| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
RESEARCH REPORT |
1 Eastman Department of Dentistry, and
2 Department of Neurobiology & Anatomy, School of Medicine & Dentistry, University of Rochester, 625 Elmwood Ave., Rochester, NY 14620;
* corresponding author, stephanos_kyrkanides{at}urmc.rochester.edu
 |
ABSTRACT |
|---|
 TOP ABSTRACT INTRODUCTIONMATERIALS & METHODSRESULTSDISCUSSIONREFERENCES |
|---|
KEY WORDS: gene therapy • immunodeficiency virus • feline • beta-galactosidase • mouse • temporomandibular joint • trigeminal ganglion
|
INTRODUCTION |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS & METHODSRESULTSDISCUSSIONREFERENCES |
|---|
and C nerve fibers, whose cell bodies are located in the posterolateral part of the trigeminal ganglion (Yoshino et al., 1998), project distally and terminate as non-encapsulated free nerve endings dispersed throughout the posterolateral part of the TMJ capsule (Bernick, 1962; Thilander, 1964; Frommer and Monroe, 1966; Klineberg, 1971), the posterior band of the meniscus, and the posterior attachment (Dreessen et al., 1990; Kido et al., 1991, 1993; Wink et al., 1992). In the quest for the development of new therapies for orofacial pain, gene therapy appears to be an emerging treatment method (Kuboki et al., 1999; Pohl and Braz, 2001; Baum et al., 2002). For example, it has been previously suggested that delivery of antisense oligonucleotides developed against nociceptive genes to appropriate tissues may offer alternatives in the design of novel treatments for pain management (Wu et al., 2001). We hypothesize that transfer of anti-nociceptive genes to sensory trigeminal neurons innervating the orofacial region may be achieved after injection of lentiviral vectors at the painful site, such as the TMJ, resulting in their uptake by free nerve endings and retrograde transport to the sensory cells’ nuclei. Previous studies demonstrated axonal retrograde transport of horseradish peroxidase from the TMJ to the central nervous system (Romfh et al., 1979; Capra, 1987), including the trigeminal ganglia (Yoshino et al., 1998). In evaluating lentiviral vectors as the basis for TMJ gene therapy, we used VSV-G pseudotyped feline immunodeficiency viral vectors (FIV) capable of stably transducing dividing, growth-arrested, as well as post-mitotic cells, since they are capable of transgene integration into the host’s genome (Poeschla et al., 1998). VSV-G pseudotyping of viral vectors confers a broad range of host specificity, including human and murine cells, since infection is mediated by the interaction of the viral envelope protein and a phospholipid component of the cell membrane, leading to membrane-fusion-mediated entry (Burns et al., 1993; Carneiro et al., 2002). Therefore, FIV vectors can potentially mediate sustained gene expression in non-dividing terminally differentiated trigeminal sensory neurons, a property unique to lentiviral vectors. The aim of the present study was to investigate the effects of viral-mediated gene transfer to neurons located in the trigeminal ganglion following local TMJ administration of a non-primate lentiviral vector.
|
MATERIALS & METHODS |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS & METHODSRESULTSDISCUSSIONREFERENCES |
|---|
. In addition, we constructed a control FIV(
'lac) vector carrying an inactive ß-galactosidase by deleting the first 1000 bp of the lacZ gene (3.75 kb in total), including the transcription initiation site (Fig. 1A). Specifically, the FIV(lacZ) vector was digested in vitro with the SstII and Cla I restriction enzymes overnight at 37°C, followed by agarose gel purification. The ends of the backbone DNA were blunted with the T4 DNA polymerase (Invitrogen, Carlsbad, CA, USA) and ligated with T4 ligase (Invitrogen) according to manufacturer’s instructions. The FIV(lacZ) and FIV('lac) vectors were transiently co-transfected, along with the packaging and VSV-G vectors, into 293H cells (GIBCO/BRL) cultured in DMEM (Invitrogen) plus 10% FBS (Gemini, Woodland Hills, CA, USA), with the Lipofectamine 2000 reagent per manufacturer’s instructions (Invitrogen), and followed by a fresh medium change supplemented by non-essential amino acids (Invitrogen). Sixty hrs post-transfection, the supernatant was collected, filtered through a 0.45-mm Surfil®-MF filter (Corning Separations Division, Acton, MA, USA), aliquoted, and frozen until further use. Titering was performed on CrfK cells (American Tissue Culture Collection, Manassas, VA, USA) cultured in 24-well tissue culture plates, and assessed at at 5 x 107 blue forming units (bfu)/mL by X-gal histochemistry.
|
'lac) infectious particles (100 µL of stock solution) in the joint space of the right TMJ. In brief, the hair of the skin covering the right TMJ was shaved, and the skin was cleaned with Betadine solution. An antero-posterior incision was made in the joint between the posterior end of the zygomatic arch and the ear cartilage, followed by a blunt dissection to expose the zygomatic arch and the posterior margin of the articular eminence. The joint space was not exposed during this procedure. The posterior margin of the eminence was identified by palpation, and a 1-mL tuberculin syringe with a 27
-gauge needle was used to inject the experimental solutions into the joint. This surgically assisted intra-articular injection technique was utilized to minimize leakage or spreading of the injectable solution beyond the articular space (Kyrkanides et al., 2002a). In addition, 2 mice that received 100 µL saline injection served as controls. Forty-five days following treatment, the mice were deeply anesthetized by pentobarbital (100 mg/Kg IP) and killed by transcardial perfusion of 4% paraformaldehyde in phosphate-buffered saline (PBS) (Kyrkanides et al., 2002a,b). The trigeminal ganglia and brain stem were dissected and sectioned at 20 µm by means of a freezing microtome. The TMJ joints were also dissected, decalcified in an EDTA-buffered solution, embedded in paraffin, and cut at 8-µm sections. All tissues were stored at -20°C until processed further.
X-Gal Histochemistry
Sections of trigeminal ganglia were processed by X-gal histochemistry and evaluated under light microscopy. Specifically, the sections were washed in 0.15 M phosphate-buffered saline (PBS), pH 7.2, for 60 min, followed by overnight processing in a staining solution containing 5-bromo-4-chloro-3-indolyl-ß-D-galactopyranoside (1 mg/mL), potassium ferricyanide (3mM), potassium ferrocyanide (3 mM), NP-40 (0.02%) in 0.1 M PBS, pH 7.2 (Invitrogen), and MgCl2 (1.3 mM). The tissue was then washed in PBS for 30 min and briefly rinsed with dH2O. Considerable attention was given so that only the bacterial form of ß-galactosidase was detected. The slides were cover-slipped with DPX mounting medium (Fluka, Neu-Ulm, Switzerland) and examined under a light microscope (BX51 Olympus; Tokyo, Japan). Color microphotographic images were captured in TIFF 16-bit format by means of a SPOT RT Color CCD digital camera attached to the microscope and connected to a PC computer.
Cell Counting
The mouse ganglia (1.5 x 2 x 3 mm) were sectioned sagittally on a freezing cryotome, along their long axis, into 20-µm-thick sections. Approximately 42 sections were produced from each ganglion and were sequentially collected onto 3 glass slides, each containing representative ganglion sections 60 µm from each other. One glass slide of each ganglion was processed by X-gal histochemistry and was used in cell counting. All X-gal-positive (blue) cells were counted on each tissue section on the slides. Since the tissue sections were 60 µm apart, counting all blue cells on a single slide gave a representative number of infected cells in each ganglion while avoiding overlap between sections and subsequently any "double counting".
Immunocytochemistry
Tissue sections from trigeminal ganglia were analyzed by immunocytochemistry, with the use of rabbit anti-ß-galactosidase polyclonal antibody (Chemicon INTL, Temecula, CA, USA). In brief, sections were washed in PBS for 60 min, followed by a 30-minute blocking step in normal goat serum (4% in PBS) and overnight incubation in the primary antibody solution containing rabbit anti-ß-galactosidase polyclonal antibody (1:2500), 0.5% Triton-X, 4% normal goat serum (Invitrogen), and 1% bovine serum albumin (Sigma, St. Louis, MO, USA) in PBS. The next morning, the tissue was washed in PBS for 60 min, followed by a 30-minute blocking step, then incubated for 90 min in the secondary antibody solution containing a goat anti-rabbit polyclonal antibody (1:2000), Triton-X (0.5%), and normal goat serum (0.15%) in PBS. Subsequently, the tissue was washed in PBS for 30 min and incubated in a avidin-biodin complex solution (ABC kit; Vector Laboratories, Burlingame, CA, USA), and then was washed in 0.1 M sodium-acetate-buffered solution (pH 7.4) for 30 min. The tissue was then reacted in a DAB (3,3' diaminobenzidine)-nickel solution in 0.1 M sodium-acetate-buffered solution (pH 7.4) for 5 min, followed by a 15-minute wash in PBS (Kyrkanides et al., 2002a,b). The glass slides were then dehydrated through multiple ethanol solutions, cleared through xyaline, and cover-slipped by means of DPX permanent mounting medium. The tissue sections were then studied under a light microscope, and microphotographic images were captured as described above.
Tissue sections from the temporomandibular joints were first deparaffinized by immersion in a series of xylines and alcohols, followed by antigen retrieval processing (95°C heating for 15 sec in 0.1 M Tris-HCl buffer, pH 8.9) and processing according to the aforementioned immunocytochemical method.
Polymerase Chain-reaction (PCR)
The DNA from the left and right trigeminal ganglia of 8 mice (4 control and 4 experimental) was extracted with use of the Trizol reagent (Invitrogen) according to manufacturer’s instructions. The concentrations of the recovered DNA ranged between 17 and 50 ng/µL and were analyzed for the presence of viral DNA by PCR, with the following primer sets: detection of FIV viral DNA (Fig. 1A
), 5' TTT TTC CAG TTC CGT TTA TCC and TTT ATC GCC AAT CCA CAT CT 3' (TA = 58°C; 40 total cycles); detection of active ß-galactosidase gene (Fig. 2A), 5' CCC ATA GTA ACG CCA ATA GG and AAA TGT GAG CGA GTA ACA ACC 3' (TA = 59.6°C; 45 total cycles). Detection of genomic DNA was performed with the use of primers designed for the murine G3PDH housekeeping gene: ACC ACA GTC CAT GCC ATC AC and TCC ACC ACC CTG TTG CTG TA (TA = 58°C; 30 cycles). A 400-ng quantity was used as the DNA template in the PCR reactions. The PCR products were analyzed by agarose gel (1% w/v) electrophoresis, and the images were captured with the use of a KODAK Image Analysis system (Rochester, NY, USA).
|
|
RESULTS |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS & METHODSRESULTSDISCUSSIONREFERENCES |
|---|
). There was a lack of ß-galactosidase expression in the contralateral joints as well as the saline-injected animals. These results suggest that FIV successfully infected and stably transferred the reporter gene to cells of hard and soft TMJ tissues.
FIV Injection into the TMJ Resulted in Transduction of Trigeminal Neurons
Two FIV vectors were used in our experiment: the wild-type FIV(lacZ) and the mutated FIV('lac) (Fig. 1A). FIV('lac) is capable of transducing cells with an inactive form of the reporter gene ß-galactosidase compared with FIV(lacZ), which carries a full-length lacZ (Figs. 1B, 1C). Injection of either FIV vector into the right TMJ of mice resulted in transduction of neurons located in the ipsilateral trigeminal ganglia, as assessed by PCR (Fig. 3A). The full-length lacZ gene was detected by PCR only in the FIV(lacZ)-treated animals (Fig. 3B), accompanied by neuronal ß-galactosidase expression as assessed by X-gal histochemistry. The X-gal staining was localized primarily in the posterolateral part of the ganglion within the cell bodies of cells that appear histologically as neurons (Figs. 4A, 4B). In fact, the cell bodies of the primary sensory neurons that innervate the TMJ are known to localize in this part of the trigeminal ganglion. In contrast, FIV('lac)-injected mice did not display any X-gal-positive cells in the ganglia (Fig. 4C). Expression of bacterial ß-galactosidase in the trigeminal ganglia was also confirmed by immunocytochemistry in the FIV(lacZ)- but not the FIV('lac)-treated mice (Figs. 4D, 4E). Moreover, analysis of sections from the brain stem did not reveal any X-gal-positive staining (data not shown), as was anticipated, since the vectors are defective, do not replicate, and cannot infect second-order neurons.
|
|
|
DISCUSSION |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS & METHODSRESULTSDISCUSSIONREFERENCES |
|---|
The efficacy of VSV-G pseudotyped FIV vectors to transduce peripheral tissues (Kang et al., 2002), as well as the brain (Blömer et al., 1997) and cerebellum (Alisky et al., 2002), has been previously demonstrated. However, limited information is available on the ability of non-primate lentiviruses to infect neurons retrogradely following peripheral administration. Our observations of cells staining positively for X-gal in the trigeminal ganglion ipsilateral to the site of injection suggest that FIV virions were taken up by peripheral nerve projections of trigeminal sensory neurons that lead to infection and expression of the reporter gene lacZ by these neurons. Novel strategies for the treatment of pain have been previously discussed (Wu et al., 2001), including the use of antisense oligonucleotides delivered to target cells that can bind to mRNAs encoding for nociceptive molecules. Therefore, VSV-G pseudotyped lentiviruses, such as the defective feline or human immunodeficiency virus, may serve as the platform for the transfer of anti-nociceptive genes to temporomandibular joint tissues as well as to the neurons that innervate these structures.
|
ACKNOWLEDGMENTS |
|---|
Received March 18, 2003; Last revision August 15, 2003; Accepted October 16, 2003
|
REFERENCES |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS & METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Baum BJ, Kok M, Tran SD, Yamano S (2002). The impact of gene therapy on dentistry: a revisiting after six years. J Am Dent Assoc 133:35–44.
Bernick S (1962). The vascular and nerve supply to the temporomandibular joint of the rat. Oral Surg Oral Med Oral Pathol 15:488–498.[Medline]
Blömer U, Naldini L, Kafri T, Trono D, Verma IM, Gage FH (1997). Highly efficient and sustained gene transfer in adult neurons with a lentivirus vector. J Virol 71:6641–6649.[Abstract]
Burns JC, Friedmann T, Driever W, Burrascano M, Yee JK (1993). Vesicular stomatitis virus G glycoprotein pseudotyped retroviral vectors: concentration to very high titer and efficient gene transfer into mammalian and nonmammalian cells. Proc Natl Acad Sci USA 90:8033–8037.
Capra NF (1987). Localization and central projections of primary afferent neurons that innervate the temporomandibular joint in cats. Somatosens Res 4:201–213.[ISI][Medline]
Carneiro FA, Bianconi ML, Weissmüller G, Stauffer F, Da Poian AT (2002). Membrane recognition by vesicular stomatitis virus involves enthalpy-driven protein-lipid interactions. J Virol 76:3756–3764.
Cox BM, Ginsburg M, Osman OH (1968). Acute tolerance to narcotic drugs in rats. Br J Pharmacol 33:245–256.[ISI][Medline]
Dreessen D, Halata Z, Strasmann T (1990). Sensory innervation of the temporomandibular joint in the mouse. Acta Anat 139:154–160.[ISI][Medline]
Frommer J, Monroe CW (1966). The morphology and distribution of nerve fibers and endings associated with the mandibular joint of the mouse. J Dent Res 45:1762–1766.
Kang Y, Stein CS, Heth JA, Sinn PL, Penisten AK, Staber PD, et al. (2002). In vivo gene transfer using a nonprimate lentiviral vector pseudotyped with Ross River virus glycoproteins. J Virol 76:9378–9388.
Kido MA, Kondo T, Ayasaka N, Terada Y, Tanaka T (1991). The peripheral distribution of trigeminal nerve fibers in the rat temporomandibular joint studied by an anterograde axonal transport method with wheat germ agglutinin horseradish peroxidase. Arch Oral Biol 36:397–400.[ISI][Medline]
Kido MA, Kiyoshima T, Kondo T, Ayasaka N, Moroi R, Terada Y, et al. (1993). Distribution of substance P and calcitonin gene-related peptide like immunoreactive nerve fibers in the rat temporomandibular joint. J Dent Res 72:592–598.
Klineberg I (1971). Structure and function of temporomandibular joint innervation. Ann R Coll Surg Engl 49:268–288.[Medline]
Kuboki T, Nakanishi T, Kanyama M, Sonoyama W, Fujisawa T, Kobayashi K, et al. (1999). Direct adenovirus-mediated gene delivery to the temporomandibular joint in guinea-pigs. Arch Oral Biol 44:701–709.[ISI][Medline]
Kyrkanides S, Tallents RH, Macher DJ, Olschowka JA, Stevens SY (2002a). Temporomandibular joint nociception: effects of capsaicin on substance P-like immunoreactivity in the rabbit brain stem. J Orofac Pain 16:229–236.[ISI][Medline]
Kyrkanides S, Moore AH, Olschowka JA, Daeschner JC, Williams JP, Hansen JT, et al. (2002b). Cyclooxygenase-2 modulates brain inflammation-related gene expression in central nervous system radiation injury. Brain Res Mol Brain Res 104:159–169.[Medline]
Martin WR, Eades CG (1961). Demonstration of tolerance and physical dependence in the dog following a short-term infusion of morphine. J Physiol 133:262–270.
Poeschla EM, Wong-Sta-Lo F, Looney DJ (1998). Efficient transduction of nondividing human cells by feline immunodeficiency virus lentiviral vectors. Nat Med 4:354–357.[ISI][Medline]
Pohl M, Braz J (2001). Gene therapy of pain: emerging strategies and future directions. Eur J Pharmacol 429:39–48.[ISI][Medline]
Romfh JH, Capra NF, Gatipon GB (1979). Trigeminal nerve and temporomandibular joint of the cat: a horseradish peroxidase study. Exp Neurol 65:99–106.[ISI][Medline]
Sessle BJ, Hu JW (1991). Mechanisms of pain arising from articular tissues. Can J Physiol Pharmacol 69:617–626.[ISI][Medline]
Thilander B (1964). Innervation of the temporo-mandibular disc in man. Acta Odontol Scand 22:151–156.[Medline]
Wink CS, St. Onge M, Zimny ML (1992). Neural elements in the human temporomandibular articular disc. J Oral Maxillofac Surg 50:334–337.[ISI][Medline]
Wu CL, Garry MG, Zollo RA, Yang J (2001). Gene therapy for the management of pain. Part II: molecular targets. Anesthesiology 95:216–240.[ISI][Medline]
Yoshino K, Kawagishi S, Amano N (1998). Morphological characteristics of primary sensory and post-synaptic sympathetic neurones supplying the temporomandibular joint in the cat. Arch Oral Biol 43:679–686.[ISI][Medline]
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| IADR Journals | Advances in Dental Research ® |
| Journal of Dental Research ® | Critical Reviews (1990-2004) |
