle entry could damage surviving sensory fibers, especially those of the dorsal columns. For this reason, it may be preferable to enter the spinal cord via the dorsal root entry zone, avoiding the dorsal columns. Such cavities vary in their contents, ranging from those containing only fluid to those containing extensive mesenchymal tissue including Schwannosis, and others with preserved spanning strands of spinal cord parenchyma within which come axons have been identified [71,70]. Whether these contribute to function has not been determined, but it seems prudent to attempt to not damage these strands. To the extent the cavity

contains empty space, the injection strategy is simplified. Once within the tissue cavity, needle motion relative to the spinal cord is less concerning than within axon-containing parenchyma. Furthermore, an injection may be made into a space without creating hydrodynamic tissue dissection if an excessive volume is not used. Within cavities containing septations of tissue a more delicate technique is advisable Retention of cells within large chronic cavities may be more difficult than within parenchyma for several reasons including extensive cell death, lack of substrate for adhesion, slow vascularization. In this setting, combinations of cells with matrix biomaterials may be especially helpful to stabilize the cell implant and enhance new tissue formation [66]. In an acute or subacute injury there may be value to allowing the egress of necrotic fluid prior to, or during injection, to reduce the intraparenchymal pressure and increase compliance.

2.7. Ischemia During large volume injections into the spinal cord visible swelling may occur. The potential for spinal cord injections to cause ischemia has not been previously considered but we speculate that large volume injections could raise tissue pressure to ischemic levels that may persist until the elevated pressure can be dissipated. The normal tissue pressure of muscle is 4–5 mm Hg and the tissue pressure in the optic nerve of dogs has been recorded to be an average of 6 mm Hg [61]. A classic example of ischemia caused by edema and hematoma is compartment syndrome, which is considered imminent in muscle when tissue pressures rise to 30 mm Hg (0.6 psi). Interstitial injections have been a cause of compartment syndrome in many reported cases. Using a spinal cord distraction technique to induce tissue tension in non injured spinal cord it was observed that (somatosensory evoked potentials) SSEPs were maintained until a tissue pressure of 47 mm Hg was obtained, at which point a sudden drop in spinal cord blood flow occurred and SSEPS were lost [41]. We thus recommend that future studies measure intraparenchymal pressures and their dissipation during spinal cord injection.

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Fig. 3. Experiment illustrating hydrodynamic dissection. In this experiment an uninjured pig underwent T9 stereotaxic injection of autologous Schwann cells concentrated at 200,000 cells per ␮l. During injection the animal was not ventilated. Two injections were made, on the right 75 ␮l was injected over one minute with slow simultaneous needle removal. On the left 100 ␮l was injected over 2 min followed by 2 min of dwell time and slow simultaneous needle removal. Injectate backout occurred upon needle removal on the right but not the left. Panels A–C are MRI images obtained within 2 h of injection. Panels D–F are ex vivo MRI images obtained 7 days after injection. In A, linear tapered T2 signal is seen to extend for a total of 4 cm. Spinal cord swelling is evident. In B, cavitation with possible hemosiderin signal is observed. (C) An axial image of the surgical site shows the loss of normal grey matter signal after injection. In D, a sagittal image, the persistence of induced cavitation, cord swelling and linear T2 signal change is evident. In E, a coronal image, the cavitation is observed to be unilateral, on the right side, and appears to occupy grey matter. Focal cord swelling is evident. In F, both injection tracts are evident extending into the dorsal horns of the grey matter. The dimensions of the spinal cord are 6.1 mm × 6.7 mm. We interpret the events as follows: the primary injection of 75 ␮l caused hydrodynamic dissection as evidenced by injectate backout after needle removal. Although the second injection occurred from the left side we think the injectate crossed to the right side (the path of least resistance) and caused further dissection of the grey matter [Ex vivo MRI of the injured spinal cord was performed to obtain high-resolution standard imaging before processing the tissue for histopathology. The spinal cord was placed in fixative for a minimum of 1 week after removal to ensure adequate fixation. Twenty-four hours before ex vivo MRI, the tissue was washed and placed in phosphate-buffered saline at 4 ◦ C until scanning. Tissue was again placed in fresh phosphate-buffered saline solution just before the 24-h MRI procedure. T2-RARE experiments were collected in 3 orientations (sagittal, coronal, and axial). The coronal and sagittal data sets were collected with a slice thickness of 0.4 mm and an in-plane resolution of 68 ␮m × 70 ␮m. The axial T2-RARE data were collected with a 0.6-mm slice thickness and an in-plane resolution of 70 ␮m × 70 ␮m.]

2.8. Cell stability and survival “Stability” refers to the viability and potency of the transplanted cells within “the final container cell product” at the time when they are actually administered as compared to when the release tests are conducted. The various effects of concentration in a small media volume, time on ice, vacuum loading, exposure to medical tubing or metal, and shearing stress related to injection must be considered, in addition to the cellular stress that is induced in the process of removal from cell culture, or recovery from cryopreservation. It is important to verify that cells are viable at the time of delivery. Conventional tests such as Trypan blue or propidium iodide can show which cells are dead but do not predict which cells will die. We augment cell stability testing in our preclinical transplant studies by returning a small aliquot of injected cells to cell culture and assessing their viability. Flow of solutions through narrow channels such as a needle induces shearing stresses due to inequalities of flow velocity adjacent to the channel wall and channel midpoint. These shearing stresses can damage cell membranes possibly causing cell injury and death. Shear stress is a function of dynamic viscosity multiplied by  fluid velocity/height of boundary. As a useful reference, hemolysis of blood is induced by shear stress exceeding 1500 dynes/cm2 [52]. As an example, the passage of 0.5 cm3

of one centipoise solution (water) through a 30G one inch length needle in 1.5 s at 35 psi causes an average fluid shear stress of 7340 dynes/cm2 . Using these parameters, blood, with a viscosity of 10 centipoise would have a fluid shear stress of 73,400 dynes/cm2 , which would destroy the cells. [Cell biology: a laboratory handbook, Volume 1, Julio E. Celis, page 237]. Even small levels of shear stress (10 dynes/cm2 ) have profound effects on activation of molecular cascades in cells [24,79]. Thus high flow rates through narrow needle bores should be avoided, and the survival of the cells actually ejected should be verified. The factors that govern cell survival after injection into spinal cord tissue have not been fully determined but likely include access to oxygen, energy substrates, essential cofactors, tissue temperature, pH, and potential for substrate adherence. Studies of Schwann cells transplanted to contusion cavities showed a 22% survival with most cell death occurring in the first 24 h [35]. Cell survival after injection will depend on avascular diffusion of metabolites for a considerable period until new vascularized tissue is formed. Although seemingly improbable, the long-term survival of cells that have been encapsulated and thus fully dependent on CSF diffusion is encouraging [85]. The diffusion characteristics relevant to an acutely placed intraspinal cell mass within an injured spinal cord have not been established. The consequences of the death of injected cells on inflammatory activity in evolving lesions has

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not been adequately studied. A further issue is that due to the effects of transplanted cells on formation of new tissue, and recruitment of endogenous cells their long term survival may not be essential. An example is that extent to which transplanted olfactory ensheathing glia elicit immigration of endogenous SC to the transplant site. 2.9. Neuroprotection during injection In clinical practice two common strategies to reduce injury during procedures that involve spinal cord manipulation such as tumor removal, spinal cord untethering, or excision of vascular anomalies are the use of intravenous steroids and local or systemic hypothermia [25]. Perioperative steroids have not been proven to provide spinal cord neuroprotection despite their common use. It is harmful to administer methylprednisolone as per the NASCIS II protocol for neuroprotection if the same protocol has been administered within several days for SCI therapy [59]. Therefore, we disadvise a second course of perioperative steroids if these have been given for the primary SCI. The only proven peri-operative protective benefit of spinal cord hypothermia is for ischemic neuroprotection [8], a very important consideration when the thoracic spinal cord is manipulated. Mild systemic cooling appears safe in patients undergoing elective spinal surgery for myelopathy [25]. Because neuroprotection is known to be more effective if applied prior to, and during injury, it seems likely that a method for peri-injectional neuroprotection may be found. Anesthetic agents such as ketamine [39], or propofol [46] may have neuroprotective effects. Thus, potentially neuroprotective variables such as temperature and perioperative drugs need to be carefully controlled in studies that evaluate injection injury. 2.10. Methods to measure injection injury In our translational development toward autologous Schwann cell transplantation we have emphasized parameters that can be measured clinically such as post-injection MRI signal change, intraoperative neurophysiologic conduction, and behavioral observation. Each of these allows real time or early feedback re the impact of the injection procedures. In addition to these we study histological changes such as astrogliosis and cavitation in the post-perfusion spinal cords. It may be argued that if, at the end of the experiment the spinal cords that have received transplants show better tissue preservation than those of media- injected non-transplanted controls, the issue of injection injury is moot. We think that this is a flawed argument that only establishes that the cell effect apparently overcame the additional damage created by delivery. It has been often observed that after injection, animals show transient worsening, which recovers [73]. Again we think that the ability to create injections without causing new neurological injury is an important aim. 3. Technical issues pertinent to cell injections Spinal cord injections typically consist of the sequence of surgical exposure, sharp pial perforation, needle passage through the parenchyma, instillation of a volume of cells and medium under pressure, followed by “stabilization time,” and needle withdrawal. Injection injury may be caused by cord compression at needle entry, laceration/cavitation due to needle/cord motion, fluid dissection of tissue, hemorrhage, ischemia due to vasospasm or excessive tissue pressure, and injection-initiated secondary injury processes including inflammation. There may be direct axotomy, neuronal and glial death, microglial activation, pial and subarachnoid scarring and reactive glial scarring. Currently, spinal cord injections are made using open laminectomy techniques that

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require surgical exposure of the spinal cord. This generally means that injections occur via penetration of the dorsal surface of the spinal cord where residual sensory axons of the dorsal columns may be present. The associated morbidity of clinical injections for spinal cord repair should be considered to include those of standard spinal surgical procedures including the physiological stresses of anesthesia, tissue exposure, blood loss, infection risk including meningitis, CSF leakage, and temporary interruption of rehabilitation progress, because these procedures are usually elective. 3.1. Devices for cell injection Most neuroscience investigators are familiar with conventional stereotaxic equipment for CNS injections (Fig. 1). For animal surgery these consist of immobilization frames, micromanipulators, syringes, and syringe pumps such as those produced by Hamilton, Kopf, Stoelting, Harvard and other companies. Human application, however, requires medically approved devices, preferentially those which are disposable, which makes it more difficult to use the creative solutions often applied to experimental injections. Key considerations for human application include feasibility of use in the operating room, rapid assembly, reliability, potential for sterilization, reproducibility, non-obstruction of the visual field afforded through a surgical microscope, and resistance to accidental perturbation or displacement once deployed within the spinal cord. An equipment failure that would preclude successful transplantation would be very unfortunate and have serious consequences for a clinical experimental protocol, especially if the patient was under anesthesia and surgically exposed. It is vastly more important to anticipate a variety of failures in human surgeries than in animal experiments and all contingencies should be anticipated. Backup and failsafe plans must be constructed and appropriate replacement equipment should be on hand. Experienced surgeons know that various unexpected problems will often arise. Truly disastrous events would be, e.g. the patient waking and moving with a fixed needle within the spinal cord or an error in setting an infusion pump causing rapid injection. We recommend the use of paralytic agents during the injection procedure, even if neurophysiologic testing is to be conducted. Large animal studies that allow an extensive testing of all steps of the human procedure are invaluable and are almost certain to disclose unanticipated technical issues, and are highly recommended prior to human surgical testing. We feel that aside from the pure scientific merit of such studies, the practical ability to pre-test the interplay of anesthesia, surgical exposure, device function, cell delivery, and explore limits, is warranted, so that the lessons obtained occur in the experimental animals and not in patients. We further recommend adherence to a checklist methodology to ensure that key issues related to the safety of spinal cord injections are not overlooked. Two differing examples of injection techniques are provided by completed human studies. In the Proneuron Phase 1 and 2 trials, activated autologous macrophages were injected utilized hand-held syringe injection techniques [43], which are subject to inter-operator variability regarding depth of placement, motion during injection, and injection rates, but are simple, more rapid to deploy and more flexible regarding approach angles. A protocol for transplantation of suspensions of olfactory ensheathing glia utilized a more rigorous injection paradigm. A surgical operating table mounted stereotaxic device [20] was deployed. Then, a large number of small injections were made into the damaged cord and the normal cord above and below the lesions. Four injections of 1.1 ␮l were made during each penetration at set depths from ventral to dorsal, as many as 318 injections were made. The cell concentration was 80,000 cells/␮l, and the needle used was 28G

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with a 30 degree bevel. These small cell volumes are unlikely to create serious damage but given that each spinal cord penetration itself causes injury, we consider that fewer injections would be optimal. 3.2. Syringes Syringes are designed to allow liquid volumes to be injected at precise rates and their design and reliability is especially important for the injection of viscous fluids such as highly concentrated cell suspensions. Syringes can generate very high pressures to produce flow, and components such the plunger and barrel are susceptible to friction. When pressures are excessive they may fail, typically at points where components are joined. Because of the incompressibility of water, it often difficult to appreciate the amount of pressure being generated in a syringe other than by a sense of plunger pressure. For this reason, the use of downstream plastic tubing containing a segment of air is useful, because the air can compress indicating the extent of outflow resistance. Syringe failure could lead to loss of injectate, and possible contamination of the injectate. Some injection needles, especially smaller gauges (e.g. 27–33G) are easily occluded by a concentrated cell suspension. This may occur when the injectate is allowed to evaporate at the needle opening and is more likely when multiple sequential injections are placed. It may be helpful to use a stylet to avoid this complication by keeping injectate from entering the needle until the time of injection. In general, it is important to avoid opening syringe components to air once cells are loaded to avoid contamination. The syringe barrel and plunger is well suited to being firmly secured within apparatus such as syringe pumps, which can provide excellent control over infusion rates. 3.3. Needle design Differences in needle design may affect the ease of penetration through the pia, the degree to which tissues stick to the needle, tissue coring, inadvertent needle bending causing targeting error, and the risk of vascular damage. The passage of a needle through tissue may be associated with either sharp cutting, compression, or deflection depending on needle characteristics such as stiffness, sharpness, and bevel design. There are many variables in needle design including inner and outer diameter, bevel shape and orientation, and the alloys employed in construction. The bevel length and angle affect the injectate dispersion and direction. It is also quite common to observe that even after the pia is adequately perforated, spinal cord tissue sticks to the needle as it is being advanced forming a small depressed tissue cone. For this reason, we often over advance the needle by 1–1.5 mm in large animals such as pigs and primates and retract it back, at which point the visible surface tissue levels. Although pulled glass micropipettes are excellent for creation of small injections, the possibility of a broken small glass fragment retained in the human spinal cord is unacceptable, and needles must be sufficiently robust to withstand injection stresses. Glass pipettes offer the advantage of a small needle diameter, and a tapering shape. Our experience indicates that they advance more precisely and are associated with very small intrinsic penetration injuries, as opposed to larger metal needles. Needle technology has been quite static and there are several conceivable variations that could reduce motion related injection injury, such as needles that have the potential to be in either a rigid or flexible state. Novel smart materials such as magnetorheological elastomers and electroactive polymers [29] offer the promise of development of materials whose rigidity could be varied by magnetic fields, current, or temperature. Various hydrophilic and siliconized coatings have been proposed to reduce needle–tissue friction.

3.4. Dosage Effective cell doses for human SCI have not been established. At this point of evolution, doses are extrapolated from data with experimental animals, predominantly rodents. Such extrapolations may be quite sobering. For example, in published studies of Schwann cells a dose of 2 million rodent cells in 6 ␮l has been injected into the spinal cord injury epicenter [68]. Depending on how the extrapolation is calculated to the human, generally using relative spinal cord volume, doses of 100 million or more cells result. One hundred million human SC is 420 ␮l of packed cells. If diluted by one-half to give 120,000 cells per ␮l, a delivery volume of 840 ␮l would result. This is a massive volume that exceeds the total volume of the human T8 spinal segment [47]. Thus, it is very important that further careful dosing studies are performed in preclinical models, including large animals [73] to determine both minimum effective doses and the maximum tolerated dose [88]. Regarding injection injury, Marsala et al., were able to perform 20 injections per side of the lumbar spinal cord each of 0.5 ␮l, using an 80–100 um pulled glass pipette tip without obvious neurological deficits in uninjured rodents, when 35–45 injections per side of neural stem cells were transplanted following ischemia, histological evidence of added injury was modest [56], in a subsequent study in pigs, injections of greater than 6 ␮l into uninjured parenchyma caused obvious neuronal damage [88]. 3.5. Localization of the injury epicenter Most cell therapies aim to populate the injury epicenter with therapeutic cells and have these cells extend into the uninjured parenchyma on either side of the lesion. Therefore, accurate identification of the injury epicenter is necessary. Once the dura is opened and retracted, a region of dorsal spinal cord surface scarring is often apparent in experimental animals and humans. Subdural arachnoid adhesions may be present and require lysis. Although hemosiderin staining may be visible on the cord surface, unless a large cavity is present the actual location of the intraparenchymal epicenter of SCI may not be obvious. In this situation, an error of injection targeting may occur. Although relative landmarks based on pre-surgical MRI or other imaging may be determined, these may not be fully reliable due to shifts associated with surgical exposure and dural opening. Ultrasound is useful to evaluate the traumatized spinal cord [60,51] and can accurately localize a tissue cavity if one is present [56]. The normal spinal cord parenchyma is hypoechogenic, but the central canal is hyperechogenic as are tissue cavities and fibroglial scarring [68]. We have conducted preliminary ultrasound experiments in a gelatin model of the spinal cord containing fluid filled cysts which allowed us to verify needle placement and visualize injections in real time (Fig. 4). Use of standard ultrasound probes requires that the operative field is immersed in fluid which might increase the risk of cell leakage into the CSF, however, cord surface probes could be developed. 3.6. Lesion characteristics The risks associated with injecting into an established cavity as opposed to a subacute contusion are different. A chronic cavity represents a space that may be filled before a significant pressure rise occurs. Furthermore, the boundary of an established cavity may be gliotic and provide increased resistance to tissue dissection. Repair processes such as neoangiogenesis, gliagenesis, and remyelination may have completed and be less susceptible to perturbation. We speculate that an evolving injury is more likely to be susceptible to injection damage. Injections from a standard needle provide little control over the injectate dispersion, which typically will occupy that volume which has the least tissue resistance. If

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Fig. 4. Gelatin spinal cord cyst model spinal cord to test the ability to make injections using ultrasound guidance. (A) Collagen gel cylinders with dimensions similar to human spinal cord are produced. Citrus segments are embedded in the gelatin cylinders forming small fluid filled cysts (arrowhead). (B) Indicator dye (methylene blue) turns pale green when injected into an acidic citrus segment. Here injections have been made using a stereotaxic micromanipulator and a 100 ␮l Hamilton syringe while the collagen gel cylinder surrounded by a nitrile sheath to emulate dura is immersed in a saline bath. Each injection, control (arrowhead), and intravesicular (arrow) was 20 ␮l. (C and D) Real-time ultrasound images of gelatin spinal cord cysts being injected. The layers are indicated. A clinical ultrasound machine and probe used for tumor localization during spinal cord surgery was used. Deformation of the cord surface due to needle penetration is visible (asterisk), the needle is visible (arrowhead) extending into the citrus vesicle (second arrowhead). (D) The layers visible using ultrasound imaging are shown in the sagittal and axial plane. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.)

the mechanism of cell effect is to repair axons in the residual shell of the spinal cord the manner in which the cells are released and organize is less critical than if the goal is to promote regenerative growth through the lesion area. To illustrate this point, in humans with chronic SCI, there is often an extensive ingress of Schwann cells and sensory axons, a phenomenon called Schwannosis [13]. Such Schwannosis may naturally partially fill cavities but the fascicles are disorganized and form whorls without linearity. To reconstruct longitudinal pathways, a linear organization is needed and if cells supported only whorling axon growth, no benefit would be obtained.

3.7. Maintenance of a dry injection area There is often some fluid at the injection site including blood and CSF. If cellular extrusion occurs into the CSF, cells could potentially be widely disseminated. Thus, we are very careful to prepare the injection site so that CSF and blood will flow outwards into cottonoid wicks during the injection procedure. We also apply light suction to the cottonoid wicks to remove fluid more rapidly. This also allows injectate backout to be more accurately detected and removed.

3.9. Injection technique and reduction of extrusion An important common problem observed during spinal cord injection is that the needle causes compression of the spinal cord focally. This is due to the resistance of the pia mater to perforation. Eventually, as pressure is applied, the pia mater may be suddenly perforated and the preceeding pressure may cause substantial cord compression, with subsequent uncontrolled rapid cord penetration. To avoid this problem, it is often necessary to perforate the pia with an alternative instrument prior to inserting the injection needle. If surface vessels are avoided apparent bleeding is usually negligible. Injectate extrusion is an important observation because it indicates that the spinal tissue tolerance to volume and pressure has been exceeded. It follows that having reached this limit, injection damage due to hydrodynamic dissection forces are likely. Extrusion or back-out may have causes other than high intraspinal pressure, such as a large pial entry point, a very thinned spinal cord with little surface parenchymal resistance, or an inadequate “dwell” time. Dwell time is a period following injection in which the needle is left in position. It is assumed that the tissue pressure gradient created by the injection is partially dissipated during this period, which is generally empirically set at 1–5 min, reducing the probability of backout/extrusion after the needle is removed. Although extrusion may be frustrating, it is more desirable than hydrodynamic dissection.

3.8. Flow of the cellular suspension during injection 3.10. Dural and wound closure Very viscous cellular suspensions may be subject to uneven injection flow and intermittent resistance if clumps of cells form and transiently block the injection needle. Due to gravity, the cells may layer out within the syringe or tubing further increasing viscosity. Therefore, it is important to maintain an even cell suspension using agitation or other methods. At lower cell concentrations, these problems are less evident. We consider a cell concentration of greater than 100,000 cells per ␮l to be highly concentrated and are working with concentrations as high as 200,000 cells per ␮l in order to achieve high cell doses in smaller volumes.

It is very important that the injected spinal cord not be deformed or squeezed after cell injection. Care must be taken during dural closure, wound closure, and initial animal handling to avoid compressing or bending the cord as that could cause injectate extrusion and additional spinal cord damage. Usually, it is possible to close the dura mater primarily in large animals and humans but there may be a role for duraplasty to augment the local subarachnoid space [77] to prevent occurrence or recurrence of dural subarachnoid scarring. Muscles will show some swelling after surgery and

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should not be sutured too tightly over the laminectomy site. If the dural closure is adequate the use of a surgical drain may be beneficial to reduce the collection of inflammatory fluid and hematoma in the wound cavity. 3.11. Spinal cord conduction monitoring during injection If neurophysiological conduction is maintained after SCI, (incomplete injury) neuromonitoring techniques may be useful to monitor for, and minimize, injection-associated injury. Clear predictive criteria have been established for the clinical significance of changes in motor or sensory conduction during spinal cord surgery, however the significance of changes that occur during spinal cord injection procedures is unknown. Spinal cord conduction testing has been applied during fetal tissue transplantation in the spinal cord [83] and during spinal cord cyst aspiration [69]. Our preliminary data in a pig spinal cord injury model indicates that large injections can cause loss of motor and sensory evoked potentials correlated with new post-injection behavioral deficits (Levene, Solano, Benvenides, Guest, unpublished data). It is conceivable that membrane potential changes associated with the injectate effect of enlarging the extracellular spaces might account for such changes, but we recommend that the safety of injections be established in incomplete injury models using conduction testing correlated to other outcomes. The argument may be advanced that if there is no clinical deficit following an injection that “occult” changes such as might be observed in spinal cord conduction testing are irrelevant. We take the position that all possible advantages should be employed as experience with clinical injections is built. 3.12. Open incisional cell delivery The pia has limited elasticity and when there is extensive spinal cord edema and swelling, spinal cord infarction can occur due to elevated tissue pressures [82]. A procedure that addressed this problem is known as a myelotomy [74] and was originally suggested by Allan, inventor of the spinal cord weight drop model in 1914 [2]. Creating a myelotomy to reduce spinal cord pressure after SCI was explored in the 1960–1980s by various surgeons and was based on evidence of spinal cord swelling observed as a flow blockage to intrathecal contrast media via myelography [48]. Such a decompressive technique might obviate a role for spinal cord injections if the myelotomy site could be later accessed for placement of cell-polymer lattices. Although apparently more invasive, this approach obviates many of the concerns related to cellular injections and should be further explored [44]. 3.13. Post injection imaging MRI is a sensitive tool for imaging the spinal cord. Following an injection some changes in the MRI image, i.e. “signal changes” are expected. These might include a small amount of edema and evidence of blood–brain barrier disruption if contrast media is administered intravenously. Post-injection changes that are of concern are hemorrhage, loss of grey/white discrimination, cord swelling, and extensive longitudinal T2 signal change, indicating new cord injury. Given the quantity of information that a postinjection MRI can provide, the authors think that experimental injections into the spinal cord in the context of clinical trials should be followed by an MRI within 24 h of the injection. Many of the immediate MRI changes may regress with time, but the changes provide detailed information that may be correlated with excessive tissue pressures, possible hydrodynamic dissection and hemorrhage. In addition these changes can be correlated with the neurological outcome.

4. Alternatives to laminectomy based spinal cord injections From the foregoing, it is apparent that current methods of spinal cord injection are limited by associated injury and poor three dimensional control of the injectate dispersion. Another important consideration is that the risks and logistical demands of laminectomy-based surgical exposure preclude the placement of multiple injections over time, which might be quite useful to increase the extent of repair. Thus, development of alternative methods of cell delivery is desirable. 4.1. Delivery of cells into the CSF via lumbar puncture (LP) has been proposed as a less invasive alternative to direct cell injections for the repair of SCI [5]. In this technique, a suspension of cells is injected into the lumbar CSF cistern below the L2 level. A key concept that has been proposed is that the injected cells are able to “home” to the injury site [91,75,45,11] and participate in repair. It is unlikely that transplanted cells will show “homing” to sites of chronic disease lacking chronic inflammatory cell activity [30]. It is also important the subarachnoid space is functional and not severely occluded by scarring. Throughout the world several thousand persons have already been treated by such techniques [15], but well controlled longitudinal trials are lacking. The technique sacrifices any attempt to control cell dispersion of the injected cells to other loci in the CNS compartments, and is thus unsuitable for cells that may have even minimal risk of seeding tumors. At least one case of disseminated tumor has been reported after delivery of fetal cells in this manner [4]. However, the technique is well suited to multiple injections over an extended time period. The extent to which cells can penetrate into sites of cord injury and how they organize in the injury site requires further elucidation but is doubtful they would form lesion bridging structures. Attempts have been made to exercise more control over cell dispersion by using magnetic fields to limit the motion of cells that have incorporated iron oxide nanoparticles in vitro and within the CSF [31,78]. Also, it may be possible to use intrathecal catheter techniques to make injections closer to the target site, possibly increasing local cell concentration. 4.2. Intravenous delivery Studies have been published claiming spinal cord repair effects via intravenous delivery of therapeutic cells [1,64]. Each delivery technique has specific advantages and disadvantages. In the case of intravenous delivery the advantages are the lack of need of an operating theatre or procedural imaging, no risk of meningitis, extensive clinical experience in hematological diseases multiple studies in other diseases such as cardiac diseases and stroke and the disadvantages, risk of systemic reactions, uncontrolled cell distribution with likely cell trapping in solid organs such as the lungs [22] and liver, low percentage of injected cells at target site [32,12], and development of immunity to non-self components. 4.3. Transvascular delivery Theoretically, it should be possible to perform spinal cord cell injections via transvenous or transarterial delivery [84] using microangiographic techniques. These techniques show promise for trans-coronary cell delivery to the myocardium [23]. The anterior spinal artery could be well suited for this purpose due to its important vascular distribution if technical problems such as risk of occlusion and ischemia, or causing hemorrhage could be solved. Such a technique could avoid dispersion of cells into the CSF and allow for multiple injections spatially and temporally, in the ventral cord region where important motor pathways are present. Some

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authors have described the local infusion of bone marrow stromal cells to the injury region via placement of a local angiographic catheter in the anterior spinal artery [81]. However, these techniques have not been experimentally validated and safety concerns regarding occlusion of the anterior spinal artery exist. 5. Minimally invasive spinal cord injections Methods to perform minimal exposure injections to the spinal cord have been described. These include percutaneous access to a CSF cistern via a lumbar puncture and the direction of a small endoscope to the site of desired injection. The endoscope itself can be used as a rigid structure to allow the needle to perforate the spinal cord. The endoscope can then be retracted leaving the intraspinal needle connected via small flexible tubing [27]. This technique allows injection to occur while the spinal cord is moving without concern of a “pistoning” effect [US patent 7,666,177]. Recently, we and others have been exploring the potential to deliver injections under MRI guidance without a requirement for an extensive surgical exposure. This technique would permit procedures to be performed in a surgical MRI suite, and have the potential for repeat injections over time and injections to multiple sites. Such techniques require MRI compatible devices and specialized materials such as ceramic needles [10]. Advances in techniques of minimally invasive spine surgery have allowed for the use of robotics in combination with spinal imaging to place spinal screws percutaneously [80].

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as with drugs, rather the effects are multifold, and cumulative. Typically, the preclinical dose range should be correlated to an animal model behavioral endpoint showing improvement as compared to controls. However, there have been few, if any publications, demonstrating such a clear dose relationship [18,90], especially in traumatic SCI. Our experience indicates that, with current knowledge, it is feasible to correlate dose with safety end-points such as new neurological deficits, MRI changes, and loss of spinal cord conduction. 8. Conclusions Intraparenchymal spinal cord cell injections cause tissue injury that varies from minor to severe. Aside from needle trauma, the key variables are injection volume and rates. Pressure based injections of large volumes may be damaging because the tissue tolerances may be exceeded by raised intraparenchymal pressures leading to hydrodynamic dissection and possible ischemia. These injuries can be reduced by attention to several variables and the use of real-time imaging. Wheras, injectate backout or extrusion has previously been considered a technical failure we argue that this event is a clear indicator of excessive intraparenchymal pressure. We recommend studies of the intraparenchymal pressure gradients associated with injection. Competing interests No competing interests.

6. Alternative methods of pressure injection The transfer of cells from a syringe to the spinal cord requires pressure, and because volume, compliance, and pressure are linked, large volumes can have damaging effects if they create pressure gradients that exceed the tissue tolerance to distortion and dissection. Large volumes are currently used to achieve doses extrapolated from those used in rodent studies as based on scale up calculations. The only injection variable available to potentially reduce the creation of damaging pressure gradients if a large volume is to be delivered is time. We theorize that the spinal cord will tolerate larger injection volumes delivered over protracted times. We suggest that an alternative method to deliver suspensions is to simultaneously perform spinal cord tissue pressure measurements and manage the injection rate based on tissue pressure. 7. Discussion Considerable work remains to be done to assess key variables to optimize spinal cord injections. Current techniques are mainly limited to a single injection during an open surgical exposure. One difficulty is that methods to observe injectate dispersion and monitor tissue pressure during injection have not been established. Thus, injections are relatively blind procedures with respect to events within the parenchyma and the spatial distribution of transplanted cells is poorly controlled. Ultrasound may be of value to observe injections in real time. Because a spinal cord cell transplant is currently a major clinical event requiring an open surgery, a large cell dose and volume is typically delivered. If injections could be made with less surgical effort, lower risk smaller doses might be delivered over multiple sessions. There is increasing evidence that the injured spinal cord undergoes more extensive endogenous repair than was previously understood [92], and thus it is important that manipulations such as cell injections not disrupt endogenous repair. Finally, the concept of cell dose as regards direct intraparenchymal cell delivery to sites of SCI is complex and influenced by numerous factors. There may be no threshold for an effect of delivered cells, such

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