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Reversal of neurochemical changes and pain-related behavior in a model of neuropathic pain using modified lentiviral vectors expressing GDNF
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doi:10.1016/j.ymthe.2005.11.026
Reversal of Neurochemical Changes and Pain-Related Behavior in a Model of Neuropathic Pain Using Modified Lentiviral Vectors Expressing GDNF Sophie Pezet,1,* Agnieszka Krzyzanowska,1 Liang-Fong Wong,2 John Grist,1 Nicholas D. Mazarakis,2 Biljana Georgievska,3 and Stephen B. McMahon1 1
The London Pain Consortium, Neurorestoration, The Wolfson Centre for Age-Related Diseases, KingTs College London, Hodgkin Building, Wolfson Wing, GuyTs Campus, London SE1 1UL, UK 2 Oxford BioMedica (UK) Ltd., Medawar Centre, Robert Robinson Avenue, The Oxford Science Park, Oxford OX4 4GA, UK 3 Neurobiology Group, Department of Experimental Medical Science, Wallenberg Neuroscience Center, Lund University, BMC A11, 221 84 Lund, Sweden *To whom correspondence and reprint requests should be addressed. Fax: +44 0207 848 6165. E-mail: [email protected].
Available online 28 February 2006
In this study, we evaluated the possible use of lentiviral vectors in the treatment of neuropathic pain. We chose to administer GDNF-expressing vectors because of the known beneficial effect of this trophic factor in alleviation of neuropathic pain in adult rodents. Lentiviral vectors expressing either GDNF or control, green fluorescent protein or B-galactosidase, were injected unilaterally into the spinal dorsal horn 5 weeks before a spinal nerve ligation was induced (or sham surgery for the controls). We observed that intraspinally administered lentiviral vectors resulted in a large and sustained expression of transgenes in both neurons and glial cells. Injection of GDNF-expressing viral vectors induced a significant reduction of ATF-3 up-regulation and IB4 down-regulation in damaged DRG neurons. In addition, it produced a partial but significant reversal of thermal and mechanical hyperalgesia observed following the spinal nerve ligation. In conclusion, our study suggests that lentiviral vectors are efficient tools to induce a marked and sustained expression of trophic factors in specific areas of the CNS and can, even if with some limitations, be efficient in the treatment of neuropathic pain. Key Words: survival, neuroprotection, trophic factor, ATF-3, IB4, spinal cord
INTRODUCTION Neuropathic pain can arise from lesions of the peripheral or central nervous system (CNS). Peripheral nerve injury elicits morphological, metabolic, and biochemical changes in the damaged dorsal root ganglion (DRG) neurons. Such changes in the DRGs include downregulation of Griffonia simplicifolia, isolectin IB4 (IB4) binding, P2X3 receptors [1], and some sodium channel subunits [2–4]; decreased activity of the enzyme thiamine monophosphate (TMP; [5]); as well as up-regulation of galanin, neuropeptide Y [6–9], and the activating transcription factor 3 (ATF-3; [10]). Glial cell line-derived neurotrophic factor (GDNF) is a member of the transforming growth factor-h superfamily. It is expressed in a wide variety of structures of the central and peripheral nervous systems, where it regulates neuronal survival, differentiation, and gene expression. In the DRG, many cells responsive to GDNF are the smalldiameter cells positive for IB4. As previously mentioned,
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nerve injury results in dysregulation of peptides, receptors, and channels, including a down-regulation of GDNF itself [11]. Exogenous administration of GDNF has previously been shown to be successful in preventing the down-regulation of IB4 [6,12], CGRP [13], P2X3 [1,6,14], sodium channels [12,15], and TMP activity [5] and up-regulation of ATF-3 [6,14], galanin, and NPY [6]. The therapeutic value of GDNF has been tested in models of neurodegenerative diseases. For example in animal models of Parkinson disease (PD) direct infusion of GDNF to the midbrain promoted dopaminergic neuron survival and improved neurological function [16–18], while in neuropathic pain models delivery of GDNF to the lumbar DRG/spinal cord reversed sensory abnormalities arising from neuropathic pain states [12]. However, in a clinical trial in which PD patients received GDNF via intracerebroventricular catheters, GDNF-treated patients suffered from many side effects and did not show improvements in parkinsonism [19]. The failure to
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demonstrate clinical improvement was probably due to the spread of GDNF to other brain areas and the weak penetration of GDNF in the target tissue, suggesting that a more specific targeted route of administration is required. More recently, focal delivery of GDNF in the putamen induced a good clinical improvement and limited side effects, suggesting that locally delivered GDNF can have a therapeutic value [20,21]. Consequently, those studies suggest that new methods to induce a local up-regulation of GDNF are likely to be successful therapies. Targeted delivery of GDNF to specific CNS regions can be achieved by viral vectors and may be exploited as a therapeutic strategy in the treatment of PD and neuropathic pain [6,12]. Lentiviral vectors are ideal gene delivery tools as they induce limited inflammation, mediate long-term and stable expression even in nondividing cells, and have a relatively large cloning capacity. In this study we investigated the possible use of lentiviral vectors encoding GDNF to achieve targeted delivery to the spinal cord and evaluated these vectors in neuropathic pain models.
RESULTS Cellular Transduction Using Lentiviral Vectors in the Spinal Cord In this study, we compared the transduction efficiency and potential beneficial effect of two different lentiviral vectors (human immunodeficiency virus (HIV) and equine infectious anemia virus (EIAV) based) in the development of neuropathic pain. Spinal injection of lentivirus synthesizing the green fluorescent protein (GFP; HIV-based vector) induced a minor local injury, and we performed direct observation of GFP immunofluorescence 2, 4, and 6 weeks postinjection. At 2 weeks, GFP was expressed in many cells of the gray and the white matter and this expression increased at 4 and 6 weeks after injection (Fig. 1A). Quantification using quantitative RTPCR showed that 4 weeks after intraspinal injection of the EIAV-based vector expressing LacZ or GDNF a strong expression of LacZ or GDNF mRNA, respectively, was induced (Fig. 1D). Six to eight weeks after the injection of viral vector, we observed transduced cells (GFP or LacZ, i.e., HIV- or EIAV-based vectors) rostrocaudally at least one spinal segment away from the site of injection. Fig. 1B shows an example of rostrocaudal distribution of LacZ staining in the lumbar cord. Following vector injection at the L5 level, we observed transduced cells in the white and gray matter of the cord from L3 to L6, with a limited expression in L3 and L4 and maximum expression in L5 and L6 (Fig. 1B). Despite the fact that the virus was injected unilaterally in laminae IV–V of the cord, LacZ or GFP staining appeared in deep laminae of the cord (V) and spread more superficially (I–IV) and deeply (ventral horn). We observed some LacZ staining (but never any GFP staining) as well in the ventral horn of the contralateral site (Figs. 1A–1C). Using immunostaining for GDNF, we
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observed that the rostrocaudal spread of GDNF transduction was similar to that of control viruses coding for GFP or LacZ (Fig. 1C, a). One striking difference, however, was the hypertrophy of the white matter at the level of the injection site (Fig. 1C, a). Examination of GFP- (Figs. 2A–2G) and h-galactosidase- (Figs. 2H–2J) expressing cells revealed that cells of different morphologies were transduced by the EIAVbased LacZ and HIV-based GFP vectors. Both vectors transduced neurons, as shown in Figs. 2A and 2B for the GFP-expressing vector by their typical morphology and size (Fig. 2B taken at the level of motor neurons in the ventral horn). This was confirmed by colocalization with the NeuN marker and demonstrated that neurons in superficial laminae were also transduced (Figs. 2C and 2J). The majority of cells transduced by the HIV-based vector were astrocytes, as shown in Figs. 2D and 2E. However, some microglial cells were seen to be transduced by this viral vector as well (Figs. 2F and 2G). In contrast the EIAVbased vector transduced mainly neurons (Figs. 2H–2J). At the level of the lateral funiculus, most of the h-galactosidase-expressing cells were neurons, even if a few astrocytes were transduced as well. Using OX42, GFAP, and ED1 immunostaining, we analyzed the potential microglial, astrocytic, or inflammatory reaction induced by intraspinal injection of the different viral vectors. We observed a limited microglial, astrocytic, and inflammatory reaction at the level of the injection site, but we did not observe any increased reaction induced by either h-galactosidase nor GDNF themselves compared to GFP. Reversal of Neurochemical Changes Associated with Spinal Nerve Ligation (SNL) Primary sensory neurons are a heterogeneous population. Approximately two-thirds are small- and medium-sized neurons and code for noxious stimulation. Half of this nociceptive population are NGF sensitive and synthesize neuropeptides (substance P, CGRP). The other half are bnonpeptidergicQ and express GDNF receptors, i.e., Ret and GFRa1, and are responsive to GDNF [5,22]. Following a peripheral nerve injury, damaged neurons of the DRG show a drastic dysregulation of their peptides, receptors, and channels. The exact mechanism underlying the dysregulation of these molecules is not completely understood, but decreased trophic support by NGF and GDNF induced by the nerve injury seems to play a role. The content of both NGF [23] and GDNF [11] is reduced in the DRG following nerve injury due to a lack of retrograde transport of these factors and the down-regulation of their receptors [24–26]. However, if intrathecally administered, NGF and GDNF reverse the changes in IB4, ATF-3, TMP, and CGRP expression. NGF and GDNF act on different cell populations: NGF reverses these changes in the NGFresponsive peptidergic population, whereas GDNF acts on the TMP-, P2X3-, and IB4-positive population [5,14].
MOLECULAR THERAPY Vol. 13, No. 6, June 2006 Copyright C The American Society of Gene Therapy
doi:10.1016/j.ymthe.2005.11.026
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FIG. 1. Intraspinally injected, modified viral vectors induce a strong and sustained transfection of numerous cells in the spinal cord. (A) Immunostaining for GFP following transfection with the HIV-based viral vector, showing the time course and spread of the GFP transduction in the spinal cord ipsilateral to the injection. Note that the amount of GFP produced increases with time. (B) X-gal revelation showing a representative rostrocaudal spread of LacZ-transduced cells in the spinal cord, 6 weeks after the injection of the EIAV-based viral vector. (C) GDNF immunostaining showing the spread of GDNF-transduced cells 6 weeks after the injection of the EIAV-based viral vector. (b) is a high-power magnification of (a). Note the bright labeling for GDNF in the dorsal and ventral horns and in the hypertrophic dorsal root (arrow). (D) Relative LacZ or GDNF expression (measured by quantitative RT-PCR) in the spinal dorsal horn 4 weeks after intraspinal injection of EIAV-based vectors, represented as means F SEM. Scale bar: (A) 340 Am, (B) 1 mm, (C) a, 600 Am, b, 75 Am.
In this study, once we established that the viral vectors could be used to deliver GDNF to the spinal cord, we investigated the efficiency of this viral-vectormediated expression of GDNF in reversing neurochemical and behavioral changes induced by a spinal nerve ligation.
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In naRve animals the percentage of IB4-positive DRG neurons was 40%. IB4 staining was not significantly modified in sham animals (39.3 F 0.7%). Two weeks after the spinal nerve ligation, immunostaining for IB4 was completely lost in the L5 DRGs in LacZ- (0 F 0%) or GFP(1.7 F 1.5%) treated animals. In contrast, such a lack of
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FIG. 2. Lentiviral vectors transduce both spinal glial cells and neurons. (A–G) Spinal cells transduced by the HIV-based viral vector. Examples of GFP staining in the spinal dorsal cord in an animal that received an intraspinal injection of the HIV-based vector. (A and B) An example of the spread and shape of the GFP-positive cells in the dorsal horn. Many cells have glial cell morphology. (A) The dotted line shows the border between the spinal gray and white matter. (B) Example of motor neurons transduced with GFP in the spinal ventral horn. (C) Double staining with NeuN (red) and GFP (green) in the dorsal spinal cord of an animal that received the GFP lentiviral vector. The inset shows a highpower magnification in superficial laminae where costained cells are found, suggesting that a subset of these GFP-positive cells are neurons. (D and E) Double staining for GFP (D) and GFAP (E) showing that a proportion of GFPpositive cells are astrocytes (arrows). (F and G) Double staining for GFP (F) and OX-42 (G) showing that a proportion of GFP-positive cells are microglial cells (arrows). (H–J) Spinal cells transduced with the EIAV-based vector. Examples of h-galactosidase staining in the spinal dorsal cord in an animal that received an intraspinal injection of the EIAV-based vector. (I) A high-power magnification of the white rectangle in (H). The dotted line shows the border between the spinal gray and white matter. Note that the shape, size, and location of transduced cells in the spinal gray matter suggest that they have neuronal morphology. (J) Double immunofluorescent staining for h-galactosidase (green) and NeuN (red). The inset is a higher power magnification showing an example of doubly labeled cells (arrows). All pictures were taken 6 weeks after injection. Scale bar: (A) 65 Am; (B) 110 Am; (C) 125 Am, inset, 45 Am; (D, E) 150 Am; (F, G) 80 Am; (H) 105 Am; (I) 10 Am; (J) 90 Am, inset, 35 Am. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
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staining was partially prevented in GDNF virus-treated animals (HIV-based vector, 28.2 F 5.7%; EIAV vector, 29.3 F 3.9%; Figs. 3C and 3D). In naRve animals and in DRGs contralateral to SNL, there was a complete absence of expression of the transcription factor ATF-3. In sham-treated animals in contrast, a small percentage of cells was positive for ATF-3 (2 F 1.1%). Two weeks after SNL, as previously described [10], ATF-3 was expressed in most neurons of the control DRGs (HIV-based vector 93.5 F 0.9%, EIAV-based vector 97.9 F 0.4%; Fig. 3B). Such up-regulation was partially reversed by intraspinal injection of the GDNF-producing vector (HIV-based vector 34.2 F 3.12%, EIAV-based vector 36.7 F 6.6%; Fig. 3C). These results suggest that spinal expression of GDNF through viral vectors induced neuroprotection of a population of DRG neurons. Immunostaining for IB4 in the spinal cord of naRve or sham animals was located in lamina II of the dorsal horn (Fig. 4A). Two weeks after SNL, as previously described [26], there was a lack of immunostaining for IB4 in the lumbar spinal cord at the L5 level (Figs. 4B and 4E). This lack of staining was completely reversed at the injection site in GDNF virus-injected animals and was even much higher than the contralateral side or in sham animals (HIV-based vector 66 F 30% increase over contralateral values; EIAV-based vector 40 F 22% increase over contralateral values; Figs. 4C and 4E). However, when looking away from the injection site (in L5–L6), such increased staining and rescue of IB4 fibers had disappeared (Figs. 4D and 4E) and a partial lack of IB4 was observed (Figs. 4D and 4E). In conclusion, the GDNF-synthesizing viruses injected intraspinally partially rescued the reduction in IB4 staining in areas in proximity to the injection site. However, directly at the injection site, virally administered GDNF induced an enormous increase in IB4 fibers as well as a hypertrophy in the dorsal root. Results shown in Fig. 4 are those obtained with the EIAV virus. Use of the HIV-based vector gave similar results. These results are in agreement with our observations in the DRG that spinal overexpression of GDNF rescues a population of sensory neurons. However, the large amount of GDNF expressed induced aberrant changes in the dorsal root as well. Behavioral Data Following injection of the vectors, we followed the behavior of the rats for 6 weeks and established a baseline thermal and mechanical threshold. There were no marked differences in the sensitivity of the rats after the spinal injection compared to values before the injection, nor were there any differences between the ipsilateral and the contralateral paws. The rats had normal appearance and level of activity and were feeding regularly. After the spinal nerve ligation, control rats showed a marked reduction in mechanical threshold on the ipsi-
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and significantly different from the baseline only after 10 days postsurgery. At three time points (3 and 6 days postligation) the GDNF-treated rats showed a reduced mechanical sensitivity in the ipsilateral paws compared to the LacZ-treated group in the first 6 days after SNL (P V 0.01, two-way ANOVA, Student Newman–Keuls post hoc test). The heat threshold of control animals significantly decreased by 33.8 F 10.4% (P V 0.05) at 3 days postligation on the ipsilateral side (Fig. 5A). Shamoperated animals showed no change in thermal latency. The thermal sensitivity of the ipsilateral hind paw of GDNF-treated rats was significantly reduced compared to LacZ virus-treated animals, in particular in the first week after SNL (P V 0.05, two-way ANOVA, Student Newman–Keuls post hoc test, Fig. 5A). The contralateral side was not significantly different from baseline in both treatments. Animals treated with the HIV vector expressing GDNF also showed on average a decreased mechanical and thermal hypersensitivity compared to the GFP-treated group, but due to variations in certain animals of the GDNFtreated group, these variations were not statistically different.
DISCUSSION FIG. 3. Intraspinal injection of lentiviral vector expressing GDNF reverses ATF-3 up-regulation and IB4 down-regulation in the DRGs induced by spinal nerve ligation (SNL). (A–C) Double labeling of IB4 (green) and ATF-3 (red) representing an example of sham-operated animals that received GFP- or LacZ-expressing virus (A) or SNL-treated animals that received either GFP- or LacZ-expressing virus (B) or GDNF-expressing virus (C). Note that the GDNF treatment reduces but does not prevent the up-regulation of ATF-3 and the down-regulation of IB4. (D and E) Quantification of changes in the percentage of IB4 (D) or ATF-3 (E) in the DRGs of sham- or SNL-operated animals treated with either the LacZ- or the GDNF-expressing viral vector (EIAV-based vector), represented as means F SEM. *P b 0.05, statistically different from sham- and LacZ-treated animals. Statistical test used was one way ANOVA on ranks, followed by Tukey post hoc test. Scale bar: 50 Am. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
lateral side, a drop of 41.1 F 3.2% from baseline in the EIAV vector-treated animals by day 3 (Fig. 5B) and a 38.9 F 3% drop in the HIV vector animals, significantly different from baseline values at all time points (P V 0.01). Contralateral paw values did not differ from baseline. Sham-operated animals showed no change in sensitivity from baseline after the surgery in either ipsilateral or contralateral paws. Animals treated with the EIAV vector expressing GDNF also showed a decreased threshold to mechanical stimulation; however, it was less pronounced than that of LacZ virus animals (drop of 33.4 F 5% by day 3, Fig. 5B)
MOLECULAR THERAPY Vol. 13, No. 6, June 2006 Copyright C The American Society of Gene Therapy
In the past 5 years, several laboratories used viral vectors as new strategies in the study of pain. Most of the studies used peripheral injections of viral vectors such as adeno-associated viral vectors up-regulating mu opioid receptors [27,28] or nerve growth factor or herpes simplex virus vectors expressing proenkephalin [29,30] in peripheral tissues such as the skin or the sciatic nerve. Other studies used intraspinal injections of trophic factors such as BDNF in the treatment of neuropathic pain [31]. Among all viral vectors available, we chose lentiviral vectors for their ability to transduce neuronal cells, their long-term expression patterns, and the limited inflammation they induce. Both vectors we used were pseudotyped with the vesicular stomatitis virus glycoprotein (VSVG), which recognizes specific host cell receptors [32] and reduces the possibility of side effects on nontarget cells [33]. As observed by collaborators using the same types of viruses [34], we detected a strong transduction and synthesis of GFP, LacZ, or GDNF for up to 8 weeks (the maximal time course of our experiment) following the initial injection of viral vectors. However, these types of lentiviral vectors have been observed to be expressed for up to 9 months after the injection [35,36]. We have found that the two types of vectors used were capable of transducing both glial and neuronal cells, even though the EIAV-based vector transfected mainly neuronal cells and the HIV-based vector transduced mainly glial cells. Previous work using the same vectors showed that they
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routinely transduce mainly neuronal cells [34,37]. We do not have any explanation at the moment for the fact that in the spinal cord, the HIV-based vector transduced mainly glial cells, a feature not observed in the striatum [37]. However, because in our study transduced cells in the spinal cord were used as a source of overexpressed GDNF to protect injured sensory neurons, the nature of
FIG. 5. The (A) thermal and (B) mechanical hyperalgesia associated with spinal nerve ligation is reduced in rats that received intraspinal injection of EIAVbased vector expressing GDNF compared to LacZ-expressing virus. Stars indicate statistically different data points between the two groups (P b 0.05, two-way ANOVA, Student Newman–Keuls post hoc test).
the transduced cells (neurons or glial cells) did not influence the efficacy of the neuroprotection induced by both viruses. Only one study to date examined viral administration of GDNF in pain models; however, it was based on the herpes simplex virus (HSV) system [38]. Applied at the periphery, HSV is retrogradely transported to the DRG
FIG. 4. Intraspinal injection of lentiviral vector (EIAV-based vector) expressing GDNF reverses IB4 down-regulation in the spinal cord induced by spinal nerve ligation (SNL). (A–D) IB4 staining in the spinal cord of LacZ-treated (A, B) or GDNF-treated animals (C, D) who had a sham lesion (A) or a spinal nerve ligation (denoted by arrows, B–D). (C) At the level of the injection site, not only does GDNF reverse IB4 down-regulation (illustrated in B), but IB4 staining is even higher than in the contralateral site. The black stars in (C) illustrate the hypertrophy of the dorsal root induced by the GDNF injection. Note that this hypertrophy contains many IB4-positive fibers. (D) In contrast, away from the injection site of GDNF-secreting virus, the recovery of IB4 staining is incomplete. (E) Quantification of the percentage of changes in IB4 staining in the spinal cord ipsilateral versus contralateral to the SNL and intraspinal viral injection, represented as means F SEM. Dotted line indicates 100%, i.e., no variations between ipsilateral and contralateral sides. #P b 0.05, statistically different from sham- and LacZ-treated animals. *P b 0.05, statistically different from sham and GDNF (both at the injection site and away from the injection site; one-way ANOVA on ranks, Tukey post hoc test). Scale bar: 220 Am.
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and such administration of GDNF successfully alleviated pain and reversed some neurochemical changes in the DRGs and the spinal cord after SNL [38]. This type of viral vector has the advantage of easy administration and is minimally invasive, but the toxicity [39], the immune response [40], and the short latency of expression of these vectors [33] make them far from perfect. Our study is the first to investigate the potential use of lentiviral vectors synthesizing GDNF administered intraspinally in the treatment of neuropathic pain. Our results suggest that both EIAV and HIV VSVG-coated viruses are successful tools to transduce cells for a long time with limited side effects. However, our study shows some limitations to the use of such vectors. The reversal of neurochemical and behavioral changes following SNL was less pronounced in our study using GDNF delivered by viral vectors than the neuroprotection previously observed after intrathecal infusion of GDNF [6,12]. The reduced diffusion of the viral vectors and of the trophic factor itself in the tissue is likely to have induced this restricted neuroprotection. Using minipumps, the trophic factor diffuses in the CSF throughout the spinal cord. Microinjected, viral vectors induce a local transduction with limited spread. We tried to counteract such limited diffusion by injecting at two sites instead of one. The increased content of GDNF was observed in one segment only, whereas a spread into three levels (L4 to L6) is probably necessary for complete neuroprotection. The increased intraspinal content of GDNF seems to have had another deleterious effect: we observed hypertrophy of the dorsal root following GDNF (but not GFP or LacZ) injection. Such hypertrophic action of GDNF has already been reported in other contexts [41] and it is possible that it caused the lack of stronger behavioral effect in our study, as spinal hypertrophy is likely to cause hypersensitivity. It is possible that a lower titer of this vector may have circumvented this problem.
MATERIALS
AND
METHODS
All reagents were purchased from Sigma–Aldrich (UK) or as otherwise stated. All experiments were performed in accordance with institutional and Home Office regulations. Viral Vectors Used The HIV-based vector. The recombinant lentiviral vectors based on HIV encoding GDNF and GFP were kind gifts from Dr. Biljana Georgievska and Dr. Anders Bjorklund, Lund University, Sweden. The transfer construct encoding GFP or human GDNF, a packaging construct (pCMVDR8.91), and a VSVG envelope were cotransfected into HEK293T cells. Virions were collected 48 and 72 h after transfection. The titer of the concentrated rLV-GFP vector stock was determined on HEK293T cells as described in [37] to be 1.0 108 transducing units (TU)/ml. The titer of the GDNF-expressing virus (1.8 108 TU/ml) was estimated based on comparison with the GFP vector TU values. The EIAV-based vector. The EIAV lentiviral vectors were manufactured and supplied by Oxford BioMedica, UK. The genomes of the EIAV vector containing the LacZ gene were constructed from starting plasmids pONY8.0Z or pONY8.0G as described in [42]. Instead of the LacZ gene, a 636-bp fragment of human GDNF cDNA was cloned into the EIAV transfer vector together with a Kozak consensus sequence for initiation of translation as in [43]. Vector preparations were pseudotyped with the VSVG envelope (Indiana strain) and were generated by transient transfection of HEK293T cells as described previously by [44]. The titer of the concentrated EIAV-LacZ viral vector, as determined by transduction of osteosarcoma D17 cells, was 2.9 109 TU/ml, while real-time quantitative PCR was used to estimate the titer of the EIAV-GDNF (1.7 109 TU/ml) [45]. Microinjection of Viral Vectors into the Spinal Cord and Induction of Spinal Nerve Ligation Wistar rats (225–250 g; Harlan, n = 34) were anesthetized with medetomidine (250 mg/kg) and ketamine (Parke Davis; 60 mg/kg ip). A small (4 mm) laminectomy was performed at the spinal cord L5 level. After incision of the dura matter, two slow microinjections of viral vector (2 1.5 Al at 0.25 Al/min) were made, using the HIV-based vector (GFP 8 8 1.0 10 TU/ml, n = 10, or GDNF 1.8 10 TU/ml, n = 10) or EIAVbased vector (LacZ, n = 5, or GDNF, n = 9), 1 mm apart using a Hamilton syringe with a glass tip attached (50 Am tip diameter). Microinjections were aimed at the L4–L5 level at a depth of 500 Am into the left dorsal horn. Following the end of the second injection, the animals were sutured, received a subcutaneous injection of saline (10 ml) to avoid dehydration, and were left to recover. Four weeks later, under the same anesthesia (see above), the spinal nerve at the L5 level was exposed, tied, and cut. Sham animals had the spinal nerve exposed only.
TABLE 1 Type of labeling ATF3, IB4, h-tubulin
Primary antibodies or lectin Rabbit anti-ATF-3 (1:500; Santa Cruz) Peroxidase-labeled IB4 (10 Ag/ml; Sigma) Mouse anti-III tubulin (1:2000; Promega)
IB4 GDNF
Peroxidase-labeled IB4 (10 Ag/ml; Sigma) Goat anti-GDNF (1:100; R&D, UK)
Lac Z
Incubation overnight in 1 mg/ml X-gal, 3 mM K3Fe(CN)6, 3 mM K4Fe(CN)6, 1.3 mM MgCl2, 0.1 M phosphate buffer Rabbit anti-h-galactosidase (1:200; Cortex Biochem, San Leandro, CA, USA) Mouse anti-NeuN (1:300; Chemicon)
h-Galactosidase, NeuN
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Secondary antibodies or reagent Goat anti-rabbit Alexa Fluor 543 (1:2000; Molecular Probes) Extravidin FITC (1:400; Sigma) Anti-mouse AMCA (1:200; Molecular Probes) Extravidin FITC (1:400; Sigma) Hs anti-goat biotinylated 1:400 ABC Elite (1:500; Vector) DAB Revelation Kit (Vector)
Goat anti-rabbit Alexa Fluor 488 (1:1000; Molecular Probes) Goat anti-mouse Alexa Fluor 543 (1:1000; Molecular Probes)
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Behavioral Study
Densitometric Analysis of IB4 Staining in the Spinal Cord
The animals were behaviorally tested after the SNL [46] surgery on postoperative days 1, 3, 6, 10, and 13. Prior to the surgery, the rats were habituated to the testing apparatus and a baseline behavior was established. The experimenter was blinded to the treatment at the time of testing. Rats were placed in Plexiglas boxes on a glass floor and acclimatized for 30 min before testing. Noxious thermal sensitivity was assessed by measuring the latency of paw withdrawal in response to an infrared beam applied to the plantar surface of the paw through the glass floor (bHargreavesQ apparatus from Ugo Basile). Each foot was tested three times, alternating left and right paws; each trial was separated by at least 1 min. Mechanical threshold was assessed using an electronic Von Frey apparatus (Ugo Basile). The rats were placed in boxes with a mesh floor and acclimatized for 30 min. The metal filament was pressed against the plantar surface of the hind paw at an increasing force until the animal withdrew its foot. Three measurements were taken from each paw from each animal. Only animals with viral injections at the correct level were used for behavioral data analysis (n = 5). Statistical analysis was performed using a two-way ANOVA followed by the Student Newman–Keuls post hoc test.
Five sections of the dorsal horn (right and left) were analyzed per animal. The images were acquired using 10 Plan-Neofluoro objectives (Zeiss, Germany), an AxioCam Hrm digital camera (Zeiss, Germany), and AxioVision software (Imaging Associates). All images were captured the same day with the same illumination and acquisition parameters. The average gray level in lamina II was determined using Sigma Scan software. The value of the gray level was measured in three different areas of lamina II in each hemicord and was averaged. In each hemicord, the background level was measured in lamina V, in which no IB4 staining was present, and this value was subtracted from the averaged values in lamina II. In each animal, the percentage of the gray level on the ipsilateral versus the contralateral side was calculated and averaged for the five sections analyzed. Results are expressed as mean percentages (FSEM) of the variation of IB4 staining in the ipsilateral site versus the contralateral site. Because of the differential result in GDNF-treated animals at the level of the injection site or away from the injection site, analysis was performed on both. Variations between groups were statistically analyzed using oneway ANOVA on ranks, followed by Tukey post hoc test.
Immunohistochemistry One day following the last behavioral time point, animals were deeply anesthetized with pentobarbital (140 mg/kg) and then transcardially perfused with 100 ml heparanized saline (0.9% w/v NaCl) followed by 400 ml of 4% w/v paraformaldehyde in 0.1 M phosphate buffer, pH 7.4. The lumbar spinal cord and DRGs L4 and L5 right and left were dissected out and postfixed overnight (cord) or for 2 h (DRGs). They were cryoprotected in 20% w/v sucrose in 0.1 M PB for 24 h at 48C. Tissues were embedded in OCT embedding compound (BDH) on liquid nitrogen. Transverse sections were cut serially (20 Am thickness) on a cryostat and mounted onto Superfrost slides (BDH). Every section (for the lumbar cords and DRGs) or every three sections for the cords were collected. Among the collected sections of cord and DRG, every three sections were processed for immunohistochemistry as follows: for DRG, triple labeling of IB4, ATF-3, and h III tubulin or double labeling of ATF-3 and 4,6diamidino-2-phenylindole; for cord sections, single labeling for IB4 or GDNF immunostaining or LacZ revelation. After several washes in phosphate-buffered saline (PBS; 0.01 M, 0.9% NaCl, pH 7.4), sections were incubated overnight with primary antibodies or lectin (see Table 1). After three washes in PBS, they were incubated 2 h with secondary antibodies (see Table 1) and then covered with Vectashield medium (Vector Laboratories). To maintain consistency, each type of immunostaining was done simultaneously for the animals we wanted to compare. Omission of the primary antibody or omission of any stage in the protocol did not result in labeling. All antibodies and sera were diluted in 0.01 M PBS, 0.1% w/v sodium azide, and 0.3% v/v Triton X-100 and incubation of slides was done at room temperature. Sections were viewed under an Axioplan 2 Imaging microscope (Imaging Associates) fitted with 10, 20, and 40 Plan-Neofluoro objectives (Zeiss, Germany) and images were taken using an AxioCam Hrm digital camera (Zeiss, Germany) and AxioVision software (Imaging Associates). Quantification of IB4- and ATF-3-Positive Neurons in the DRG Quantification of the proportion of ATF-3- and IB4-expressing DRG cells was done by counting the number of positive and negative cells with visible nuclei. The number of cells positive for ATF-3 and IB4 was counted in images acquired using a 40 objective. All acquisitions were performed the same day with the same setup of acquisition. The total number of cells was manually counted using the h-III tubulin staining as a neuronal marker, counting only the cells with visible nuclei. At least 200 cells were counted per DRG (over three to five sections). Results are expressed as the percentage of IB4-positive or ATF-3-positive cells in all cells of the DRG. Variations between groups were statistically analyzed using one-way ANOVA on ranks, followed by Tukey post hoc test.
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Quantitative RT-PCR At 4 weeks after microinjection of viral vectors, transduced spinal cord tissue was collected from animals and frozen on dry ice immediately. RNA was extracted from spinal cord tissue using the RNeasy kit (Qiagen, UK) and reverse transcribed to cDNA using Superscript II (Ambion, UK). Real-time quantitative PCR was performed using the ABI Prism 7700 detection system with SYBR green dye (Applied Biosystems, UK) and primers specific to the LacZ and human GDNF genes. Expression of the housekeeping gene GAPDH was also quantified and used for normalization. All primers were designed using Primer Express 2.0 (Applera, USA) and used at a concentration of 300 nM. Standard cycling conditions were used.
ACKNOWLEDGMENTS The authors thank Dr. Anders Bjorklund (Lund University, Sweden) for his generous gift of viral vectors. This work was supported by the Wellcome Trust. RECEIVED FOR PUBLICATION JULY 11, 2005; REVISED NOVEMBER 8, 2005; ACCEPTED NOVEMBER 25, 2005.
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Reversal of Neurochemical Changes and Pain-Related Behavior in a Model of Neuropathic Pain Using Modified Lentiviral Vectors Expressing GDNF Sophie Pezet,1,* Agnieszka Krzyzanowska,1 Liang-Fong Wong,2 John Grist,1 Nicholas D. Mazarakis,2 Biljana Georgievska,3 and Stephen B. McMahon1 1
The London Pain Consortium, Neurorestoration, The Wolfson Centre for Age-Related Diseases, KingTs College London, Hodgkin Building, Wolfson Wing, GuyTs Campus, London SE1 1UL, UK 2 Oxford BioMedica (UK) Ltd., Medawar Centre, Robert Robinson Avenue, The Oxford Science Park, Oxford OX4 4GA, UK 3 Neurobiology Group, Department of Experimental Medical Science, Wallenberg Neuroscience Center, Lund University, BMC A11, 221 84 Lund, Sweden *To whom correspondence and reprint requests should be addressed. Fax: +44 0207 848 6165. E-mail: [email protected].
Available online 28 February 2006
In this study, we evaluated the possible use of lentiviral vectors in the treatment of neuropathic pain. We chose to administer GDNF-expressing vectors because of the known beneficial effect of this trophic factor in alleviation of neuropathic pain in adult rodents. Lentiviral vectors expressing either GDNF or control, green fluorescent protein or B-galactosidase, were injected unilaterally into the spinal dorsal horn 5 weeks before a spinal nerve ligation was induced (or sham surgery for the controls). We observed that intraspinally administered lentiviral vectors resulted in a large and sustained expression of transgenes in both neurons and glial cells. Injection of GDNF-expressing viral vectors induced a significant reduction of ATF-3 up-regulation and IB4 down-regulation in damaged DRG neurons. In addition, it produced a partial but significant reversal of thermal and mechanical hyperalgesia observed following the spinal nerve ligation. In conclusion, our study suggests that lentiviral vectors are efficient tools to induce a marked and sustained expression of trophic factors in specific areas of the CNS and can, even if with some limitations, be efficient in the treatment of neuropathic pain. Key Words: survival, neuroprotection, trophic factor, ATF-3, IB4, spinal cord
INTRODUCTION Neuropathic pain can arise from lesions of the peripheral or central nervous system (CNS). Peripheral nerve injury elicits morphological, metabolic, and biochemical changes in the damaged dorsal root ganglion (DRG) neurons. Such changes in the DRGs include downregulation of Griffonia simplicifolia, isolectin IB4 (IB4) binding, P2X3 receptors [1], and some sodium channel subunits [2–4]; decreased activity of the enzyme thiamine monophosphate (TMP; [5]); as well as up-regulation of galanin, neuropeptide Y [6–9], and the activating transcription factor 3 (ATF-3; [10]). Glial cell line-derived neurotrophic factor (GDNF) is a member of the transforming growth factor-h superfamily. It is expressed in a wide variety of structures of the central and peripheral nervous systems, where it regulates neuronal survival, differentiation, and gene expression. In the DRG, many cells responsive to GDNF are the smalldiameter cells positive for IB4. As previously mentioned,
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nerve injury results in dysregulation of peptides, receptors, and channels, including a down-regulation of GDNF itself [11]. Exogenous administration of GDNF has previously been shown to be successful in preventing the down-regulation of IB4 [6,12], CGRP [13], P2X3 [1,6,14], sodium channels [12,15], and TMP activity [5] and up-regulation of ATF-3 [6,14], galanin, and NPY [6]. The therapeutic value of GDNF has been tested in models of neurodegenerative diseases. For example in animal models of Parkinson disease (PD) direct infusion of GDNF to the midbrain promoted dopaminergic neuron survival and improved neurological function [16–18], while in neuropathic pain models delivery of GDNF to the lumbar DRG/spinal cord reversed sensory abnormalities arising from neuropathic pain states [12]. However, in a clinical trial in which PD patients received GDNF via intracerebroventricular catheters, GDNF-treated patients suffered from many side effects and did not show improvements in parkinsonism [19]. The failure to
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demonstrate clinical improvement was probably due to the spread of GDNF to other brain areas and the weak penetration of GDNF in the target tissue, suggesting that a more specific targeted route of administration is required. More recently, focal delivery of GDNF in the putamen induced a good clinical improvement and limited side effects, suggesting that locally delivered GDNF can have a therapeutic value [20,21]. Consequently, those studies suggest that new methods to induce a local up-regulation of GDNF are likely to be successful therapies. Targeted delivery of GDNF to specific CNS regions can be achieved by viral vectors and may be exploited as a therapeutic strategy in the treatment of PD and neuropathic pain [6,12]. Lentiviral vectors are ideal gene delivery tools as they induce limited inflammation, mediate long-term and stable expression even in nondividing cells, and have a relatively large cloning capacity. In this study we investigated the possible use of lentiviral vectors encoding GDNF to achieve targeted delivery to the spinal cord and evaluated these vectors in neuropathic pain models.
RESULTS Cellular Transduction Using Lentiviral Vectors in the Spinal Cord In this study, we compared the transduction efficiency and potential beneficial effect of two different lentiviral vectors (human immunodeficiency virus (HIV) and equine infectious anemia virus (EIAV) based) in the development of neuropathic pain. Spinal injection of lentivirus synthesizing the green fluorescent protein (GFP; HIV-based vector) induced a minor local injury, and we performed direct observation of GFP immunofluorescence 2, 4, and 6 weeks postinjection. At 2 weeks, GFP was expressed in many cells of the gray and the white matter and this expression increased at 4 and 6 weeks after injection (Fig. 1A). Quantification using quantitative RTPCR showed that 4 weeks after intraspinal injection of the EIAV-based vector expressing LacZ or GDNF a strong expression of LacZ or GDNF mRNA, respectively, was induced (Fig. 1D). Six to eight weeks after the injection of viral vector, we observed transduced cells (GFP or LacZ, i.e., HIV- or EIAV-based vectors) rostrocaudally at least one spinal segment away from the site of injection. Fig. 1B shows an example of rostrocaudal distribution of LacZ staining in the lumbar cord. Following vector injection at the L5 level, we observed transduced cells in the white and gray matter of the cord from L3 to L6, with a limited expression in L3 and L4 and maximum expression in L5 and L6 (Fig. 1B). Despite the fact that the virus was injected unilaterally in laminae IV–V of the cord, LacZ or GFP staining appeared in deep laminae of the cord (V) and spread more superficially (I–IV) and deeply (ventral horn). We observed some LacZ staining (but never any GFP staining) as well in the ventral horn of the contralateral site (Figs. 1A–1C). Using immunostaining for GDNF, we
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observed that the rostrocaudal spread of GDNF transduction was similar to that of control viruses coding for GFP or LacZ (Fig. 1C, a). One striking difference, however, was the hypertrophy of the white matter at the level of the injection site (Fig. 1C, a). Examination of GFP- (Figs. 2A–2G) and h-galactosidase- (Figs. 2H–2J) expressing cells revealed that cells of different morphologies were transduced by the EIAVbased LacZ and HIV-based GFP vectors. Both vectors transduced neurons, as shown in Figs. 2A and 2B for the GFP-expressing vector by their typical morphology and size (Fig. 2B taken at the level of motor neurons in the ventral horn). This was confirmed by colocalization with the NeuN marker and demonstrated that neurons in superficial laminae were also transduced (Figs. 2C and 2J). The majority of cells transduced by the HIV-based vector were astrocytes, as shown in Figs. 2D and 2E. However, some microglial cells were seen to be transduced by this viral vector as well (Figs. 2F and 2G). In contrast the EIAVbased vector transduced mainly neurons (Figs. 2H–2J). At the level of the lateral funiculus, most of the h-galactosidase-expressing cells were neurons, even if a few astrocytes were transduced as well. Using OX42, GFAP, and ED1 immunostaining, we analyzed the potential microglial, astrocytic, or inflammatory reaction induced by intraspinal injection of the different viral vectors. We observed a limited microglial, astrocytic, and inflammatory reaction at the level of the injection site, but we did not observe any increased reaction induced by either h-galactosidase nor GDNF themselves compared to GFP. Reversal of Neurochemical Changes Associated with Spinal Nerve Ligation (SNL) Primary sensory neurons are a heterogeneous population. Approximately two-thirds are small- and medium-sized neurons and code for noxious stimulation. Half of this nociceptive population are NGF sensitive and synthesize neuropeptides (substance P, CGRP). The other half are bnonpeptidergicQ and express GDNF receptors, i.e., Ret and GFRa1, and are responsive to GDNF [5,22]. Following a peripheral nerve injury, damaged neurons of the DRG show a drastic dysregulation of their peptides, receptors, and channels. The exact mechanism underlying the dysregulation of these molecules is not completely understood, but decreased trophic support by NGF and GDNF induced by the nerve injury seems to play a role. The content of both NGF [23] and GDNF [11] is reduced in the DRG following nerve injury due to a lack of retrograde transport of these factors and the down-regulation of their receptors [24–26]. However, if intrathecally administered, NGF and GDNF reverse the changes in IB4, ATF-3, TMP, and CGRP expression. NGF and GDNF act on different cell populations: NGF reverses these changes in the NGFresponsive peptidergic population, whereas GDNF acts on the TMP-, P2X3-, and IB4-positive population [5,14].
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FIG. 1. Intraspinally injected, modified viral vectors induce a strong and sustained transfection of numerous cells in the spinal cord. (A) Immunostaining for GFP following transfection with the HIV-based viral vector, showing the time course and spread of the GFP transduction in the spinal cord ipsilateral to the injection. Note that the amount of GFP produced increases with time. (B) X-gal revelation showing a representative rostrocaudal spread of LacZ-transduced cells in the spinal cord, 6 weeks after the injection of the EIAV-based viral vector. (C) GDNF immunostaining showing the spread of GDNF-transduced cells 6 weeks after the injection of the EIAV-based viral vector. (b) is a high-power magnification of (a). Note the bright labeling for GDNF in the dorsal and ventral horns and in the hypertrophic dorsal root (arrow). (D) Relative LacZ or GDNF expression (measured by quantitative RT-PCR) in the spinal dorsal horn 4 weeks after intraspinal injection of EIAV-based vectors, represented as means F SEM. Scale bar: (A) 340 Am, (B) 1 mm, (C) a, 600 Am, b, 75 Am.
In this study, once we established that the viral vectors could be used to deliver GDNF to the spinal cord, we investigated the efficiency of this viral-vectormediated expression of GDNF in reversing neurochemical and behavioral changes induced by a spinal nerve ligation.
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In naRve animals the percentage of IB4-positive DRG neurons was 40%. IB4 staining was not significantly modified in sham animals (39.3 F 0.7%). Two weeks after the spinal nerve ligation, immunostaining for IB4 was completely lost in the L5 DRGs in LacZ- (0 F 0%) or GFP(1.7 F 1.5%) treated animals. In contrast, such a lack of
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FIG. 2. Lentiviral vectors transduce both spinal glial cells and neurons. (A–G) Spinal cells transduced by the HIV-based viral vector. Examples of GFP staining in the spinal dorsal cord in an animal that received an intraspinal injection of the HIV-based vector. (A and B) An example of the spread and shape of the GFP-positive cells in the dorsal horn. Many cells have glial cell morphology. (A) The dotted line shows the border between the spinal gray and white matter. (B) Example of motor neurons transduced with GFP in the spinal ventral horn. (C) Double staining with NeuN (red) and GFP (green) in the dorsal spinal cord of an animal that received the GFP lentiviral vector. The inset shows a highpower magnification in superficial laminae where costained cells are found, suggesting that a subset of these GFP-positive cells are neurons. (D and E) Double staining for GFP (D) and GFAP (E) showing that a proportion of GFPpositive cells are astrocytes (arrows). (F and G) Double staining for GFP (F) and OX-42 (G) showing that a proportion of GFP-positive cells are microglial cells (arrows). (H–J) Spinal cells transduced with the EIAV-based vector. Examples of h-galactosidase staining in the spinal dorsal cord in an animal that received an intraspinal injection of the EIAV-based vector. (I) A high-power magnification of the white rectangle in (H). The dotted line shows the border between the spinal gray and white matter. Note that the shape, size, and location of transduced cells in the spinal gray matter suggest that they have neuronal morphology. (J) Double immunofluorescent staining for h-galactosidase (green) and NeuN (red). The inset is a higher power magnification showing an example of doubly labeled cells (arrows). All pictures were taken 6 weeks after injection. Scale bar: (A) 65 Am; (B) 110 Am; (C) 125 Am, inset, 45 Am; (D, E) 150 Am; (F, G) 80 Am; (H) 105 Am; (I) 10 Am; (J) 90 Am, inset, 35 Am. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
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staining was partially prevented in GDNF virus-treated animals (HIV-based vector, 28.2 F 5.7%; EIAV vector, 29.3 F 3.9%; Figs. 3C and 3D). In naRve animals and in DRGs contralateral to SNL, there was a complete absence of expression of the transcription factor ATF-3. In sham-treated animals in contrast, a small percentage of cells was positive for ATF-3 (2 F 1.1%). Two weeks after SNL, as previously described [10], ATF-3 was expressed in most neurons of the control DRGs (HIV-based vector 93.5 F 0.9%, EIAV-based vector 97.9 F 0.4%; Fig. 3B). Such up-regulation was partially reversed by intraspinal injection of the GDNF-producing vector (HIV-based vector 34.2 F 3.12%, EIAV-based vector 36.7 F 6.6%; Fig. 3C). These results suggest that spinal expression of GDNF through viral vectors induced neuroprotection of a population of DRG neurons. Immunostaining for IB4 in the spinal cord of naRve or sham animals was located in lamina II of the dorsal horn (Fig. 4A). Two weeks after SNL, as previously described [26], there was a lack of immunostaining for IB4 in the lumbar spinal cord at the L5 level (Figs. 4B and 4E). This lack of staining was completely reversed at the injection site in GDNF virus-injected animals and was even much higher than the contralateral side or in sham animals (HIV-based vector 66 F 30% increase over contralateral values; EIAV-based vector 40 F 22% increase over contralateral values; Figs. 4C and 4E). However, when looking away from the injection site (in L5–L6), such increased staining and rescue of IB4 fibers had disappeared (Figs. 4D and 4E) and a partial lack of IB4 was observed (Figs. 4D and 4E). In conclusion, the GDNF-synthesizing viruses injected intraspinally partially rescued the reduction in IB4 staining in areas in proximity to the injection site. However, directly at the injection site, virally administered GDNF induced an enormous increase in IB4 fibers as well as a hypertrophy in the dorsal root. Results shown in Fig. 4 are those obtained with the EIAV virus. Use of the HIV-based vector gave similar results. These results are in agreement with our observations in the DRG that spinal overexpression of GDNF rescues a population of sensory neurons. However, the large amount of GDNF expressed induced aberrant changes in the dorsal root as well. Behavioral Data Following injection of the vectors, we followed the behavior of the rats for 6 weeks and established a baseline thermal and mechanical threshold. There were no marked differences in the sensitivity of the rats after the spinal injection compared to values before the injection, nor were there any differences between the ipsilateral and the contralateral paws. The rats had normal appearance and level of activity and were feeding regularly. After the spinal nerve ligation, control rats showed a marked reduction in mechanical threshold on the ipsi-
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and significantly different from the baseline only after 10 days postsurgery. At three time points (3 and 6 days postligation) the GDNF-treated rats showed a reduced mechanical sensitivity in the ipsilateral paws compared to the LacZ-treated group in the first 6 days after SNL (P V 0.01, two-way ANOVA, Student Newman–Keuls post hoc test). The heat threshold of control animals significantly decreased by 33.8 F 10.4% (P V 0.05) at 3 days postligation on the ipsilateral side (Fig. 5A). Shamoperated animals showed no change in thermal latency. The thermal sensitivity of the ipsilateral hind paw of GDNF-treated rats was significantly reduced compared to LacZ virus-treated animals, in particular in the first week after SNL (P V 0.05, two-way ANOVA, Student Newman–Keuls post hoc test, Fig. 5A). The contralateral side was not significantly different from baseline in both treatments. Animals treated with the HIV vector expressing GDNF also showed on average a decreased mechanical and thermal hypersensitivity compared to the GFP-treated group, but due to variations in certain animals of the GDNFtreated group, these variations were not statistically different.
DISCUSSION FIG. 3. Intraspinal injection of lentiviral vector expressing GDNF reverses ATF-3 up-regulation and IB4 down-regulation in the DRGs induced by spinal nerve ligation (SNL). (A–C) Double labeling of IB4 (green) and ATF-3 (red) representing an example of sham-operated animals that received GFP- or LacZ-expressing virus (A) or SNL-treated animals that received either GFP- or LacZ-expressing virus (B) or GDNF-expressing virus (C). Note that the GDNF treatment reduces but does not prevent the up-regulation of ATF-3 and the down-regulation of IB4. (D and E) Quantification of changes in the percentage of IB4 (D) or ATF-3 (E) in the DRGs of sham- or SNL-operated animals treated with either the LacZ- or the GDNF-expressing viral vector (EIAV-based vector), represented as means F SEM. *P b 0.05, statistically different from sham- and LacZ-treated animals. Statistical test used was one way ANOVA on ranks, followed by Tukey post hoc test. Scale bar: 50 Am. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
lateral side, a drop of 41.1 F 3.2% from baseline in the EIAV vector-treated animals by day 3 (Fig. 5B) and a 38.9 F 3% drop in the HIV vector animals, significantly different from baseline values at all time points (P V 0.01). Contralateral paw values did not differ from baseline. Sham-operated animals showed no change in sensitivity from baseline after the surgery in either ipsilateral or contralateral paws. Animals treated with the EIAV vector expressing GDNF also showed a decreased threshold to mechanical stimulation; however, it was less pronounced than that of LacZ virus animals (drop of 33.4 F 5% by day 3, Fig. 5B)
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In the past 5 years, several laboratories used viral vectors as new strategies in the study of pain. Most of the studies used peripheral injections of viral vectors such as adeno-associated viral vectors up-regulating mu opioid receptors [27,28] or nerve growth factor or herpes simplex virus vectors expressing proenkephalin [29,30] in peripheral tissues such as the skin or the sciatic nerve. Other studies used intraspinal injections of trophic factors such as BDNF in the treatment of neuropathic pain [31]. Among all viral vectors available, we chose lentiviral vectors for their ability to transduce neuronal cells, their long-term expression patterns, and the limited inflammation they induce. Both vectors we used were pseudotyped with the vesicular stomatitis virus glycoprotein (VSVG), which recognizes specific host cell receptors [32] and reduces the possibility of side effects on nontarget cells [33]. As observed by collaborators using the same types of viruses [34], we detected a strong transduction and synthesis of GFP, LacZ, or GDNF for up to 8 weeks (the maximal time course of our experiment) following the initial injection of viral vectors. However, these types of lentiviral vectors have been observed to be expressed for up to 9 months after the injection [35,36]. We have found that the two types of vectors used were capable of transducing both glial and neuronal cells, even though the EIAV-based vector transfected mainly neuronal cells and the HIV-based vector transduced mainly glial cells. Previous work using the same vectors showed that they
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routinely transduce mainly neuronal cells [34,37]. We do not have any explanation at the moment for the fact that in the spinal cord, the HIV-based vector transduced mainly glial cells, a feature not observed in the striatum [37]. However, because in our study transduced cells in the spinal cord were used as a source of overexpressed GDNF to protect injured sensory neurons, the nature of
FIG. 5. The (A) thermal and (B) mechanical hyperalgesia associated with spinal nerve ligation is reduced in rats that received intraspinal injection of EIAVbased vector expressing GDNF compared to LacZ-expressing virus. Stars indicate statistically different data points between the two groups (P b 0.05, two-way ANOVA, Student Newman–Keuls post hoc test).
the transduced cells (neurons or glial cells) did not influence the efficacy of the neuroprotection induced by both viruses. Only one study to date examined viral administration of GDNF in pain models; however, it was based on the herpes simplex virus (HSV) system [38]. Applied at the periphery, HSV is retrogradely transported to the DRG
FIG. 4. Intraspinal injection of lentiviral vector (EIAV-based vector) expressing GDNF reverses IB4 down-regulation in the spinal cord induced by spinal nerve ligation (SNL). (A–D) IB4 staining in the spinal cord of LacZ-treated (A, B) or GDNF-treated animals (C, D) who had a sham lesion (A) or a spinal nerve ligation (denoted by arrows, B–D). (C) At the level of the injection site, not only does GDNF reverse IB4 down-regulation (illustrated in B), but IB4 staining is even higher than in the contralateral site. The black stars in (C) illustrate the hypertrophy of the dorsal root induced by the GDNF injection. Note that this hypertrophy contains many IB4-positive fibers. (D) In contrast, away from the injection site of GDNF-secreting virus, the recovery of IB4 staining is incomplete. (E) Quantification of the percentage of changes in IB4 staining in the spinal cord ipsilateral versus contralateral to the SNL and intraspinal viral injection, represented as means F SEM. Dotted line indicates 100%, i.e., no variations between ipsilateral and contralateral sides. #P b 0.05, statistically different from sham- and LacZ-treated animals. *P b 0.05, statistically different from sham and GDNF (both at the injection site and away from the injection site; one-way ANOVA on ranks, Tukey post hoc test). Scale bar: 220 Am.
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and such administration of GDNF successfully alleviated pain and reversed some neurochemical changes in the DRGs and the spinal cord after SNL [38]. This type of viral vector has the advantage of easy administration and is minimally invasive, but the toxicity [39], the immune response [40], and the short latency of expression of these vectors [33] make them far from perfect. Our study is the first to investigate the potential use of lentiviral vectors synthesizing GDNF administered intraspinally in the treatment of neuropathic pain. Our results suggest that both EIAV and HIV VSVG-coated viruses are successful tools to transduce cells for a long time with limited side effects. However, our study shows some limitations to the use of such vectors. The reversal of neurochemical and behavioral changes following SNL was less pronounced in our study using GDNF delivered by viral vectors than the neuroprotection previously observed after intrathecal infusion of GDNF [6,12]. The reduced diffusion of the viral vectors and of the trophic factor itself in the tissue is likely to have induced this restricted neuroprotection. Using minipumps, the trophic factor diffuses in the CSF throughout the spinal cord. Microinjected, viral vectors induce a local transduction with limited spread. We tried to counteract such limited diffusion by injecting at two sites instead of one. The increased content of GDNF was observed in one segment only, whereas a spread into three levels (L4 to L6) is probably necessary for complete neuroprotection. The increased intraspinal content of GDNF seems to have had another deleterious effect: we observed hypertrophy of the dorsal root following GDNF (but not GFP or LacZ) injection. Such hypertrophic action of GDNF has already been reported in other contexts [41] and it is possible that it caused the lack of stronger behavioral effect in our study, as spinal hypertrophy is likely to cause hypersensitivity. It is possible that a lower titer of this vector may have circumvented this problem.
MATERIALS
AND
METHODS
All reagents were purchased from Sigma–Aldrich (UK) or as otherwise stated. All experiments were performed in accordance with institutional and Home Office regulations. Viral Vectors Used The HIV-based vector. The recombinant lentiviral vectors based on HIV encoding GDNF and GFP were kind gifts from Dr. Biljana Georgievska and Dr. Anders Bjorklund, Lund University, Sweden. The transfer construct encoding GFP or human GDNF, a packaging construct (pCMVDR8.91), and a VSVG envelope were cotransfected into HEK293T cells. Virions were collected 48 and 72 h after transfection. The titer of the concentrated rLV-GFP vector stock was determined on HEK293T cells as described in [37] to be 1.0 108 transducing units (TU)/ml. The titer of the GDNF-expressing virus (1.8 108 TU/ml) was estimated based on comparison with the GFP vector TU values. The EIAV-based vector. The EIAV lentiviral vectors were manufactured and supplied by Oxford BioMedica, UK. The genomes of the EIAV vector containing the LacZ gene were constructed from starting plasmids pONY8.0Z or pONY8.0G as described in [42]. Instead of the LacZ gene, a 636-bp fragment of human GDNF cDNA was cloned into the EIAV transfer vector together with a Kozak consensus sequence for initiation of translation as in [43]. Vector preparations were pseudotyped with the VSVG envelope (Indiana strain) and were generated by transient transfection of HEK293T cells as described previously by [44]. The titer of the concentrated EIAV-LacZ viral vector, as determined by transduction of osteosarcoma D17 cells, was 2.9 109 TU/ml, while real-time quantitative PCR was used to estimate the titer of the EIAV-GDNF (1.7 109 TU/ml) [45]. Microinjection of Viral Vectors into the Spinal Cord and Induction of Spinal Nerve Ligation Wistar rats (225–250 g; Harlan, n = 34) were anesthetized with medetomidine (250 mg/kg) and ketamine (Parke Davis; 60 mg/kg ip). A small (4 mm) laminectomy was performed at the spinal cord L5 level. After incision of the dura matter, two slow microinjections of viral vector (2 1.5 Al at 0.25 Al/min) were made, using the HIV-based vector (GFP 8 8 1.0 10 TU/ml, n = 10, or GDNF 1.8 10 TU/ml, n = 10) or EIAVbased vector (LacZ, n = 5, or GDNF, n = 9), 1 mm apart using a Hamilton syringe with a glass tip attached (50 Am tip diameter). Microinjections were aimed at the L4–L5 level at a depth of 500 Am into the left dorsal horn. Following the end of the second injection, the animals were sutured, received a subcutaneous injection of saline (10 ml) to avoid dehydration, and were left to recover. Four weeks later, under the same anesthesia (see above), the spinal nerve at the L5 level was exposed, tied, and cut. Sham animals had the spinal nerve exposed only.
TABLE 1 Type of labeling ATF3, IB4, h-tubulin
Primary antibodies or lectin Rabbit anti-ATF-3 (1:500; Santa Cruz) Peroxidase-labeled IB4 (10 Ag/ml; Sigma) Mouse anti-III tubulin (1:2000; Promega)
IB4 GDNF
Peroxidase-labeled IB4 (10 Ag/ml; Sigma) Goat anti-GDNF (1:100; R&D, UK)
Lac Z
Incubation overnight in 1 mg/ml X-gal, 3 mM K3Fe(CN)6, 3 mM K4Fe(CN)6, 1.3 mM MgCl2, 0.1 M phosphate buffer Rabbit anti-h-galactosidase (1:200; Cortex Biochem, San Leandro, CA, USA) Mouse anti-NeuN (1:300; Chemicon)
h-Galactosidase, NeuN
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Secondary antibodies or reagent Goat anti-rabbit Alexa Fluor 543 (1:2000; Molecular Probes) Extravidin FITC (1:400; Sigma) Anti-mouse AMCA (1:200; Molecular Probes) Extravidin FITC (1:400; Sigma) Hs anti-goat biotinylated 1:400 ABC Elite (1:500; Vector) DAB Revelation Kit (Vector)
Goat anti-rabbit Alexa Fluor 488 (1:1000; Molecular Probes) Goat anti-mouse Alexa Fluor 543 (1:1000; Molecular Probes)
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Behavioral Study
Densitometric Analysis of IB4 Staining in the Spinal Cord
The animals were behaviorally tested after the SNL [46] surgery on postoperative days 1, 3, 6, 10, and 13. Prior to the surgery, the rats were habituated to the testing apparatus and a baseline behavior was established. The experimenter was blinded to the treatment at the time of testing. Rats were placed in Plexiglas boxes on a glass floor and acclimatized for 30 min before testing. Noxious thermal sensitivity was assessed by measuring the latency of paw withdrawal in response to an infrared beam applied to the plantar surface of the paw through the glass floor (bHargreavesQ apparatus from Ugo Basile). Each foot was tested three times, alternating left and right paws; each trial was separated by at least 1 min. Mechanical threshold was assessed using an electronic Von Frey apparatus (Ugo Basile). The rats were placed in boxes with a mesh floor and acclimatized for 30 min. The metal filament was pressed against the plantar surface of the hind paw at an increasing force until the animal withdrew its foot. Three measurements were taken from each paw from each animal. Only animals with viral injections at the correct level were used for behavioral data analysis (n = 5). Statistical analysis was performed using a two-way ANOVA followed by the Student Newman–Keuls post hoc test.
Five sections of the dorsal horn (right and left) were analyzed per animal. The images were acquired using 10 Plan-Neofluoro objectives (Zeiss, Germany), an AxioCam Hrm digital camera (Zeiss, Germany), and AxioVision software (Imaging Associates). All images were captured the same day with the same illumination and acquisition parameters. The average gray level in lamina II was determined using Sigma Scan software. The value of the gray level was measured in three different areas of lamina II in each hemicord and was averaged. In each hemicord, the background level was measured in lamina V, in which no IB4 staining was present, and this value was subtracted from the averaged values in lamina II. In each animal, the percentage of the gray level on the ipsilateral versus the contralateral side was calculated and averaged for the five sections analyzed. Results are expressed as mean percentages (FSEM) of the variation of IB4 staining in the ipsilateral site versus the contralateral site. Because of the differential result in GDNF-treated animals at the level of the injection site or away from the injection site, analysis was performed on both. Variations between groups were statistically analyzed using oneway ANOVA on ranks, followed by Tukey post hoc test.
Immunohistochemistry One day following the last behavioral time point, animals were deeply anesthetized with pentobarbital (140 mg/kg) and then transcardially perfused with 100 ml heparanized saline (0.9% w/v NaCl) followed by 400 ml of 4% w/v paraformaldehyde in 0.1 M phosphate buffer, pH 7.4. The lumbar spinal cord and DRGs L4 and L5 right and left were dissected out and postfixed overnight (cord) or for 2 h (DRGs). They were cryoprotected in 20% w/v sucrose in 0.1 M PB for 24 h at 48C. Tissues were embedded in OCT embedding compound (BDH) on liquid nitrogen. Transverse sections were cut serially (20 Am thickness) on a cryostat and mounted onto Superfrost slides (BDH). Every section (for the lumbar cords and DRGs) or every three sections for the cords were collected. Among the collected sections of cord and DRG, every three sections were processed for immunohistochemistry as follows: for DRG, triple labeling of IB4, ATF-3, and h III tubulin or double labeling of ATF-3 and 4,6diamidino-2-phenylindole; for cord sections, single labeling for IB4 or GDNF immunostaining or LacZ revelation. After several washes in phosphate-buffered saline (PBS; 0.01 M, 0.9% NaCl, pH 7.4), sections were incubated overnight with primary antibodies or lectin (see Table 1). After three washes in PBS, they were incubated 2 h with secondary antibodies (see Table 1) and then covered with Vectashield medium (Vector Laboratories). To maintain consistency, each type of immunostaining was done simultaneously for the animals we wanted to compare. Omission of the primary antibody or omission of any stage in the protocol did not result in labeling. All antibodies and sera were diluted in 0.01 M PBS, 0.1% w/v sodium azide, and 0.3% v/v Triton X-100 and incubation of slides was done at room temperature. Sections were viewed under an Axioplan 2 Imaging microscope (Imaging Associates) fitted with 10, 20, and 40 Plan-Neofluoro objectives (Zeiss, Germany) and images were taken using an AxioCam Hrm digital camera (Zeiss, Germany) and AxioVision software (Imaging Associates). Quantification of IB4- and ATF-3-Positive Neurons in the DRG Quantification of the proportion of ATF-3- and IB4-expressing DRG cells was done by counting the number of positive and negative cells with visible nuclei. The number of cells positive for ATF-3 and IB4 was counted in images acquired using a 40 objective. All acquisitions were performed the same day with the same setup of acquisition. The total number of cells was manually counted using the h-III tubulin staining as a neuronal marker, counting only the cells with visible nuclei. At least 200 cells were counted per DRG (over three to five sections). Results are expressed as the percentage of IB4-positive or ATF-3-positive cells in all cells of the DRG. Variations between groups were statistically analyzed using one-way ANOVA on ranks, followed by Tukey post hoc test.
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Quantitative RT-PCR At 4 weeks after microinjection of viral vectors, transduced spinal cord tissue was collected from animals and frozen on dry ice immediately. RNA was extracted from spinal cord tissue using the RNeasy kit (Qiagen, UK) and reverse transcribed to cDNA using Superscript II (Ambion, UK). Real-time quantitative PCR was performed using the ABI Prism 7700 detection system with SYBR green dye (Applied Biosystems, UK) and primers specific to the LacZ and human GDNF genes. Expression of the housekeeping gene GAPDH was also quantified and used for normalization. All primers were designed using Primer Express 2.0 (Applera, USA) and used at a concentration of 300 nM. Standard cycling conditions were used.
ACKNOWLEDGMENTS The authors thank Dr. Anders Bjorklund (Lund University, Sweden) for his generous gift of viral vectors. This work was supported by the Wellcome Trust. RECEIVED FOR PUBLICATION JULY 11, 2005; REVISED NOVEMBER 8, 2005; ACCEPTED NOVEMBER 25, 2005.
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