Báo cáo sinh học: "Transplanted astrocytes derived from BMP- or CNTF-treated glialrestricted precursors have opposite effects on recovery and allodynia after spinal cord injury" docx

adult rat spinal cord promoted first, a 39% efficiency of endogenous ascending dorsal column axon regeneration across sites of injury; second, protection of axotomized red nucleus neuron

Research article

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gglliiaall rre essttrriicctte ed d p prre eccu urrsso orrss h haavve e o op pp po ossiitte e e effffe eccttss o on n rre ecco ovve erryy aan nd d aallllo od dyyn niiaa

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Addresses: *Department of Neurosurgery, Anschutz Medical Campus, University of Colorado Denver, 12800 East 19th Ave, Aurora, CO

80045, USA †Department of Biomedical Genetics, University of Rochester Medical Center, 601 Elmwood Avenue, Rochester, NY 14642, USA

Correspondence: Stephen JA Davies Email: Stephen.Davies@UCHSC.edu

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Baacckkggrrooundd:: Two critical challenges in developing cell-transplantation therapies for injured or

diseased tissues are to identify optimal cells and harmful side effects This is of particular

concern in the case of spinal cord injury, where recent studies have shown that transplanted

neuroepithelial stem cells can generate pain syndromes

R

Reessuullttss:: We have previously shown that astrocytes derived from glial-restricted precursor

cells (GRPs) treated with bone morphogenetic protein-4 (BMP-4) can promote robust axon

regeneration and functional recovery when transplanted into rat spinal cord injuries In

contrast, we now show that transplantation of GRP-derived astrocytes (GDAs) generated by

exposure to the gp130 agonist ciliary neurotrophic factor (GDAsCNTF), the other major

signaling pathway involved in astrogenesis, results in failure of axon regeneration and

functional recovery Moreover, transplantation of GDACNTF cells promoted the onset of

mechanical allodynia and thermal hyperalgesia at 2 weeks after injury, an effect that persisted

through 5 weeks post-injury Delayed onset of similar neuropathic pain was also caused by

transplantation of undifferentiated GRPs In contrast, rats transplanted with GDAsBMPdid not

exhibit pain syndromes

C

Coonncclluussiioonnss:: Our results show that not all astrocytes derived from embryonic precursors are

equally beneficial for spinal cord repair and they provide the first identification of a

differentiated neural cell type that can cause pain syndromes on transplantation into the

damaged spinal cord, emphasizing the importance of evaluating the capacity of candidate cells

to cause allodynia before initiating clinical trials They also confirm the particular promise of

GDAs treated with bone morphogenetic protein for spinal cord injury repair

Open Access

Published: 19 September 2008

Journal of Biology 2008, 77::24 (doi:10.1186/jbiol85)

The electronic version of this article is the complete one and can be

found online at http://jbiol.com/content/7/7/24

Received: 31 December 2007 Revised: 14 June 2008 Accepted: 19 August 2008

© 2008 Davies et al.; licensee BioMed Central Ltd

This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited

Baacck kggrro ou und

Two critical challenges that must be addressed in the

development of cell-based tissue repair strategies are the

identification of optimal cell types and the identification of

instances in which cell transplantation may create severe

adverse side effects The first problem is important because

of the considerable resources that will be required to

establish clinical efficacy of putative treatments The second

problem is perhaps of even greater importance, because

adverse outcomes in clinical trials could seriously hinder

the development of stem cell technology for tissue repair

Diseases of the central nervous system (CNS) are of

particular interest as candidates for clinical evaluation of

cell transplantation therapies, with the treatment of spinal

cord injury being one of the primary targets for early

translation of laboratory efforts to clinical trials A variety

of cell types of both non-CNS and CNS origin, such as

Schwann cells [1], olfactory ensheathing glia [2], marrow

stromal cells [3,4] and oligodendrocyte progenitor cells

[5], are being considered for clinical trial to treat spinal

cord injuries One of the most attractive reasons for

considering the use of non-CNS cells such as Schwann

cells, olfactory ensheathing cells and marrow stromal cells

for CNS repair has been their relative ease of isolation

compared to cells of CNS origin However, continuing

advances in stem cell technology are making the goal of

utilizing CNS cell types to repair the injured CNS more

readily attainable

One new potential candidate for use in CNS repair is a

population of astrocytes that is derived by treatment of

glial progenitor cells (GRPs) of the embryonic spinal cord

with bone morphogenetic protein (BMP) before

The replacement of damaged neurons and

oligo-dendrocytes in the injured or diseased spinal cord has

been pursued by a number of laboratories (reviewed in

[6]), but less attention has been given to the development

of astrocyte replacement therapies, despite the fact that

astrocytes account for the majority of cells in the adult

CNS [7] and are critical to normal CNS function [8] This

relative lack of attention is probably due to the modest

levels of axon regeneration and lack of functional recovery

seen after transplantation into the injured CNS of

astro-cytes isolated from the immature cortex [9-12] Factors

such as contamination with microglia and

undifferen-tiated progenitors, isolation from cortex rather than spinal

cord, and a phenotype that is less supportive of axon

growth (resulting from the prolonged in vitro growth

required to generate postnatal astrocyte cultures) [13],

may have rendered these glial cultures suboptimal for

repairing the injured adult spinal cord

In contrast to the lack of effect of astrocyte transplantation

regeneration, neuroprotection and functional recovery after acute spinal cord injury [14] The ability to generate specific subtypes of astrocytes from defined glial precursors provides

a new platform for the development of astrocyte-based transplantation therapies for the injured adult CNS

adult rat spinal cord promoted first, a 39% efficiency of endogenous ascending dorsal column axon regeneration across sites of injury; second, protection of axotomized red nucleus neurons; third, a significant reduction of inhibitory scar formation; and fourth, a degree of behavioral recovery from dorsolateral funiculus injuries that enabled rats to generate an average score by 4 weeks after transplantation that was statistically indistinguishable from that obtained for uninjured animals on a stringent test of volitional foot placement [14] Moreover, this strategy allows the rapid generation of astrocytes directly from embryonic precursor cells, thus eliminating the use of the prolonged in vitro purification procedures that result in a phenotype that is less supportive of axon growth [13]

Recent studies demonstrating the ability of transplanted neuroepithelial stem cells (NSCs) to cause pain syndromes

in animals with spinal cord injury have, however, raised concerns that the astrocytes generated by transplanted stem

or progenitor cells might cause adverse effects that outweigh any benefits Two recent studies have shown that trans-plantation of NSCs into acute spinal cord injuries in rats promotes the onset of both mechanical allodynia (a painful response to normally non-painful touch stimuli) and thermal hyperalgesia (abnormal sensitivity to heat) [15,16] These adverse side effects correlated with the differentiation

of the transplanted NSCs into astrocytes, and were prevented by the suppression of astrocyte generation by overexpression of the transcription factor neurogenin-2 in the transplanted NSCs [15] It was therefore very important

to determine whether transplantation of astrocytes, or of precursor cells capable of generating astrocytes, would promote the onset of allodynia, or whether this is a problem unique to the transplantation of NSCs

The study reported here was carried out to determine whether all astrocytes generated from GRPs [17] were equally able to promote repair of adult injured spinal cord Two types of astrocytes can be generated from embryonic

from the gp130 receptor agonist ciliary neurotrophic factor (CNTF)) We found that transplantation of these two types

of astrocytes into acute spinal cord injuries (Figure 1) yielded significantly different outcomes In contrast to

and, more importantly, transplantation of either GDAsCNTF

or undifferentiated GRPs caused neuropathic pain Our

results also confirm earlier work [14] showing that

pre-differen-tiation of GRPs can provide substantial benefits after spinal

cord injury and that this pre-differentiation can avoid the

problem of transplanted glial precursors themselves causing

pain syndromes

R

Reessuullttss

C

Chhaarraacctteerriizzaattiioonn ooff GGDDAAss iinn vviittrroo

a flat, type-1 antigenic phenotype that express glial fibrillary

acidic protein (GFAP) and do not label with the A2B5

anti-body [14] In contrast, GRPs grown in the presence of the

seeking to use GDAs for repairing the injured spinal cord, it

is critical to know whether the favorable properties of

identity of the glial precursor cell from which they are derived, or whether it is necessary to generate a very specific population of astrocytes from these precursor cells to promote repair

axon-growth-F

Fiigguurree 11

Schematic illustration of the adult rat models of spinal cord injury used in this study ((aa)) Dorsal view of rat brain and spinal cord Dorsal column

white matter on the right side was transected (shaded area) at the C1/C2 spinal level, and the ability of either BDA-labeled endogenous axons or axons from microtransplanted GFP-expressing adult sensory neurons (DRGs) to cross injuries bridged with GDAs or GRPs was assayed

((bb)) Horizontal and ((cc)) sagittal views of the dorsal column white-matter pathways at the C1/C2 cervical vertebrae of the spinal cord (b) Injections of GDAs or GRPs (black diamonds) suspended in medium were made directly into the centers of the injury sites as well as their rostral and caudal

margins in the cervical spinal cord ((cc)) A discrete population of endogenous ascending axons within the cuneate and gracile white-matter pathways of dorsal columns was labeled by BDA injection at the C4/C5 spinal level (5 mm caudal to the injury site, shaded) Alternatively, microtransplants of

GFP+DRGs were injected 500 µm caudal to the injury site ((dd)) The right-side dorsolateral funiculus white matter containing descending axons of the rubrospinal tract was transected at the C3/C4 spinal level and GDAs or GRPs were transplanted as described for dorsal column injuries CC, central canal; Cf, cuneate fasciculus; CST, corticospinal tract; DF, dorsolateral funiculus; Gf, gracile fasciculus; GM, gray matter; RN, red nucleus; RST,

rubrospinal tract; T1, level of the first thoracic vertebra

(b)

(c) Rostal

C1/C2

GDAs

(horizontal view)

(sagittal view)

Cf/Gf Cf/Gf

Dorsal midline

GM

GM

C1/C2

C4/C5

C8

T1

GDAs

or GRPs

BDA

cc

Cf/Gf CST

C1/C2

C4/C5

BDA

C8 T1

DF/RST

or GRPs

RN RN

inhibitory chondroitin sulfate proteoglycans (CSPGs) NG2

(Figure 2a,b) and phosphacan (Figure 2c,d), both of which

are also expressed at high levels in glial scar tissue [18] We

their regulation of the transcriptional regulator Olig2 in vitro

(Figure 3a,b) In agreement with previous observations of

the effects of BMP on Olig2 expression in cortical neural

progenitors [19], GRPs exposed to BMP-4 downregulated

high levels of Olig2 in their nuclei (Figure 3b) Several

recent studies have reported the natural generation of cells

that coexpress Olig2 and GFAP in vivo after injury to the

brain [20,21] Although those studies described cytoplasmic

rather than nuclear localization of Olig2, our examination

of control injured spinal cords at 8 days revealed the

localization of Olig2 (Figure 3c)

F

Fiigguurree 22

GDAsBMP, GDAsCNTFand GRPs express different levels of NG2 and

phosphacan in vitro GRPs were induced to differentiate into

astrocytes by exposure to BMP or CNTF Relative levels of expression

of NG2 and phosphacan proteins were determined by quantitative

Western blot and immunocytochemical analysis ((aa,,cc)) Western blot

analysis of whole-cell lysates demonstrates that GDAsCNTFexpress

higher levels of (a) NG2 and (c) phosphacan The graph shows fold

change in protein levels for GDAs compared to GRPs Error bars

represent 1 standard deviation (SD) *p < 0.05 ((bb,,dd))

Immuno-fluorescent labeling of cells using (b) NG2 antibodies and (d)

anti-phosphacan Scale bars 50µm

Phosphacan

Phosphacan

Phosphacan

(c)

B-tubulin

Phosphacan

(d)

GDA BMP

NG2

GDA CNTF

B-tubulin

NG2

CNTF BMP GRP

1.0

0

0.2

0.4

0.6

0.8

1.2

1.4

*

CNTF BMP GRP

1

2

3

4

5

*

*

DAPI DAPI

F Fiigguurree 33 Differential expression of Olig2 protein by different astrocyte populations ((aa)) GDAsBMPdo not express Olig2 ((bb)) In sharp contrast, GDAsCNTFare uniformly immunopositive for Olig2 in vitro ((cc)) A subset of endogenous GFAP+astrocytes in the margins of untreated dorsal column spinal cord injuries is also Olig2-immunoreactive Survival, 8 days post-injury Note the nuclear localization of Olig2 in GDAsCNTFin vitro and in reactive, endogenous GFAP+astrocytes in vivo Scale bars: (a,b) 50 µm; (c) 25 µm

GFAP Olig2

GDACNTF

GFAP Olig2

Olig2

GFAP

GDABMP

(a)

(b)

(c)

DAPI

DAPI

Chhaarraacctteerriizzaattiioonn ooff ttrraannssppllaanntteedd GGDDAAssC CN NT TF Fiinn vviivvoo

able to completely span sites of injury (Figures 4-7) We

markedly different from those previously observed for

their GFAP immunoreactivity after transplantation to acute

spinal cord injury, particularly for those cells adjacent to

also displayed immunoreactivity for the

axon-growth-inhibitory proteoglycan neurocan at 4 and 8 days

retained their in vitro immunoreactivity for NG2 (Figure 5)

not retain GFAP immunoreactivity after transplantation to

identical acute spinal cord injuries [14] More importantly,

remained negative for neurocan and NG2 immunoreactivity

at 8 days after transplantation [14]

E

Effffeeccttss ooff GGDDAAssC CN NT TF Faanndd GGRRPPss oonn ssccaarr ffoorrmmaattiioonn

different effects on the reactivity of host astrocytes at sites of

injury We previously showed that transplantation of

within injury margins and promoted a remarkable

contrast, did not suppress astrogliosis, nor did these cells

align host astrocytes in injury margins Instead, the margins

(Figure 4a), similar to that observed in both control

un-treated injuries and the margins of GRP-transplanted injuries

in a suppression of neurocan and NG2 expression by host

tissue at sites of injury at 4 days post-injury, an effect we

[14] At 4 days after injury, the margins of control, untreated

injuries displayed a high density of neurocan

-processes that we previously showed to be associated with

(Additional data file 1), neurocan immunoreactivity within

injury margins was mainly associated with the cell bodies of

similar to that observed for neurocan at 2 days after injury

in untreated control animals [18] However, by 8 days after

immunoreactivity at sites of injury was similar in intensity

and distribution to that seen in untreated control injuries

promoted transient suppression of axon-growth-inhibitory

host astrocytes within injury margins

G

GDDAAssC CN NT TF Fddoo nnoott ssuuppoorrtt aaxxon rreeggeenerraattiioonn iinn vviivvoo

regeneration in vivo, both of endogenous ascending dorsal column axons and of axons emanating from transplanted

F Fiigguurree 44 GDAsCNTFexpress GFAP and neurocan after transplantation into spinal cord injuries ((aa)) Intra-injury GDAsCNTFare uniformly GFAP+within acute dorsal column injuries Note the co-localization (yellow) of human placental alkaline phosphatase (hPAP, red) with GFAP (green) GDAsCNTFhave also failed to align host astrocytic processes within injury margins Survival, 8 days post-injury/transplantation ((bb)) High-magnification confocal image of neurocan immunoreactivity at the injury margin and within a GDACNTF-transplanted injury site at 8 days after injury/transplantation Note that some GDAsCNTFare immunoreactive for neurocan (green) In contrast, intra-injury transplanted GDAsBMP

(not shown) do not express GFAP or neurocan, and can align host astrocytic processes within injury margins [14] Scale bars 100 µm

(a)

(b)

GFAP

Neurocan

adult dorsal root ganglion (DRG) neurons For analysis of

endogenous axon regeneration, a discrete population of

ascending axons aligned with the injury site was traced with

a single injection of biotinylated dextran amine (BDA) at a

GRP-trans-planted or control transection injuries of the right-hand

dorsal column cuneate and gracile white-matter pathways

This minimized the labeling of spared axons Previous

studies have shown that around 30-40% of ascending

dorsal column axons projecting to the dorsal column nuclei

arise from postsynaptic dorsal column neurons in spinal

lamina IV and that 25% of ascending dorsal column axons

are also propriospinal in origin [22,23] It has been shown

that only 15% of primary afferents of DRG neurons entering

the spinal cord at lumbar levels reach the cervical spinal

cord and that most leave dorsal column white matter within

two to three segments of entering [24] Therefore, our en

passage labeling of dorsal column axons at the cervical level

would have included significant proportions of axons from

both CNS spinal neurons and DRG neurons To further test

across an acute spinal cord injury in a model that eliminates

the possibility of axon sparing, we examined their ability to

support the growth of adult sensory axons across identical

stab injuries in an adult DRG neuron/GDA transplant

spinal cord injury model [14] In these experiments, a

separate series of animals received microtransplants of adult

mouse sensory neurons labeled with green fluorescent protein (GFP) acutely into dorsal column white matter at a

injuries (Figure 1c)

column transection injuries failed to improve the regenera-tion of endogenous ascending dorsal column axons above that observed in untreated injuries (Figure 7) There was also a complete failure of axons grown from adjacent micro-transplanted adult mouse DRG neurons expressing enhanced

-trans-planted injuries (Additional data file 2) In both experi-mental models, the majority of axons instead formed dystrophic endings within the caudal injury margins of

data file 2a), an axon morphology well known as the hallmark of failure of axon regeneration in CNS injury [25,26] Quantitative analysis of the efficiency of ascending

GRP-trans-planted rats at 8 days after transplantation/injury showed that only 7% (SD ± 2.0) and 5.3% (SD ± 3.0), respectively,

of BDA-labeled axons within white matter 0.5 mm caudal

to injury sites had reached injury centers; 6.2% (SD ± 3.5) and 4.7% (SD ± 3.9) of axons had extended 0.5 mm beyond injury sites into distal white matter, with 4.6% (SD ± 2.3) and 4.2% (SD ± 3.6) reaching 1.5 mm beyond

F

Fiigguurree 55

NG2 immunoreactivity in GDACNTF-transplanted dorsal column injuries ((aa)) Transplanted hPAP+GDAsCNTF(arrowheads) at 8 days post

injury/transplantation ((bb)) The same slide stained for NG2 (green) showing that the transplanted cells (arrowheads) show immunoreactivity for NG2 ((cc)) Co-localization (yellow) of NG2 and hPAP immunoreactivity in regions containing higher densities of GDAsCNTF(arrowheads) In general, regions

of the injury site that contained higher densities of hPAP+GDAsCNTFhad a higher density of NG2 immunoreactivity Scale bars 50 µm

NG2

injury sites (Figure 7c) No BDA-labeled axons were

detected beyond 1.5 mm in distal white matter or within

GRP-transplanted rats (Figure 7c) All the percentages of

BDA-labeled axons within injury sites and at all points beyond

were not statistically different from those quantified for

BDA-labeled endogenous ascending dorsal column axons in

identical control, untransplanted injuries [14] (ANOVA,

p > 0.05)

support axon regeneration is in stark contrast to the ability

endogenous dorsal column axons across spinal cord

labeled axons extended to the injury center, 36.5% (SD ± 11.0) extended to 0.5 mm beyond the injury site, and 30.4% (SD ± 9.2) had extended to 1.5 mm beyond the injury site (Figure 7c) Furthermore, 12.6% (SD ± 9.0) of labeled axons were detected within white matter at 5 mm beyond the injury site, and 2.1% (SD ± 1.4) were observed within the dorsal column nuclei (Figure 7c) This is consistent with our

cells promote regeneration of 60% (SD ± 11.0) of labeled

F

Fiigguurree 66

Transplanted GDAsCNTFexpress neurocan and NG2, but suppress host expression of these two CSPGs at 4 days post-injury/transplantation ((aa)) At

4 days after injury, control dorsal column injury margins express dense neurocan immunoreactivity (green) mainly associated with GFAP-processes Note the absence of neurocan immunoreactivity in the injury center (to the left) ((bb,,cc)) While neurocan immunoreactivity in host white matter was markedly lower and mainly associated with astrocyte cell bodies, many intra-injury GDAsCNTFwithin injury centers displayed neurocan

immunoreativity ((dd)) NG2 immunoreactivity in control injuries is high in both injury centers and margins ((ee,,ff)) Although overall levels of NG2

immunoreactivity were reduced within injury centers and margins of GDACNTF-transplanted injury sites compared to untreated control injuries

(compare (d) and (f)), levels of NG2 immunoreactivity were still higher than that previously observed for identical dorsal column injuries

transplanted with GDAsBMP[14] Scale bars 200 µm

Neurocan

GDACNTF

GDACNTF

(b)

(e)

NG2

Fiigguurree 77

Failure of axons to regenerate across GDACNTF or GRP transplanted dorsal column injuries ((aa)) Biotinylated dextran amine (BDA)-labeled endogenous, ascending dorsal column axons (green) fail to cross GDACNTF-transplanted injury sites and instead form dystrophic endings within caudal injury margins While a few axons sprout towards the injury center, BDA+axons are rarely detected beyond the injury/transplantation site at 8 days post-injury/transplantation Scale bar 200µm ((bb)) In contrast, transplanted GDAsBMPsupport extensive axon growth across dorsal column injuries at 8 days after injury/transplantation Scale bar 200 µm ((cc)) Quantification of numbers of regenerating BDA+axons in GDA- or GRP-transplanted dorsal column white matter at 8 days after injury and transplantation BDA-labeled axons were counted in every third sagittally oriented section within the injury center and at points 0.5 mm, 1.5 mm and 5 mm rostral to the injury site and within the dorsal column nuclei (DCN) Note that 55% of BDA+axons reached the centers of GDABMP-transplanted injuries, and 36% to 0.5 mm beyond the injury site After GDACNTFor GRP transplantation, however, only 7% and 5.3% of BDA+axons, respectively, were observed within injury centers, with only 4.6% and 4.2% of the axons observed at 0.5 mm beyond the injury site No BDA+axons were detected beyond 1.5 mm rostral to the injury site in GDACNTF- or GRP-transplanted spinal cords Error bars represent 1 SD

0

2 0

4 0

6 0

8 0

1 0 0

1 2 0

–0.5 mm Center 0.5 mm 1.5 mm 5 mm DCN

GDABMP GDACNTF GRP

(a)

(b)

(c)

GDABMP

BDA

GDACNTF

BDA

endogenous ascending dorsal column axons into the center of

injury sites, and more than two-thirds of these axons were

within white matter beyond the injury site by 8 days after

transplantation/injury [14]

F

Faaiilluurree ooff GGDDAAssC CN NT TF Fttoo pprroomottee llooccoomottoorr ffuunnccttiioonnaall

rreeccoovveerryy aafftteerr ssppiinnaall ccoorrdd iinnjjuurryy

funiculus transection injuries to the spinal cord, an analysis of

rats versus rats injected with control medium was carried out at

times ranging from 3 to 28 days after injury/transplantation

Transection of the dorsolateral funiculus severs descending

supraspinal axons and results in chronic deficits in both

fore-and hindlimb motor function [27] that can be detected by the

grid-walk behavioral test [28] We have previously shown that

injuries resulted in robust improvements in grid-walk locomotor

function compared to media-injected control injured animals at

all time points ranging from 3 to 28 days post-injury In contrast,

transplantation of undifferentiated GRPs failed to improve scores

to greater than those observed for control injured animals [14]

medium alone (controls) made an average of 6.2 (SD ± 0.5) and 6.0 (SD ± 0.3) mistakes, respectively, at 3 days after injury/transplantation and showed no statistically significant improvement at any later time point, with an average of 5.2 (SD ± 0.3) and 5.0 (SD ± 0.9) mistakes at 28 days post-injury (Figure 8) Thus, despite receiving transplants of astrocytes

did not show any recovery of locomotor function when compared with controls In contrast, at 3 days after

already making an average of 4.5 (SD ± 0.3) mistakes; that is,

animals (Figure 8) Consistent with our previous report [14],

continued to improve significantly between 3 and 28 days after injury (two-way repeated measures ANOVA, p < 0.05) At 28

1.7 (SD ± 0.3) mistakes on the grid walk apparatus (Figure 8), a score that was statistically indistinguishable from their pre-injury baseline scores

T Trraannssppllaannttaattiioonn ooff GGDDAAssC CN NT TF Foorr GGRRPPss ffaaiillss ttoo ssuupprreessss aattrroophyy ooff rreedd nnuucclleeuuss nneurroonnss

Transection of axons of the rubrospinal tract (RST) in the dorsolateral funiculus of the spinal cord causes atrophy of significant numbers of red nucleus neurons, a process that begins approximately 1 week after spinal cord injury [29]

F

Fiigguurree 88

Grid-walk analysis of locomotor recovery Graph showing the average

number of missed steps per experimental group from 1 day before

injury (baseline pre-injury) to 28 days after injury for all

GDA-transplanted/dorsolateral funiculus injured rats versus the

control-injured animals GDABMP-transplanted animals (green) performed

significantly better than GDACNTF-transplanted animals and injured

control animals at all post-injury time points (p < 0.05) Note that the

performance of GDACNTF-transplanted animals was not different from

untreated control injured rats at all time points (two-way repeated

measures ANOVA, *p < 0.05) N = 9 rats per group

0

1

2

3

4

5

6

7

8

Days

Control

GDA BMP GDA CNTF

*

*

* *

* * * *

F Fiigguurree 99 Neuroprotection of red nucleus neurons Injured left-side red nuclei contained an average of 52% of the neurons counted in uninjured right-side red nuclei at 5 weeks after transection of the right-right-side rubrospinal tract The numbers of neurons in the injured left-side red nuclei of GRP- and GDACNTF-transplanted animals were no different from controls, and contained an average of 55% and 51%, respectively, of the neurons counted in the uninjured right-side nuclei In contrast, the number of neurons in the injured left-side red nuclei of GDABMP -transplanted animals was 81% of the total number of neurons in uninjured right-side nuclei *p < 0.01 Error bars represent 1 SD

0 25 50 75 100

Control GRP GDACNTF GDABMP

*

In the absence of intervention, the number of neurons with

left-side red nucleus in control, untreated RST-injured animals

fell to 52% (SD ± 4.2%) of the values in the uninjured

right-side nucleus at 5 weeks after injury (Figure 9)

Consistent with our previous findings [14], animals that

(Figure 9) once again showed a significant suppression of red

nucleus neuron atrophy with 82% (SD ± 6.1) of neurons in

the injured left-side red nucleus having cell body diameters

right-side nucleus (Figure 9) In contrast, transplantation of

dorso-lateral funiculus injuries completely failed to suppress neuron atrophy in the injured left-side red nucleus (Figure 9; see also Additional data file 3) Counts of neurons with a

only 51% (SD ± 8.7%) and 55% (SD ± 8.0%), respectively,

of the values in uninjured right-side red nucleus at 5 weeks after injury and did not differ statistically from untreated injured animals (ANOVA, p < 0.05) Thus, despite the fact

from the same embryonic precursor cells, they do not share the same ability to rescue red-nucleus neuronal populations from atrophy

G

GDDAAssC CN NT TF Foorr GGRRPPss,, bbuutt nnoott GGDDAAssB BMP,, iinnduccee mmeecchhaanniiccaall aallllooddyynniiaa aanndd tthheerrmmaall hhyyppeerraallggeessiiaa wwhhen ttrraannssppllaanntteedd iinnttoo ssiitteess ooff ssppiinnaall ccoorrdd iinnjjuurryy

GRPs might promote the induction of mechanical allodynia and thermal hyperalgesia in acute spinal cord injuries, initial experiments were carried out to test for increases in mechanical and thermal sensitivity in control rats receiving injections of medium into transection injuries of the right-side dorsolateral funiculus at 2, 3, 4, and 5 weeks after injury Importantly, compared with pre-injury scores, injured medium-injected control rats did not show statistically significant increases in gram force withdrawal thresholds for right-side forepaws in response to application of graded Von Frey filaments at all time points after injury and trans-plantation (Figure 10a) Similarly, analysis of paw-withdrawal response latencies to an experimental heat source pre- and post-injury revealed no statistically signifi-cant induction of thermal hyperalgesia in injured controls at all time points post-injury (Figure 10b) These results enable a direct comparison of the effects of intra-injury

of mechanical allodynia and thermal hyperalgesia in rats with identical dorsolateral funiculus transection injuries

-transplanted animals did not show a statistically significant increase in sensitivity to mechanical or heat stimuli at any times (2, 3 and 4 weeks) up to 5 weeks post-injury (Figure 10a,b) compared to pre-injury responses (two-way

animals showed a significant increase in sensitivity to both mechanical and heat stimuli by 2 weeks after injury, an effect that persisted to 5 weeks after injury, the last time point tested (Figure 10a,b) Animals that received

intra-F

Fiigguurree 1100

Von Frey filament and hot-plate analysis of mechanical and thermal

allodynia ((aa)) Withdrawal threshold of the right front paw to a

mechanical stimulus (force in grams) Measurements were made on

GRP- or GDA-transplanted and injured control animals at 2, 3, 4 and 5

weeks after dorsolateral funiculus injury/transplantation ((bb)) Latency (in

seconds) to paw withdrawal from a heat source Note that injury alone

and GDABMPtransplantation do not induce statistically significant

mechanical or thermal allodynia at any time point However, the

mechanical threshold and latency to withdrawal from a heat source are

significantly lower in GDACNTF- and GRP-transplanted rats beginning at

2 and 3 weeks, respectively, post-injury/transplantation Asterisks

denote a statistical difference from time-matched control animals

(two-way repeated measures, ANOVA, p < 0.05) Error bars represent 1 SD

Mechanical allodynia

0

5

10

15

20

25

30

Week 2 Week 3 Week 4 Week 5

Thermal hyperalgesia

0

1

2

3

4

5

6

7

8

9

Week 2 Week 3 Week 4 Week 5

(a)

(b)

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