Human adult olfactory neural progenitors rescue axotomized rodent rubrospinal neurons and promote functional recovery

Experimental Neurology 194 (2005) 12 – 30 www.elsevier.com/locate/yexnr

Human adult olfactory neural progenitors rescue axotomized rodent rubrospinal neurons and promote functional recovery Ming Xiaoa,b, Kathleen M. Kluebera, Chengliang Lua, Zhanfang Guoa, Charles T. Marshalla, Heming Wanga,b, Fred J. Roisena,T a

Department of Anatomical Sciences and Neurobiology, School of Medicine, University of Louisville, Louisville, KY 40292, USA b Nanjing Medical University, Jiangsu 210029, China Received 9 September 2004; revised 15 December 2004; accepted 11 January 2005 Available online 9 March 2005

Abstract Previously, our lab reported the isolation of patient-specific neurosphere-forming progenitor lines from human adult olfactory epithelium from cadavers as well as patients undergoing nasal sinus surgery. RT-PCR and ELISA demonstrated that the neurosphere-forming cells (NSFCs) produced BDNF. Since rubrospinal tract (RST) neurons have been shown to respond to exogenous BNDF, it was hypothesized that if the NSFCs remained viable following engraftment into traumatized spinal cord, they would rescue axotomized RS neurons from retrograde cell atrophy and promote functional recovery. One week after a partial cervical hemisection, GFP-labeled NSFCs suspended in MatrigelR matrix or MatrigelR matrix alone was injected into the lesion site. GFP-labeled cells survived up to 12 weeks in the lesion cavity or migrated within the ipsilateral white matter; the apparent number and mean somal area of fluorogold (FG)-labeled axotomized RST neurons were greater in the NSFC-engrafted rats than in lesion controls. Twelve weeks after engraftment, retrograde tracing with FG revealed that some RST neurons regenerated axons 4–5 segments caudal to the engraftment site; anterograde tracing with biotinylated dextran amine confirmed regeneration of RST axons through the transplants within the white matter for 3–6 segments caudal to the grafts. A few RST axons terminated in gray matter close to motoneurons. Matrix alone did not elicit regeneration. Behavioral analysis revealed that NSFC-engrafted rats displayed better performance during spontaneous vertical exploration and horizontal rope walking than lesion MatrigelR only controls 11 weeks post transplantation. These results emphasize the unique potential of human olfactory neuroepithelial-derived progenitors as an autologous source of stem cells for spinal cord repair. D 2005 Elsevier Inc. All rights reserved. Keywords: Axonal regeneration; BDNF; Functional recovery; Olfactory progenitors; Red nucleus; Spinal cord injury

Introduction The limited regeneration of the adult CNS has been attributed to a variety of factors including glial scaring (Geller and Fawcett, 2002), inhibitory factors (Fawcett, 1997; Niederost et al., 1999), and lack of trophic support (Beattie et al., 1997). Strategies for spinal cord repair have included exogenous growth factors (Bregman et al., 1997; Coumans et al., 2001; Kim et al., 1999; Selzer, 2003),

T Corresponding author. Fax: +1 502 852 6228. E-mail address: [email protected] (F.J. Roisen). 0014-4886/$ - see front matter D 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.expneurol.2005.01.021

implants of artificial bridges (Bunge, 2001; Teng et al., 2002), peripheral nerves (Carter et al., 1989), embryonic tissue (Bunge, 2001; Murray, 2004; Murray and Fischer, 2001), Schwann cells (Kocsis et al., 2002; Xu et al., 1995), embryonic stem cells (McDonald and Howard, 2002; McDonald et al., 1999), and engineered fibroblasts (Liu et al., 1999, 2002). Recently, the regenerative capacity of the olfactory system has attracted attention. Olfactory ensheathing cells (OECs) from adult olfactory bulbs have been used to provide limited axonal regeneration (Li et al., 1998; Ruitenberg et al., 2003; Ramer et al., 2004) and repair demyelinated regions of the CNS (Franklin and Barnett,

M. Xiao et al. / Experimental Neurology 194 (2005) 12–30

2000; Nieto-Sampedro, 2003; Santos-Benito and RamonCueto, 2003). However, the harvest of OECs from the olfactory bulb involves highly invasive surgery, making this a problematic source for clinical therapy (Barnett et al., 2000; Lu et al., 2002). The olfactory neuroepithelium (ONe) undergoes lifelong repair by progenitors, which are capable of replacing both neuronal and supporting cells (Calof and Chikaraishi, 1989). Previously, our lab developed the procedures for isolation and culture of human ONe-derived neurosphere-forming cells (NSFCs) from cadavers and patients undergoing nasal sinus surgery that remain mitotically active and have characteristics of progenitors (Roisen et al., 2001;Winstead et al., 2005). The location of ONe makes it possible to harvest cells without lasting damage to the donor. Importantly, our pilot studies have shown that the NSFC lines constitutively produce brain-derived neurotrophic factor (BDNF); irrespective of passage number, donor age, or sex (Marshall et al., 2003) as demonstrated by ELISA and RT-PCR studies (Marshall et al., 2004). To evaluate the therapeutic potential of NSFCs, the red nucleus (RN) and rubrospinal tract (RST) in the adult rat, which respond to exogenous BDNF (Kwon et al., 2002), were chosen as an ideal in vivo model. Infusion of exogenous BDNF (Houle and Ye, 1999; Kobayashi et al., 1997; Namiki et al., 2000; Novikova et al., 2000) or transplants of BDNF-engineered fibroblasts/OECs (Liu et al., 1999, 2002; Ruitenberg et al., 2003; Tobias et al., 2003) prevented atrophy in RN neurons or enhanced regenerative axonal growth by rubrospinal axons in the RST. The current study aimed to determine if engrafted human adult NSFCs would survive and function sufficiently to rescue axotomized adult rat RN neurons from retrograde atrophy and promote RST axonal regeneration following cervical spinal cord dorsolateral/lateral funiculotomy. Our long-term goal is to develop procedures through which a victim of spinal cord injury or other neurological degenerative conditions could serve as the donor of progenitors for his/her own restorative grafts without the need for immunosuppression or ethical controversy.

Materials and methods Transduction of NSFCs with LERS-EGFP vector Our lab has reported the culture procedure for establishing ONe-derived NSFC lines from postmortem as well as from patients undergoing nasal sinus surgery (Roisen et al., 2001; Winstead et al., 2005, respectively). Both ELISA and RT-PCR studies have shown that 15 evaluated NSFC lines produce BDNF irrespective of passage number, donor age, or sex (Marshall et al., 2004). For the present experiments, two of these well-characterized NSFC lines were selected randomly and transfected with green fluorescent protein (GFP) using the LERS-EGFP vector (Kinsella and Nolan, 1996).

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After transfection, the GFP-labeled cells were maintained for 7 DIV in medium which consisted of 90% Minimal Essential Medium (MEM) with Hanks’ salts with lglutamine, 10% fetal bovine serum (FBS), and 10 mg% gentamycin and incubated at 378C in an atmosphere of humidified 5% CO2 in air. These two GFP-labeled NSFC lines were cultured for up to 40 passages with a feeding schedule of every other day and passaged on alternate feedings. Representative passages were frozen in liquid nitrogen for future use. Preparation of NSFCs for engraftment One week prior to transplantation, GFP-labeled NSFCs were rapidly thawed and cultured in MEM + 10% FBS, and weaned from FBS 10%, 5% to 2.5% every 2 DIV, then maintained in MEM + 2.5% FBS. Fifteen to thirty minutes prior to injection, NSFCs were detached, pelleted by low-speed centrifugation (900 rpm for 5 min), carefully resuspended in MEM + 2.5% FBS and MatrigelR matrix (1:1; BD Biosciences) at a concentration of 2  104/Al and were maintained at 48C. The remaining cells were replated and cultured for 3–4 days for in vitro immunocytochemical characterization, and BDNF RT-PCR, and ELISA analysis. BDNF RT-PCR and ELISA analysis The RNA was isolated from NSFCs from the two selected GFP-labeled NSFC-lines, using RNA Stat-60 reagent (Tel-Test, Inc). The GeneAmp RNA PCR Kit (Applied Biosystems, N808-0017) was used to detect BDNF RNA. The BDNF primers were derived on the basis of published sequence (298 bp) (Yamamoto et al., 1996) prepared commercially (Integrated DNA Technologies, Coralville, IA) as indicated (5V: 5V-AGCCTCCTCTGCTCTTTCTGCTGGA-3V; 3V: 5V-CTTTTGTCTATGCCCCQ TGCAGCCTT-3V). The PCR procedures were followed as described in protocol for the kit, including positive (pAW109 RNA) and negative controls (Eppendorf Mastercycler Gradient). The procedure consisted of an initial step of (958C, 2 m), 35 cycles of melt (958C, 1 m), annealextend (608C, 1 m) and the final step (728C, 7 m). Separation was completed by using 2% agarose gel, stained with 0.05% ethidium bromide. Band intensities were scanned with Alpha Innotech TMM-20 Transilluminator. To determine BDNF protein levels in these two GFPlabeled NSFCs, ELISA analysis of cell lysates for BDNF (Emax ImmunoAssay system, part #TB257, Promega, Madison, WI) was performed. Cells were counted and broken by lysis buffer (1  106 cells/100 Al lysis buffer) for 10 min on ice. Acid treatment was performed to dissociate the protein; and the protein content was measured using a Bio-Rad protein assay (Bio-Rad laboratories, Hercules, CA). Samples containing the same amounts of protein were examined using the procedures for the BDNF ELISA

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following the manufacturer’s protocol. Tertiary antibodies were conjugated to horseradish peroxidase. Wells were developed with tetramethylbenzidine and measured at 450 nm on a standard microplate reader. BDNF content was quantified against a standard curve calibrated with known amounts of protein. The detection limit was less than 2.9 pg/ml for BDNF. Experimental design Ninety-four female Sprague–Dawley rats (220–270 g; Charles River, Wilmington, MA) were divided into a control group (n = 12) and 3 experimental groups (n = 82) (Table 1). Body weight of experimental animals was monitored during the experiment as a general measure of health. The control animals were used to establish baseline information concerning the size and number of cells within both the left and right RNs as well as for anterograde or retrograde tracing to and from the RN (Table 1). All rats in the experimental groups received a dorsolateral/lateral funiculotomy of the right side of the mid-cervical spinal cord and either a transplant of GFP-labeled NSFCs suspended in MatrigelR matrix or MatrigelR matrix alone 1 week after the lesion was made. Rats in experimental group I (n = 12) were sacrificed 1 or 2 weeks postengraftment to evaluate the survival and migration of the transplanted NSFCs in the injured spinal cord (Table 1). Rats in experimental group II (n = 40) were used to evaluate the effects of engrafted NSFCs on the axotomized RN neurons and functional recovery (Table 1). The RNs of

Table 1 Animal groups Animal groups

Normal control groups Toluidine blue staining FG BDA Experimental group I IA: PCHX + NSFCs IB: PCHX + NSFCs Experimental group II IIA: PCHX + FG + Matrigel IIA: PCHX + FG + NSFCs IIB: PCHX + FG + Matrigel + Behavioral test IIB: PCHX + FG + NSFCs + Behavioral test Experimental group III IIIA: PCHX + Matrigel + BDA IIIA: PCHX + NSFCs + BDA IIIB: PCHX + Matrigel + FG IIIB: PCHX + NSFCs + FG

Survival weeks post transplantation

Number of animals

2 2 12

12 (Total) 4 4 4 12 (Total) 6 6 40 (Total) 8 8 12

12

12

14 14 12 12

30 (total) 9 9 6 6

1 2

FG, Fluorogold retrograde tracing; BDA, biotinalyted dextran amine anterograde tracing; NSFCs, neurosphere-forming cells; PCHX, partial cervical hemisection.

these animals were prelabeled by FG injection after the cervical spinal cord partial hemisection and either were sacrificed 2 weeks (IIA, n = 16) or 12 weeks (IIB, n = 24) post transplantation for RN histological and image analysis. In addition, animals in group IIB were used to evaluate the effect of engrafted NSFCs on functional recovery by spontaneous vertical exploration and horizontal ropewalking tests. Rats in experimental group III (n = 30) were used to evaluate the potential of engrafted NSFCs to promote axotomized RST axonal regeneration (Table 1). The RST of some animals (IIIA, n = 18) was labeled by anterograde transport of biotinylated dextran amine (BDA) 12 weeks post transplantation. The animals were allowed to continue to survive an additional 2 weeks. Other animals (IIIB, n = 12) were given an injection of FG into the right side of the spinal cord at T1–2 level and were sacrificed after 3 days. All procedures were approved by the University of Louisville Institutional Animal Care and Use Committee and were in accord with the National Institutes of Health guidelines for the care and use of laboratory animals. Prevention of inflammation with penicillin and immunosuppression with cyclosporine For all experimental rats (NSFCs engrafted and lesion MatrigelR matrix only), daily Penicillin G (subcutaneous, 100,000 units/kg; G.C. Hanford Mfg., NY) and cyclosporine (intramuscular, 10 mg/kg; Ben Venue Labs, OH) injections were administered from the day of spinal cord injury to 1 week after transplantation. Cyclosporine (10 mg/kg) was then administered daily up to 6 weeks post transplantation, after which the dose was changed to 5 mg/kg daily throughout the remainder of the experimental period (Liu et al., 1999; Ruitenberg et al., 2003). At the end of the 12-week experimental period, all animals had body weights of 450–500 g, suggesting that the cyclosporine treatment did not impact negatively on the animals’ health. Surgical procedures Unilateral C3–4 cervical spinal cord dorsolateral/lateral funiculotomy model, involving the RST, was preformed as previously described (Liu et al., 1999). Briefly, rats were deeply anesthetized by an intraperitoneal injection of ketamine (100 mg/kg body weight; Ben Venue Labs, OH) and xylazine (10 mg/kg; Butler, OH). The spinal cord was exposed by dorsal laminectomy of the C3 and C4 vertebrae, and a cavity 1–2 mm in length was formed with microscissors and gentle aspiration in the lateral funiculus on the right side of the cervical spinal cord. The lesion completely severed the axons of the RST in the ipsilateral dorsolateral funiculus, partially lesioned the ipsilateral ventral funiculus and gray matter but left the dorsal columns intact. In experimental group II, the cavity was filled with a small piece of Gelfoam soaked with 1 Al 2% FG (Fluorochrome,

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Englewood, CO). The dura mater was covered with a piece of deep fascia removed from the subcutaneous tissue of the neck. The muscles were closed in layers while the skin was closed with wound clips. After surgery, the animals were given a subcutaneous injection of 5 ml physiological saline to compensate for blood loss, maintained on heating pads, closely observed until awake, and then returned to their home cages. After 1 week, the animals were anesthetized; the lesion site was exposed. The cavity was debreded with micro-scissors and cellular debris was removed by aspiration. A piece of gel foam was placed into the cavity to stop bleeding, and then removed after 5–10 min. The GFPlabeled NSFCs (8–10 Al, 2  104 /Al cells) suspended in MEM + 2.5% FBS and MatrigelR matrix (1:1) or MEM + 2.5% FBS and MatrigelR matrix alone was drawn into a 25Al Hamilton syringe attached to a glass pipette (50 Am tip diameter). The syringe was left undisturbed for 1–2 min to allow the MatrigelR to partially solidify, and then the gel was slowly injected into the lesion site. The wound was closed following the procedures outlined above. For anterograde tracing, 2 weeks before termination of the experiment, animals in experimental group IIIA were anesthetized as described above and positioned in a stereotaxic apparatus. A burr hole was made on the left surface of the skull over the RN with a dental drill at the coordinates described previously (Mori et al., 1997) and 1 Al of 10% BDA (10,000 molecular weight in double distilled water; Molecular Probes, Leiden, The Netherlands) was slowly injected over 2–3 min in five 200 nl pulses using a 10 Al Hamilton syringe. The needle was left in place for an additional 20 min and gradually withdrawn over 2–3 min. For retrograde tracing, animals in experimental group IIIB were anesthetized and the right side of their spinal cords was exposed at T1–2 level for pressure-injection of 0.5 Al 2% FG into the right spinal cord and animals were sacrificed 3 days later. Behavioral testing Examining forelimb use during spontaneous vertical exploration is a highly sensitive test to monitor limb use asymmetries (Kozlowski et al., 1996; Liu et al., 1999; McDermott et al., 1995; Schallert and Jones, 1993; Schallert and Lindner, 1990). Horizontal rope walking has been shown to be a sensitive test for partial spinal cord hemisection models to assess specific aspects of behavioral deficits such as posture and balance during gait (Murray and Fischer, 2001; Ruitenberg et al., 2003; Shumsky et al., 2003). Therefore, the animals were trained before surgery and baseline behavior measurements were obtained. The animals in experimental group IIB were tested 11 weeks after surgery using the 2 behavioral tests, spontaneous vertical exploration, and horizontal rope walking. Two individuals completed the scoring of all behavioral performance tests in a double-blind study.

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For spontaneous vertical exploration testing at 11 weeks after transplantation, the rats were placed in a clear glass cylinder (20 cm in diameter and 30 cm high) for 5 min and scored for independent use of the left (non-impaired) forelimb, right (impaired) forelimb, and/or both (left and right) forelimbs for contacting the wall of the cylinder during vertical posturing along the wall. Each behavior was expressed in terms of percentage forelimb use of the left, right, or both relative to the total number of limb usages (left, right, and both). For horizontal rope walking at 11 weeks after engraftment, the animal’s gait was studied during 3 untimed separate walks across a 1.25-m-long rope (4 cm diameter) from one platform to another. Rats were scored for their general performance during rope locomotion using a deficit rating scale: (0) normal rope locomotion; (1) close to normal locomotion with occasional deficits such as inability to maintain consistent weight-supported limb placement during traverse; (2) able to cross the rope but with frequent deficits; and (3) great difficulty to cross the rope, frequent slips (loss of balance but remains on the rope) and falls. If the animal fell off the rope, it was restarted only three times. During rope traverse, the number of slips and falls, and total steps for each limb were counted to determine the btotal error/stepQ ratio, which was calculated and defined as the following: [number of slips + 2 (number of falls)]/total number of steps. Each rat was observed three times, and final scores were the mean of the 6 scores obtained by the two independent observers. Tissue preparation Animals were deeply anesthetized with an overdose of ketamine (200 mg/kg body weight) and xylazine (20 mg/kg body weight), perfused through the heart with 0.01 M PBS (pH 7.4, 200 ml) followed by 4% paraformaldehyde in 0.1 M PB, pH 7.4 (48C, 500 ml). The midbrain plus cervical and thoracic segments of the spinal cord were dissected out and immersed in the same fixative at 48C overnight followed by cytoprotection at 48C in PB containing 30% sucrose for 1–2

Table 2 Primary antibodies Antibodies

Target

Source

A2B5 monoclonal 1:100

Glia progenitor/ some neurons BDNF Neurons Astrocytes Transfected GFP Neurons NGF receptor BDNF receptor BDNF receptor Neurotrophin receptor Neurons

Chemicon

BDNF polyclonal 1:250 GAP-43 monoclonal 1:100 GFAP monoclonal 1:100 GFP Polyclonal 1:100 MAP2 monoclonal 1:100 Trk A polyclonal 1:50 Trk B polyclonal 1:50 Trk C polyclonal 1:50 Trk pan monoclonal 1:100 h-tubulin III monoclonal 1:100

Sigma Chemicon Chemicon Chemicon Roche Santa Cruz Santa Cruz Santa Cruz Santa Cruz Sigma

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Fig. 1. NSFCs were transfected with green fluorescent protein (GFP) using the LERS-EGFP vector. After 4 DIV 60% of the cells were fluorescent. The level of fluorescence of individual cells varied reflecting the length of time from the previous division (DIC, confocal optics).

Fig. 3. RT-PCR and ELISA analysis revealed that both of the NSFC lines used in these studies expressed BDNF mRNA and protein, respectively. (A) RT-PCR for BDNF exhibited a clear PCR product of 298 bp in the cDNA samples from each of the two GFP-labeled NSFC lines; PCR for BDNF in the absence of reverse transcriptase (control) yielded no reaction product, confirming that the BDNF amplification product did not arise from contaminating or genomic DNA. (B) ELISA of cell lysates also demonstrated that BDNF protein was produced in each of the two NSFC lines.

days. Spinal cord and brain tissue were embedded in OCT compound (Fisher Scientific, Pittsburgh, PA), and cut on a cryostat. The spinal cord was serially cut in either transverse

Fig. 2. After transplantation, the remaining GFP-labeled NSFCs were replated, cultured for 3–4 DIV and found to be heterogeneous and produce BDNF. (A) The majority of the cells with nuclei vitally stained with 4V, 6-diamidino-2-phenylindole dihydrochloride were immunopositive for the neuronal marker, h-tubulin III. (B) A few cells were immunoreactive for A2B5. (C) Trk pan immunoreactivity was identified on some GFP positive and negative cells. (D) Trk B immunoreactivity was observed on some cellular surfaces and frequently appeared as an intensely stained region (arrow). (E) A few NSFCs were Trk A positive. (F) Most of the NSFCs were immunopositive for BDNF (DIC), confocal optics.

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or horizontal sections at 20 Am and mounted onto gelatincoated slides. To ensure that the entire RN was collected, the midbrains were serially sectioned transversely at 20 Am from caudal to rostral extent. Every third serial section (70–80) was mounted onto gelatin-coated slides in sequence. The first series of sections was used for toluidine blue staining and analysis of total neuronal number and size for both RN magnocellular regions. The other series of sections were used for analysis of the number and size of FG-labeled RN neurons. Histology and immunocytochemistry Toluidine blue staining and immunocytochemical procedures have been established in our laboratory (Othman et al., 2003). Briefly, the NSFCs on coverslips were vitally stained with 4V, 6-diamidino-2-phenylindole dihydrochloride (DAPI, 1:500; Molecular Probes, Eugene, OR) to localize the nuclei (378C, 30 min) prior to staining. After extensive washing, both coverslips and the tissue on slides were immunostained overnight at 48C with the primary antibodies listed in Table 2. After washing, secondary antibodies Texas Red-conjugate goat anti-mouse IgG and Texas Red-conjugate goat anti-rabbit IgG (all diluted 1:100, FITC, Cappel, West Chester, PA; Texas Red, Molecular Probes, Eugene, OR) were applied. Omission of primary or secondary antibodies insured the specificity of the reaction. The BDA staining procedures were modified from protocols by Kelly et al. (2003). Briefly, slides with RN and spinal cord sections were rinsed three times, 10 min each, in 0.1 M PB, pH 7.4, and preincubated in 10% normal goat serum (NGS) in phosphate-buffered saline PBS, pH 7.4 (30 min). The slides were incubated overnight at room temperature in avidin and biotinylated horseradish peroxidase (1:50; ABC; Vector, Burlingame, CA) in PBS, with 1% NGS and 0.5% Triton X-100. After three 10 min rinses in PB and a 10 min rinse in 0.1 M sodium acetate, pH 6.0, the sections were reacted with Sigma Fast-DAB compounds (Sigma, St. Louis, MO) for 5–10 min. Following PB washes, some sections were toluidine blue counterstained, then dehydrated in ETOH, and coverslipped with DPX (Fluka Chemie AG, Buchs, Switzerland).

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sections (20 Am) of cervical spinal cord containing the lesion site were stained with GFP antibody. The sections containing the caudal and rostral extent of the graft were found. Micrographs (200) of sections at 50 Am, 100 Am, 250 Am, 500 Am, and 1000 Am, 1500 Am, 2000 Am distance from the rostral or caudal host–graft interface were used for cell counts. Only those GFP-labeled cells with distinct cytoplasm and nuclei were counted. For analysis of the RN, the first toluidine-blue-stained section containing 3–10 neurons was selected as beginning of the caudal pole of the RN. Only sections from the magnocellular region of the RN, which project to the spinal cord, were included in this study (Mori et al., 1997; Kwon et al., 2002). The number and size of neurons on both sides of RN were measured from Sections 4, 7, 10, and 13, 16, 19 of set I; the number and size of FG-labeled RN neurons were measured on Sections 5, 8, 11, and 14, 17, 20 of set II. For toluidine blue staining, only those cells with identifiable nuclei, nucleoli, and characteristic neuronal morphology were counted. For FG-labeled neuron counts, to avoid overestimation of neuronal numbers, adjacent sections (section sets II and III) were examined and only those cells appearing on 1 section were counted (Liu et al., 1999). The cross-sectional area of toluidine-blue-stained or FG-labeled RN neurons was measured under 200 counting box using an NIH image macro program. The maximal cross-sectional area for each neuron was measured. A total of 150–200 RN neurons was measured for each RN. Statistical analysis Data obtained from the histological assessments and behavioral tests were analyzed for significant differences between animal groups using either Student’s t test or oneway ANOVA. The Pearson correlation test was used to investigate whether there were possible relationships between different outcome parameters and were considered statistically significant if P b 0.01. All quantitative data were presented as mean F standard deviation (SD). The graphs were prepared using Microsoft Excel software.

Results Cell counts and image analysis Characterization of GFP-labeled NSFCs in vitro To ensure consistency between animals and groups, sequential sections were collected from the spinal cord lesion site as well as the brain stem for each animal. Images of NSFCs in culture, spinal cord, and midbrain were captured using the Olympus confocal microscope or the Nikon microscope with Olympus video camera system, and processed using Photoshop Adobe software. Cell size was determined using NIH image analysis software. For analysis of GFP-labeled NSFC migration into the host spinal cord 2 weeks after engraftment, serial cross

The two selected NSFC lines were transfected with GFP using the LERS-EGFP vector; approximately 60% of viable cells 4 days after transfection were fluorescent (Fig. 1). The expression rate of GFP in NSFCs was relatively stable, undergoing only a slight decrease over the initial 10 passages, and remaining at over 40% after 35 additional passages. This heterogeneous population was immunoreactive for a variety of lineage specific markers. In MEM + 2.5% FBS, the majority (~95%) of the GFP-labeled NSFCs were positive for

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h-tubulin III (Fig. 2A), while ~5% were positive for A2B5, an antibody against a ganglioside-enriched in glial membranes (Fig. 2B). No immunoreactivity was observed for GFAP (glial fibrillary acidic protein). Approximately 20% of the NSFCs were immunopositive for Trk pan, an antibody that reacts promiscuously with Trk A, B, or C (Fig. 2C).

Additional studies demonstrated that most (65%) of that immunoreactivity was for Trk B (Fig. 2D) with fewer cells (35%) reactive for Trk A (Fig. 2E) and none reactive for Trk C. Immunoreactivity for BDNF was detected throughout the cytoplasm and on the surface of approximately 90% of the NSFCs (Fig. 2F).

Fig. 4. Photomicrographs of horizontal sections from an engrafted rat cervical spinal cord reveal NSFCs within the lesion cavity and the host spinal cord (A–C) 1 week or (D–G) 2 weeks after engraftment. The orientation of all sections was left-caudal and right-rostral. (A) Note the large number of GFP-labeled NSFCs (green) in the lesion cavity as well as a few cells within the host spinal cord bridging the injury site. Several GFP-labeled cells can be seen within the adjacent host spinal cord (arrows). (B and C) Higher-magnification images of area outlined in A, showing the host–graft interface (dashed line) (B). A modest GFAPpositive astrocytic presence was observed, with few astrocytes being detected in the graft (C). (D and E) Low and high power of rostral cervical spinal cord horizontal sections at the level of the dorsolateral columns showing numerous GFP-labeled cells distributed rostrally in the white matter for up to 2.0 mm. A few of these cells had long processes that extended into the surrounding tissue (box in D). (F) Most of the GFP-labeled cells were positive for Trk B receptors. (G) The majority of the GFP labeled cells were h-tubulin III positive.

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Furthermore, RT-PCR demonstrated the expression of mRNA for BDNF in both GFP-labeled NSFC lines used in the present experiments. Specific bands of RNA encoding BDNF (298 bp) were detectable in the cDNA samples from the two GFP-labeled NSFC lines (Fig. 3A). Control reactions were negative for the amplification products, demonstrating that the PCR method and reagents used yielded specific amplified products only when a cDNA

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source was included. ELISA of cell lysates from each NSFC line demonstrated 40 pg BDNF/mg protein in both lines (Fig. 3B). Although two different lines were used in our initial studies, no differences were observed between the two different neurosphere-forming lines. Since equivalent responses of the NSFCs to engraftment as well as the response of the host tissues to the engraftment were similar, data from only one line are presented for clarity.

Fig. 5. Photomicrographs of cervical spinal cord cross sections showing the lesion and GFP-labeled NSFCs at the lesion site 2 weeks after transplantation. (A–H) Caudal to rostral, cross sections from a single rat at 750-Am intervals were serially reconstructed to show the extent of the lesion and the distribution of engrafted NSFCs. (I–N) High-magnification images of corresponding outlined areas in A–D and F–H. (A and B) NSFCs were noted caudally up to 1.5 mm from the interface of the lesion cavity within the host spinal cord white matter. Some cells were in the corticospinal tract (cst) (I); however, most cells were found in the dorsolateral funiculus (dlf) (J), and a few cells in the ventrolateral funiculus (vlf) or the ventral columns (vc). (C) The caudal host–graft interface contained numerous blood vessels that penetrated into the transplant site (arrows in K). (D and E) The epicenter of lesion cavity contained many engrafted NSFCs. The lesion (dashed line) completely disrupted the dlf (containing the rubrospinal tract), the lateral funiculus (lf), and partially lesioned the vlf and the gray matter (GM). (F) Cells were also found at the rostral interface of lesion cavity. (G and H) NSFCs were observed rostrally in the white matter up to 2.0 mm from the lesion cavity. They were most frequent in the dlf and lf (L), some in the fasciculus gracilis (fg), fasciculus cuneatus (fc), and cst (N), and a few at the vlf and vc (M). The scale bar in H applies to A–G. The scale bar in N applies to I, K–M.

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Survival and integration of GFP-labeled NSFCs after engraftment into injured host spinal cord A cavity 1.5–2.0 mm long in the caudal rostral direction formed in the right side of the C3–4 spinal cord 1 week after the lesion (Fig. 4A). The lesion was limited to the right side of the cervical spinal cord and completely severed the ipsilateral dorsolateral and lateral funiculi, partially lesioned the ipsilateral dorsal and ventral funiculi plus gray matter, but left the dorsal columns intact (Figs. 5D and E). One week after engraftment, transplanted GFP-labeled NSFCs were seen within the lesion cavity and attached to the surrounding host tissue at the interface of the injury site (Fig. 4A). A few GFP-labeled cells migrated into the adjacent host spinal cord (Fig. 4B). A relatively mild astrocytic response was visible along the graft–host interface (Fig. 4C). Two weeks after engraftment, GFP-positive cells remained in the lesion cavity (Figs. 5C–E and K). These cells were supported by host blood vessels that were present throughout the transplant site. In addition, many cells were observed in the host spinal cord dispersed in a longitudinal direction (Figs. 4D and E). The majority of these cells were located in the white matter of spinal cord, primarily in the dorsolateral and lateral funiculi (Figs. 5B and J, G and L), some in the dorsal columns (Figs. 5A and I, H and N), and a few in the ventral columns and ventrolateral funiculus (Figs. 5G and M). In contrast, only a few of the engrafted cells were located in the gray matter adjacent to the injury site (Figs. 5B, C, and F). The quantitative analyses demonstrated that the number of engrafted cells in the rostral white matter was almost 2 times that in the caudal regions of all animals evaluated (Fig. 6). The cells were found in the rostral white matter up to 2 mm from the lesion; while caudally, they were seen up to a maximum of 1.5 mm (Fig. 6). The majority of GFP-labeled cells were positive for the Trk B receptor (Fig. 4F) or h-tubulin III, and some had developed characteristic neuronal shapes with long processes that extended into the surrounding tissue (Fig. 4G). Twelve weeks after transplantation, toluidine blue histological staining revealed profound differences in the

cytoarchitecture of the lesion site between the lesion plus engrafted NSFC animals and the lesion only animals. In the lesion only animals, the cavity was filled with many cells but no axons (Figs. 7A and B). However, within the lesion site of the engrafted animals, NSFCs had extended processes throughout the site (Figs. 7C–H). Furthermore, a large population of axons was observed within the proximal end of the lesion site (Fig. 7C). Immunocytochemistry demonstrated that many GFP-labeled NSFCs remained within the lesion cavity (Figs. 7E and F). These cells were positive for GAP-43 (growth-associated-protein 43) or MAP2 (microtubule-associated-protein), assumed neuronal phenotypes (Figs. 7C–H), and formed long processes that extended into the surrounding tissue. In addition to the engrafted NSFCs, numerous GAP-43 or MAP2 positive axons from the host’s spinal cord were observed in the center of the cavity of the NSFC-engrafted animals (Figs. 7E–H). No obvious GAP-43 or MAP2 positive axons were observed within the cavity of lesion only animals. Effects of axotomy and NSFC engraftment on RN neurons In normal rats, toluidine-blue-stained sections were used to determine the number and mean soma size of RN neurons. Cell size and numbers in the right and left RN were similar (Table 3). Furthermore, no differences were observed in number or mean soma size of neurons in the right RN obtained from normal, lesion only, or lesion plus NSFCengrafted rats at 2 or 12 weeks after transplantation (P N 0.05; Table 3; Fig. 8). Compared to the normal animals, the apparent number and mean soma size of neurons in the left RN decreased more in the lesion only than in the cellengrafted group at 2 or 12 weeks post transplantation. In the lesion only animals, there was a 27% reduction in the apparent numbers of neurons in the RN, compared to a 13% reduction in the lesion plus NSFC-engrafted animals 2 weeks following engraftment (P b 0.05). Furthermore, the mean neuronal size decreased by 24% in the lesion only animals compared to a 7% decrease in the lesion plus engrafted group (P b 0.05; Table 3; Figs. 8A and B). At 12 weeks after

Fig. 6. The number of the GFP-labeled NSFCs found in the white matter of the host spinal cord 2 weeks after engraftment (n = 6; Mean F SD). The GFPlabeled cells were determined in 20 Am cross sections of the engrafted cervical spinal cords at distances approximately 50 Am, 100 Am, 250 Am, 500 Am, and 1000 Am, 1500 Am, 2000 Am from the rostral or caudal boundary of the lesion cavity. More GFP-labeled cells (P b 0.05) were in the rostral than the caudal white matter.

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Fig. 7. Histological analysis of the injury sites 12 weeks post-engraftment (A and B, toluidine blue). In the lesion only animals, the sites were filled with many cells (arrows) but no axons. (C and D) However, within the lesion site of engrafted animals, transplanted cells that extended processes (arrows) were observed throughout the site. In addition, a large population of axons was observed within the rostral end of the lesion site (arrowheads). (E–H) In the center of the lesion cavity, many engrafted cells (arrows) were immunopositive for GAP-43 and MAP-2 (arrows in E and F, and G and H, respectively); axons from the host’s spinal cord also were GAP-43 or MAP-2 positive (arrowheads in E and G and H, respectively).

engraftment, the neuronal atrophy of the left RN was further exacerbated in the lesion only group, but partially prevented in the lesion plus NSFC-engrafted group (Table 3; Figs. 8C and D). The decreases in apparent neuronal number and size were 38% and 42%, respectively, in the lesion only animals; while in the lesion plus cell-engrafted animals, both parameters were partially spared with reductions of 24% and 20%, respectively (P b 0.05; Table 3). The number of neurons projecting to the spinal cord after axotomy in the lesion only and lesion plus cell-engrafted animals were compared in sections with the FG labeling. In all groups, the mean size of FG-labeled neurons was approximately 11% larger than the mean cell size deter-

mined from adjacent sections stained for toluidine blue because immunoflorescence is more diffuse compared to visible light (Table 3; Figs. 9 and 10; Mori et al., 1997). The FG labeling demonstrated the same pattern as the data obtained from the toluidine-blue-stained sections. Two weeks after engraftment, the apparent number and size of FG-labeled RN neurons was greater in the lesion plus cellengrafted than in the lesion only animals (P b 0.001; Figs. 9, 10A and B). Furthermore, after 12 weeks, the apparent number and size of FG-labeled axotomized RN neurons was substantially decreased in the lesion only group, but only slightly decreased in the lesion plus engraftment group (P b 0.001; Figs. 9 and 10C and D).

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Table 3 Neuronal counts and mean soma size in RN (toluidine blue), mean F SD Group

Right Normal (n = 4) Lesion only 2 weeks (n = 8) Lesion + Cells 2 weeks (n = 8) Lesion only 12 weeks (n = 12) Lesion + Cells 12 weeks (n = 12)

Mean soma size (Am2)

Neuronal counts 497 492 490 495 500

F F F F F

Left 31 35 36 34 38

501 360 425 309 380

F F F F F

33 41T 38T 44T 31T

Ratio (L/R)

Right

1.01 0.73 0.87 0.62 0.76

464 469 458 459 462

F F F F F

0.03 0.04TT 0.08 0.05TT 0.07

F F F F F

Left 43 42 41 38 41

469 358 427 269 368

Ratio (L/R) F F F F F

46 51T 49T 36TT 40T

1.01 0.76 0.93 0.58 0.80

F F F F F

0.06 0.05TT 0.08 0.08TT 0.07

Sections of set I were stained by toluidine blue. Quantitative analyses were performed on six midbrain sections of each animal to count neuronal number and mean soma size on both sides of the RN. T Left differs from right (P b 0.05) using paired comparison t test. TT Significant difference among group ratio was determined by one-way ANOVA (P b 0.05).

Potential of engrafted NSFCs to promote axotomized RST axonal regeneration To determine if the NSFC engraftment improved axonal regeneration of the surviving axotomized RN neurons, the distribution of RST axons in the spinal cord after injection of the anterograde tracer BDA into the magnocellular portion of the RN was evaluated. In the upper cervical segments (C1–2), rostral to the lesion site, the distribution of the BDA-positive RST axons in lesion only and engrafted animals was similar to normal animals in the superficial dorsolateral white matter contralateral to the BDA injection site (Fig. 11). A few ipsilaterally descending RST axons could also be observed in the same location. The BDA-positive RST axons in the lesion only animals appeared sparser than in the engrafted animals, which is possibly the result of the decreased efficiency of atrophied neurons to take up and transport tracer in the lesion only animals (Fig. 11). In sagittal sections of lesion site, all BDA-labeled axons stopped at the rostral host–graft interface in lesion only animals (Figs. 12C–E). In contrast, in engrafted animals, axons crossed the rostral host–graft interface, elongated caudally, and in most cases extended past the caudal host– graft interface (Figs. 12F–I). Compared to the normal linear rostrocaudal alignment of the RS tract axons located in the dorsolateral funiculus (Figs. 12A and B), most of the regenerated axons were randomly aligned, although generally orientated in a rostral–caudal direction (Figs. 12G–I). In addition, the majority of regenerated axons were smallcaliber axons with branches or terminal bouton-like structures (Figs. 12G–J and L). These structures were rarely detected in normal RS tract axons at the middle level (C3– C4) of the cervical spinal cord. Serial cross sections of segments caudal to the lesion site were examined for BDA-labeled axons. In all lesion only animals, no BDA-labeled RST axons were detected in the dorsolateral funiculus of the host spinal cord caudal to the lesion (Figs. 13A and B). In 78% of the engrafted animals, regenerated axons were found caudal to the lesion site; in some cases, BDA-labeled RST axons were seen in the upper thoracic spinal cord (T1–3), four to six segments

caudal to the transplant, although their distribution was sparser compared to the upper cervical segments. The BDA-labeled RST axons in C6–8 spinal cord segments were not as well organized as those in the C1–2 segments (Figs. 11C and D, 13C and D). Most were present in the dorsolateral funiculus and a few were diffusely located in the adjoining dorsal horn (Figs. 13C and D). Many collaterals were observed projecting to Rexed’s laminae V–VII (Fig. 13E) with bouton-like structures near neurons in these laminae. A few BDA-labeled fibers were observed in lamina IX in very close apposition to motoneurons (Fig. 13F). These results suggest that axotomized RN neurons regenerated RST axons into segments of spinal cord caudal to the lesion site and possibly reinnervated their target gray matter. To further determine the origin of the BDA-labeled axons found in the spinal cord caudal to the lesion site, NSFCs grafted animals were injected at T1–2 for retrograde tracing of FG in the ipsiltaeral spinal cord. This injection site was selected to prevent FG from penetrating the lesion site. Within the contralateral lesioned RN, FG-labeled RST neurons were detected in all six NSFC-engrafted rats (Fig. 14C), but not in any lesion only animals (Fig. 14B). Compared to the normal control (Fig. 14A), the number of regenerated RST neurons in the RN of cell engrafted rats was small, and their cell body size was slightly smaller than normal RN neurons, indicating that some axtomoized RST neurons had regenerated at least 4–5 segments caudal to the lesion site. The detailed morphology and quantitative data for FG coexistence with GAP-43 in RST neurons are currently under evaluation. Potential of engrafted NSFCs to promote functional recovery Prior to surgery, the animals used for experimental group IIB were preconditioned for the spontaneous vertical exploration and rope-walking tests. In order to objectively evaluate functional recovery after transplantation, stable baseline data were established. Only those animals that had almost equal usage of the left and right forepaws (approximate 10% respectively, Fig. 15E) to explore the wall of the

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of the time, closer to the normal data prior to surgery. In addition, the ipsilateral (right) forelimb alone use was 8% of the time in NSFC-engrafted group, demonstrating functional recovery of the injured limb, which was not observed in the lesion only animals (Fig. 15E). Similarly, the cell-engrafted animals also exhibited better performance during the rope-walking test 11 weeks post-engraftment than the lesion only group. In all operated animals, deficits in general locomotion were observed. However, a significant difference in deficit scores was found between the lesion plus NSFC-engrafted and lesion only groups (P b 0.001; Fig. 16F). Most engrafted animals had consistent locomotor activity, better body posture, and were able to complete the task. They slipped or fell from the rope less frequently than lesion only animals (Figs. 16A–D). The calculated total error/ step ratio was consistent with the behavioral data obtained by the deficit-scoring method. A significant difference was also found between the two experimental groups (P b 0.001; Fig. 16E). In order to determine whether the surviving axotomized RN neurons contributed to the observed functional recovery, the possible correlation between the number of FG-labeled RN neurons and the total error/step was analyzed using Pearson’s correlation test. A negative correlation existed between the morphological and physiological data (v = n 2 = 14, r = 0.731, P b 0.002) (Fig. 16G). Rats

Fig. 8. Photomicrographs of midbrain toluidine blue stain cross sections showing the RNs 2 weeks (A and B) and 12 weeks (C and D) after cervical spinal cord injury and NSFCs engraftment. All sections were approximately 380 Am from the caudal pole of the RN. (A) Note in the left RN, modest atrophy and apparent neuronal loss 2 weeks after hemisection in the lesion only group. (B) Partial preservation of the size of the left RN neurons as well as apparent numbers of neurons was found following NSFCs engraftment. (C) Compared to 2 weeks, apparent neuronal loss and atrophy of the left RN were further exacerbated at 12 weeks in the lesion only group. (D) In contrast, the lesion plus engrafted NSFCs group had a survival of approximately 80% of the left RN neurons and exhibited less atrophy at 12 weeks.

cylinder and complete the rope-walking tasks with a 0 deficit score were selected for surgery. Eleven weeks after surgery, spontaneous vertical exploration by the lesion only animals clearly illustrated abnormal posture and rare use of the right forelimb while exploring a glass cylinder (Figs. 15A and B). On the other hand, the lesioned animals that received NSFC transplants freely used both paws to explore the cylinder and assumed nearly normal posture (Figs. 15C and D). Compared to pre-surgery scores of bilateral forelimb use to explore the cylinder wall (approximately 80%), the lesion only animals used both forelimbs only ~10% of the time, primarily using only the unaffected forelimb (left; approximately 90%; Fig. 15E). In contrast, the cell-engrafted group used both forelimbs 75%

Fig. 9. Histogram comparing the relative number (A) and mean soma size (B) of FG-labeled RN neurons between lesion only and lesion plus NSFCs engrafted animals 2 and 12 weeks after surgery. (A) More (*P b 0.001) RN neurons were labeled in the engrafted animals (2 weeks: 360 F 39; 12 weeks: 300 F 35) than those in the lesion only animals (2 weeks: 270 F 25; 12 weeks: 200 F 25). (B) The mean soma size of surviving RN neurons was (P b 0.001) smaller in the lesion only animals (2 weeks: 390 F 25 Am2; 12 weeks: 300 F 25 Am2) than in the engrafted animals (2 weeks: 470 F 30 Am2; 12 weeks: 405 F 25 Am2).

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Fig. 10. Fluorescence photomicrographs of midbrain cross sections show FG-labeled RN neuronal soma (A and B) 2 weeks and (C and D) 12 weeks after surgery. All sections were approximately 400 Am from the caudal pole of the RN. (A and B) Two weeks after surgery, the apparent number and mean soma area of FG-labeled RN neurons were greater in the lesion and engrafted group (B) than in the lesion only group (A). (C and D) Twelve weeks after surgery, the apparent number and soma area of FG-labeled RN neurons were further decreased in the lesion only (C) while only slightly decreased in the lesion and engrafted group (D).

that performed well during horizontal rope walking were found to have many surviving FG-labeled neurons in axotomized RN, whereas animals that slipped or fell from

the rope more frequently consistently had a fewer neurons in axotomized RN. Discussion This study demonstrates that human adult olfactory neuroepithelial-derived progenitors (NSFCs) engrafted into the injured adult rat cervical spinal cord remain viable. They integrated into the injured host spinal cord and rescued axotomized RN neurons from retrograde atrophy as well as promoted RST axonal regeneration that leads to functional recovery either directly or by modification of the microenvironment within and surrounding the lesion site. Integration of engrafted human ONe-derived NSFCs in injured rat spinal cord

Fig. 11. Photomicrographs of C2 BDA-immunostained and toluidine blue counterstained spinal cord sections illustrating the distribution of BDAlabeled RST axons rostral to the lesion site. The BDA-labeled RST axons were seen in the dorsolateral funiculus (dlf) of both lesion only (A and B) and engrafted animals (C and D) 14 weeks after injury and engraftment. BDA-labeled axons were sparse in the lesion only group compared to those found in the engrafted animals.

The engrafted NSFCs survived in the injured rat spinal cord for at least 12 weeks. They entirely filled the lesion cavity; some integrated into the adjacent host spinal cord, and formed a relatively continuous interface with the host tissues that was not interrupted by cysts or scars. Several features of NSFCs and their interaction with the spinal cord likely contributed to their survival. In vitro pilot studies indicated that the NSFC lines constitutively produce BDNF, irrespective of passage number, donor age, or sex as demonstrated by ELISA and RT-PCR studies (Marshall et al., 2004). In the present study, RTPCR, cell lysate ELISA, and immunocytochemistry demonstrated that the two GFP-transfected NSFC lines used in this study produced and secreted BDNF. Preliminary

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Fig. 12. Photomicrographs of cervical spinal cord horizontal sections showing BDA anterograde tracing RST axons in the lesion region 14 weeks after transplantation. For all sections, left is rostral (proximal to the lesion cavity), and right is caudal; dashed lines indicate graft–host interfaces. (A and B) Numerous normal BDA-labeled RST axons were present in rostrocaudal parallel alignment in the dorsolateral funiculus. (C and D) BDA-labeled RST axons were found proximal to the lesion boundary but failed to enter the lesion cavity in a lesion only animal. (E) High power of outlined area in D, showing a few terminations of RST axons ending at the proximal boundary of lesion. (F–L) RST axons sprouting in NSFC-engrafted animals. (F and G) Numerous RST axons were observed at the region rostral to the graft, some axons passed through the rostral graft–host interface, and extended into the graft. (H) Some small-caliber RST axons with branches were observed at the epicenter of the graft. (I) A considerable number of BDA-labeled axons with sprouts exited the caudal board of the graft and penetrated the caudal graft–host interface. (J) Some rostrocaudally directional RST axons were observed at the graft. (K) High power of the corresponding areas in J showing a larger-caliber axon with two branches ending in terminal bouton-likes structures. (L) Some small-caliber and larger-caliber RST axons reached or passed through the caudal graft–host interface (dashed line). Scale bar: A, C, F 1 mm, others 100 Am.

studies were performed to examine the concentration of BDNF in NSFC-conditioned media, but detection levels were low. Cultures plated at higher NSFC densities did not

exhibit increased levels of BDNF in the conditioned medium. The presence of Trk B receptors on some NSFCs coupled with the presence of BDNF in their cell lysates

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axotomized RST neurons from retrograde atrophy and possible cell loss. Another important feature was the apparent histocompatibility between the engrafted NSFCs and the host spinal cord. Only a mild astrocytic response was visible within the graft at the lesion cavity. The removal of presumed scar issue in the lesion cavity just prior to the engraftment may have played a role in the lack of scar formation. The mild host immune reaction was attributed to immunosuppression by cyclosporine. The antibiotic and possible anti-inflammatory action of Penicillin may also have contributed to the NSFCs survival. In a preliminary study, transplanted NSFCs in the normal rat spinal cord remained aggregated and did not migrate or integrate into the adjacent host tissues (Roisen et al., 2002; Xiao et al., 2003). In the present study, many of the engrafted NSFCs were found at some distance in the rostral, predominantly, and caudal cord. The majority of the NSFCs were located in the white matter among the descending supraspinal or ascending sensory fibers; some were located in the gray matter adjacent to the lesion site. In addition, most NSFCs had neuron-like morphologies with h-tubulin III immunopositive processes or Trk B receptors on their surface. They may have supported spinal cord repair by

Fig. 13. Toluidine blue counterstained photomicrographs of C6–7 spinal cord sections showing that BDA-labeled RST axons regenerated two to three segments caudal to the graft 14 weeks after engraftment. (A and B) Cross C6–7 section from a lesion only animal, showing no BDAlabeled RST axons in the dorsolateral funiculus (dlf) and past the lesion site. (C and D) Cross C6–7 section from an NSFC-engrafted animal demonstrating BDA-labeled regenerating RST axons in the dlf and gray matter. (E) A few BDA-labeled axons (arrows) in the white matter projected toward lamina VII of the gray matter. (F) Close up of BDAlabeled axons in the gray matter of lamina IX. Arrows point to boutonlike structures around motoneuron soma.

suggests a possible paracrine/autocrine mechanism (Novikova et al., 1996). In vitro studies also indicated that patient age, sex, or duration in culture did not affect the viability or polyamine synthetic activity of the NSFCs (Marshall et al., 2003; Roisen et al., 2001). The GFPtransfected NSFC lines maintained their high proliferative activity and GFP expression decreased only slightly through many passages. Importantly for engraftment, the NSFCS were adapted to the absence of serum over a period of weeks by passages through a series of dilutions from 10% to 0% serum (Zhang et al., 2004). In this manner, the engrafted NSFCs could release BDNF to support their survival and modify the local environment as they integrated into the injured adult rat spinal cord. Fibroblasts genetically engineered to release BDNF have been shown under similar conditions to decrease RST neuronal atrophy after axotomy (Liu et al., 1999). Therefore, the present studies suggest that the engrafted NSFCs not only survived but may have secreted trophic factors including BDNF that rescued the

Fig. 14. Photomicrographs of midbrain showing FG retrograde tracing of RN neurons. All sections were approximately 400 Am from the caudal pole of the RN. Compared to the normal control animals (A), FG-labeled regenerated neurons were sparse and were slightly atrophic in the left (axotomized) RN region of the NSFCs recipients (C), but no FG positive cell bodies were observed at in the left RN of lesion only group (B).

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Effects of engrafted ONe-derived NSFCs on the axotomized RN neurons Injury to the RST at C3–4 in this study resulted in an apparent reduction in neuronal numbers (27%) and somal atrophy (24%) 2 weeks after surgery, with more significant changes (38% and 42%, respectively) 12 weeks post operation. These results are similar to previous reports of spinal cord injury on RST neurons (Feringa et al., 1988; Houle and Ye, 1999; Prendergast and Stelzner, 1976). The application of fetal cell transplants (Bregman and Reier, 1986; Mori et al., 1997) and neurotrophic factors (Houle and Ye, 1999; Jin et al., 2002; Kobayashi et al., 1997) including BDNF secreting cells (Liu et al., 1999, 2002) have been shown to prevent some axotomized RN cell atrophy. In this experiment, the engrafted NSFCs prevented some perikaryal atrophy of axotomized RN as well as apparent cell loss. Two important elements may have contributed to rescue of RN neurons from axotomy-induced retrograde atrophy. One is that the engrafted NSFCs may have served as a surrogate source of BDNF for the RST axons in proximity of the lesion/graft site. The other is that the relatively high level of graft survival and graft–host interface provided a relatively permissive environment, which fostered axonal regeneration and possible synapse formation with target neurons. Many GAP-43 positive processes support this presumption as well as the presence of BDA-labeled RST axons among the NSFCs within the lesion cavity of engrafted animals. Effects of engrafted ONe-derived NSFCs on functional recovery and RST axonal regeneration

Fig. 15. At 11 weeks post-engraftment, functional recovery of injured limb usage in NSFC-engrafted animals. (A and B) The lesion only animals only used the left (uninjured) forelimb to explore the wall of cylinder. The right (injured) forepaw was strongly flexed (arrows). (C and D) In contrast, engrafted animals freely used both paws to explore the cylinder with normal posture (arrows), sometimes, with the fingers of right forepaw extended (C). (E) Behavioral analysis of forelimb use. Compared to the normal data of forelimb usage prior to surgery, the engrafted animals (n = 12) showed significant recovery of use of the injured forelimb alone or together with the uninjured forelimb, whereas lesion only animals (n = 12) showed negligible use of the injured limb alone.

partially replacing lost accessory cells (Wirth et al., 2001), releasing trophic factors such as BDNF (Liu et al., 1999), and/or by laying down growth-permissive extracellular matrix molecules that could serve to guide regenerating axons through the lesion site (Geller and Fawcett, 2002). The numerous GAP-43 and MAP2 positive host axons found in the lesion cavity of engrafted animals compared to the lesion only animals were consistent with these possibilities. In summary, it is likely that the environment provided by the injured cord facilitated the survival, migration, and differentiation of the engrafted NSFCs as reported for other models (McDonald et al., 1999).

The results demonstrated that, after NSFC engraftment, functional recovery occurred. Eleven weeks after engraftment, the NSFC-engrafted animals displayed better locomotor activity during horizontal rope walking than lesion only animals, and significant improvements in forelimb usage ipsilateral to the lesion when exploring the wall of a cylinder. Importantly, the enhanced functions were consistent with the anatomical recovery of axotomized RN neurons of engrafted animals including higher FG labeling and less atrophy of RN neurons, RST axonal regeneration and innervation of target gray matter. This suggests that the enhanced function can be attributed at least partially to repair of the RN and RST pathway. Further support for this comes from studies of anterograde BDA-labeled RST axons that demonstrated axotomized RST axons in grafted animals that had regenerated sufficiently so that they passed through the graft, and in some instances found their target motoneurons. It is likely that these contacts contributed to the forelimb functional recovery. These results were consistent with previous studies (Kuchler et al., 2002; Teng et al., 2002). Three possible mechanisms could account for the recovery: (1) The NSFCs released growth factors that directly supported RN neurons and RST axonal regeneration; (2) the transplanted cells modified the microenviron-

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Fig. 16. At 11 weeks post-engraftment, horizontal rope walking was better in engrafted animals than in lesion only animals. (A and B) The photographs demonstrate the inability of lesion only animals to perform the rope-walking test. In A, the animal has poor posture and body position. Slips and falls were common among the lesioned animals (B). (C and D) The lesion plus engrafted animals assumed near-normal posture with limbs under their bodies and uplifted tails. These animals were able to complete the ropewalk. (E) Similarly, the total error/step ratio was higher in the lesion only group compared to the lesion plus engrafted group (*P b 0.001). (F) The lesion only group (n = 12) exhibited a higher deficit score (*P b 0.001) than those receiving the NSFC transplants (n = 12). (G) Functional performance of horizontal rope-walking test correlated with RN neuronal survival. Scatter graph of calculated total error/ step ratios plotted against accompanying number of FG-labeled neurons demonstrated a negative correlation between those 2 groups of parameters using Pearson’s correlation test.

ment within or surrounding the lesion site to facilitate this recovery; or (3) since some GFP-labeled NSFCs migrated into locations in which descending supraspinal or ascending sensory tracts, the presence of the NSFCs or the secretion of BDNF from the cells may have affected other systems such as the corticospinal tract, ventrolateral funiculus, dorsal columns, and ventral columns. The presence of MAP2 positive dendrites within the region of the lesion cavity may be an indicator of other systems’ axonal response to the presence of the NSFCs. This hypothesis was further supported by the existence of FG-labeled regenerated neurons in some vestibular and reticular nuclei of engrafted animals (experimental group IIIB, data not shown). Further analysis by anterograde or retrograde tracing is underway to determine whether transplanted NSFCS promote regeneration of descending motor and ascending sensory axons after spinal cord injury. This possibility, individually or collectively, with the effects on the RST pathway may have

led to the enhanced functional recovery. Potential mechanisms of RST axonal regeneration in NSFC-engrafted cord and quantitative evaluation of anterograde-labeled axonal regeneration warrants further study. In conclusion, these studies demonstrate that neural progenitors obtained from adult human olfactory epithelium following engraftment into host spinal cord can maintain axotomized RN neurons, promote RST axonal regeneration, and lead to enhanced function. Work with olfactory bulbderived ensheathing cells has shown similar effects on spinal cord repair and axonal regeneration and repair (Franklin and Barnett, 2000; Li et al., 1998; Lu et al., 2002). However, the highly invasive surgery that is required to obtain these cells negates their practical use. In contrast, NSFCs harvested from ONe provide an adult neural progenitor source which can be obtained with minimal invasive surgery and may have a unique therapeutic potential for use in autologous grafting/transplantation

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strategies aimed at repairing spinal cord injuries and other neurodegenerative disorders.

Acknowledgments We thank Dr. S.R. Whittemore for assistance with the GFP transfection; Dr. S.M. Onifier for help in planning the behavioral studies and for reading the manuscript; Mrs. C. Ekstrom for help in preparing the manuscript; Dr. W. Tetzlaff for helpful comments and advice. This work was supported by a grant from NIH #992558.

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