- Email: [email protected]
Development of neuromodulation treatments in a large animal model—Do neurosurgeons dream of electric pigs?
J. Schouenborg, M. Garwicz and N. Danielsen (Eds.) Progress in Brain Research, Vol. 194 ISSN: 0079-6123 Copyright Ó 2011 Elsevier B.V. All rights reserved.
CHAPTER 7
Development of neuromodulation treatments in a large animal model—Do neurosurgeons dream of electric pigs? J. C. Sørensen{,*, M. S. Nielsen{, F. Rosendal{, D. Deding{, K. S. Ettrup{, K. N. Jensen{, R. L. Jørgensen{, A. N. Glud{, K. Meier{, L. M. Fitting{, A. Møller{,}, A. K. O. Alstrup{, L. Østergaard} and C. R. Bjarkam{ {
Center for Experimental Neuroscience (CENSE), Department of Neurosurgery, Aarhus University Hospital, Århus C, Denmark { PET Center, Aarhus University Hospital, Århus C, Denmark } Center for Functionally Integrated Neuroscience (CFIN), Aarhus University Hospital, Århus C, Denmark
Abstract: The Göttingen minipig has been established as a translational research animal for neurological and neurosurgical disorders. This animal has a large gyrencephalic brain suited for examination at sufficient resolution with conventional clinical scanning modalities. The large brain, further, allows use of standard neurosurgical techniques and can accommodate clinical neuromodulatory devises such as deep brain stimulation (DBS) electrodes and encapsulated cell biodelivery devices making the animal ideal for basic scientific studies on neuromodulation mechanisms and preclinical tests of new neuromodulation technology for human use. The use of the Göttingen minipig is economical and does not have the concerns of the public associated with the experimental use of primates, cats, and dogs, thus providing a cost-effective research model for translation of rodent data before clinical trials are initiated. Keywords: neuromodulation; translational neuroscience; large animal models; minipig; mini swine; neurodegenerative disorders; deep brain stimulation; spinal cord stimulation; stem cells; encapsulated cells; stereotaxic surgery; high-tesla MRI.
*Corresponding author. Tel.: þ45-8949-3461; Fax: þ45-8949-3410 E-mail: [email protected] DOI: 10.1016/B978-0-444-53815-4.00014-5
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Introduction The aim of our translational research is to examine mechanisms of action and develop new treatment paradigms of neuromodulation in a large nonprimate animal model. Over the past 14 years, the CENSE group has worked to establish the Göttingen minipig as a research animal for neurological and neurosurgical disorders. The advantage of this animal is that it has a large gyrencephalic brain that can be examined at sufficient resolution using conventional clinical scanning modalities (Andersen et al., 2005; Cumming et al., 2001, 2003; Danielsen et al., 1998, 2000; Watanabe et al., 2001). The large brain, furthermore, enables the use of deep brain stimulation (DBS) electrodes and other neuromodulatory devices for human use, making the animal ideal for preclinical tests of new neuromodulation technology (Bjarkam et al., 2005, 2008, 2010; Dalmose et al., 2004; FjordLarsen et al., 2010; Glud et al., 2010; Jensen et al., 2009). The use of the Göttingen minipig is economical and without the concerns of the public, associated with the experimental use of primates, cats, and dogs (Goodman and Check, 2002). In a minipig model of DBS toward Parkinson’s disease, we have examined mechanisms of action of this treatment with positron emission tomography (PET) imaging (Nielsen, 2010). The model has been further developed with quantitative behavioral data and neurostereology (Nielsen, 2010; Nielsen et al., 2009). Minipig models of stem cell transplantation to the central nervous system (CNS) has been established (Cumming et al., 2001; Danielsen et al., 2000), to serve as a test platform for the in vivo characterization of stem cell lines developed toward neurodegenerative diseases and CNS cancer. The model also serves as a test system for new delivery systems for stem cell transfer to the CNS (Bjarkam et al., 2010). The minipig model is also being used for preclinical testing of encapsulated cell biodelivery
(ECB) for neurotrophic factor delivery to the brain (Fjord-Larsen et al., 2010). Studies concerning the possible role of Brodmann area 25 DBS for depression, hypothalamic DBS in the treatment of adiposity, and pontine DBS toward central bladder regulation dysfunction are ongoing (Dalmose et al., 2004; Ettrup et al., 2010). Finally, we use the minipig to address the mechanisms of action of spinal cord stimulation for pain. In conclusion, the minipig provides a cost-effective research model allowing further elucidation of rodent data on neuromodulation and safety testing of neuromodulation equipment for human use before clinical patient trials are initiated. The model also allows use and safety testing of neuromodulation equipment for human use. Finally, it provides a platform for the examination of mechanisms of action of neuromodulatory treatments.
The Göttingen minipig as a neurobiological research animal Pigs have been used for surgical training and as physiological nonprimate research model for several decades at our University center. It was therefore a logical step to use this animal as a translational platform to develop and test neuromodulation treatments intended for human use. As the landrace pigs grow to an inconvenient size (250–300 kg), we chose the Göttingen minipig (20–40 kg adult weight) as our experimental animal for chronic studies, whereas young Landrace animals can be used for acute studies (Bollen et al., 2010). This also lay to rest the public and economical concerns that have hampered the use of primates for neurobiological research (Goodman and Check, 2002). The Göttingen minipig was originally bred at the University of Göttingen (Germany), from a crossing between the Minnesota minipig and the Vietnamese potbelly swine (Lind et al., 2007).
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In 1992, a small colony of the white Göttingen minipig was brought to the Ellegaard farm in Denmark where it is now bread under specific pathogen free conditions. The Göttingen minipig used for our neuromodulation studies are standardized laboratory animals, with a welldefined genetic background and health status. They are mild-tempered, shy, and easy to handle (Bollen et al., 2010).
Neuroimaging in the Göttingen minipig The minipig has a gyrencephalic, and relatively large, brain, with a weight of 60–90 g (depending on age), and 6 5 4 cm in adult dimensions (Bjarkam et al., 2005). The cranium of the minipig is pneumatized with a large frontal sinus, which can prove a challenge in both imaging and surgery (Bjarkam et al., 2005). The minipigs are anesthetized, intubated, and maintained on artificial ventilation during all imaging procedures (Ettrup et al., 2011). Human-sized scanner beds fit the minipigs and magnetic resonance imaging (MRI) compatible head holders have been developed for minipig use (Bjarkam et al., 2004, 2005, 2009). At our center, we can use CT, MRI, and PET imaging for visualizing the minipig brain (Andersen et al., 2005; Cumming et al., 2001, 2003; Danielsen et al., 1998, 2000; Rosendal et al., 2009a,b, 2010; Watanabe et al., 2001). The scanning modality depending on the hypotheses and the experimental paradigm.
Neurosurgery in the Göttingen minipig Just as for the imaging procedures, the minipigs are anesthetized, intubated, and maintained on artificial ventilation during surgical procedures (Ettrup et al., 2011). To facilitate surgery and stereotaxic procedures, the head of the minipig is fixed in a head holder that also functions as an MRI compatible localizer box (Bjarkam et al., 2004, 2005, 2008, 2009). Access to the minipig
brain is achieved with standard neurosurgical technique and instruments, such as high-speed drills, fine tipped aspirators, bipolar coagulation, etc. The equipment used is the same as for human neurosurgical procedures. For stereotaxic, high-precision placement of DBS electrodes, intracerebral microinjections of stem cells, and encapsulated biodelivery devices, a stereotaxic MRI is performed prior to the surgery followed by calculation of the stereotaxic coordinates for the target site (Bjarkam et al., 2004, 2005, 2009). The calculation can be based on an implanted fiducial marker or by external fiducials in the side plates of the head holder. The latter allows import of the MR images into standard neuronavigation systems (Fig. 1f) and a precision in the stereotaxic targeting that equals that in routine clinical use (Bjarkam et al., 2009). The minipigs tolerate the anesthesia and surgery well and complications (bleeding, infections) are few.
Neural and stem cell transplantation in the Göttingen minipig In an MPTP minipig model of Parkinson’s disease, we have tested the feasibility of neural cell transplantation (Cumming et al., 2001; Danielsen et al., 2000), grafting neural tissue from the ventral mesencephalon of 28-week old pig embryos. The grafts were stereotaxically placed in the dopamine-depleted striatum of adult parkinsonian minipigs. PET imaging revealed increased fluorodopa uptake in the transplantation sites, and after 7 months, postmortem analysis revealed surviving grafts (Cumming et al., 2001; Danielsen et al., 2000). Subsequent experimental paradigms have included implantation of stem cells, using a newly developed intracerebral microinjection device (Bjarkam et al., 2010), in the minipig striatum, and stereotaxic implantation of lentiviruses, carrying an alpha-synuclein construct, into the minipig substantia nigra in order to overexpress alpha-synuclein locally in transfected nigral neurons (Glud et al., 2010).
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Fig. 1. (a) Adult Göttingen minipig in its pen. (b) Intubated minipig in stereotaxic MRI compatible localizer box with fiducial side plates attached. (c) DBS electrode being stereotaxically inserted into the minipig hypothalamus by use of Frederic Hayer Company (FHC) micromanipulator (www.fh-co.com) attached to a Surgiplan compatible stereotaxic arch system. (d) Two burr holes with DBS electrodes fixed by titanium microplates to the scull. (e) Implantable impulse generator, with the distal ends of the DBS electrodes attached, being implanted into a subcutaneous pocket above the scapula in the neck region of the minipig. (f) Postoperative control MRI imported into the Surgiplan system showing calculated trajectories for the DBS electrodes and dark artifacts from the tips of the electrodes (seen just above the green target crosshair).
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In a series of good laboratory practice (GLP) monitored studies, we have, in collaboration with the biotech company NsGene A/S, shown that nerve growth factor (NGF) and glia derived nerve growth factor (GDNF) producing encapsulated cells survive implantation and expand in their ECB device and that the neurotrophic factors diffuse out into the brain parenchyma in therapeutic amounts (Fjord-Larsen et al., 2010). This has led to a preclinical test in Alzheimer patients.
syndromes, we have not clarified its mechanism of action. The treatment involves placement of an electrode in the epidural space over spinal cord segments innervating the dermatome inflicted by chronic pain. We have accordingly set out to map the dermatomes of the minipig, and with this neuroanatomical information, we intend to address the mechanism of action of the treatment by functional imaging modalities. Conclusion and perspectives
Deep brain stimulation in the Göttingen minipig In order to examine the mechanism of action of DBS for Parkinson’s disease, we implanted the DBS system for patients into an MPTP minipig model of Parkinson’s disease. The DBS was unilateral and resulted in improved motor performance on the side contralateral to the stimulation, leading to rotational behavior (Bjarkam et al., 2005). The PET studies revealed increased blood flow around the stimulation site in the subthalamic nucleus (STN) and increased oxygen uptake in the motor cortex leading us to the hypothesis that DBS for Parkinson’s disease results in a normalization of the neural signaling in the basal ganglia system. Newer paradigms have involved targeting the subgenual area (Brodmann area 25 analogue) in the minipig to establish a DBS treatment model of depression. We have, likewise, targeted the minipig ventral hypothalamus with DBS to induce satiety in the minipig as a potential treatment for obesity (Fig. 1). Finally, we have targeted the pontine micturition center and achieved central control of voiding in the minipig (Dalmose et al., 2004; Jensen et al., 2009). Spinal cord stimulation in the Göttingen minipig Although spinal cord stimulation is an established clinical treatment for severe chronic pain
Minipigs are increasingly recognized as useful animals for neuromodulation research. The characteristics that make them particularly useful for this purpose are: 1. Convenient body size for most clinical neuromodulatory and surgical experiments or trials involving repeated collection of blood samples, biopsies, etc. 2. Similarities with the biology of the human, in particular with respect to, for example, brain, immune mechanism, heart and blood vessels, lung, liver and kidney, and physiological stage of the newborn. 3. The ease of handling and housing under confined conditions. 4. The relatively low price at which they can be made available 5. The use of pigs in research is generally accepted in the public. 6. The pig model provides a unique translational platform to develop and test innovative therapeutic approaches whether pharmacologic or surgical. With the title reference to the novel of Philip K. Dick, we not only dream of electric minipigs but also want to emphasize the potential for translational research and preclinical testing of neuromodulatory treatments that we have uncovered by the use of this animal.
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Acknowledgments The authors gratefully acknowledge the collaboration and technical assistance of Ms. Dorete Jensen, Ms. Trine W Mikkelsen, Mr. Albert Meier, Mr. Mogens Koed, Mr. Graziano Cancian, the staff and researchers at the Institute of Clinical Medicine, Påskehøjgard, and at the animal research center Foulum, Aarhus University, Dr. Lars Wahlberg and the staff at NsGene A/S, Dr. Miles Cunningham, Mc Lean Hospital, Harvard University, Professor Jens Zimmer and the Staff at Institute of Biomedical Research, University of Southern Denmark. The studies have been supported financially by the Danish Research Council, The Lundbeck Foundation, and the Karen Elise Jensen Foundation. References Andersen, F., Watanabe, H., Bjarkam, C. R., Danielsen, E. H., & Cumming, P. The DaNeX Study Group. (2005). Pig brain stereotaxic standard space: Mapping of cerebral blood flow normative values and effect of MPTP-lesioning. Brain Research Bulletin, 66(1), 17–29. Bjarkam, C. R., Cancian, G., Glud, A. N., Ettrup, K. S., Østergaard, L., & Sørensen, J. C. (2009). Isocentric MRIguided stereotaxic procedures in pigs based on a stereotaxic localizer box fitted with an adapted Leksell frame and use of related clinical computer-planning software. Journal of Neuroscience Methods, 183(2), 119–126. Bjarkam, C. R., Cancian, G., Larsen, M., Rosendal, F., Ettrup, K. S., Zeidler, D., et al. (2004). A MRI-compatible stereotaxic localizer box enables high-precision stereotaxic procedures in quadrupeds. Journal of Neuroscience Methods, 139, 293–298. Bjarkam, C. R., Glud, A. N., Margolin, L., Reinhart, K., Franklin, R., Deding, D., et al. (2010). Safety and function of a new clinical intracerebral microinjection instrument (IMI) for stem cells and therapeutics examined in the Göttingen minipig. Stereotactic and Functional Neurosurgery, 88(1), 56–63. Bjarkam, C. R., Jorgensen, R. L., Jensen, K. N., Sunde, N. A., & Sørensen, J. C. (2008). Deep brain stimulation electrode anchoring using BioGlueÒ, a protective electrode covering, and a titanium microplate. Journal of Neuroscience Methods, 168, 151–155.
Bjarkam, C. R., Larsen, M., Watanabe, H., Røhl, L., Simonsen, C. Z., Pedersen, M., et al. (2005). A porcine model of subthalamic high-frequency deep brain stimulation in Parkinson’s disease. In M. J. Willow (Ed.), Chapter 5 in Parkinson’s disease: New research. New York: Nova Science Publishers, 1-59454-352-6. Bollen, P. J. A., Hansen, A. K., & Alstrup, A. K. O. (2010). The laboratory swine (2nd ed.). Boca Raton, London, New York, Washington: CRC Press, 0-8493-1035-0. Cumming, P., Danielsen, E. H., Vafaee, M., Falborg, L., Steffensen, E., Sorensen, J. C., et al. (2001). Normalization of markers for dopamine innervation in striatum of MPTPlesioned miniature pigs with intrastriatal grafts. Acta Neurologica Scandinavica, 103(5), 309–315. Cumming, P., Gillings, N. M., Jensen, S. B., Bjarkam, C. R., & Gjedde, A. (2003). Kinetics of the uptake and distribution of the dopamine D2/3 agonist (R)-N-[1-11C]n-propylnorapomorphine in brain of healthy and MPTP-poisoned Gottingen miniature pigs. Nuclear Medicine and Biology, 30 (5), 547–553. Dalmose, A. L., Bjarkam, C. R., Sørensen, J. C., Djurhuus, J. C., & Jørgensen, T. M. (2004). Effects of high frequency deep brain stimulation on urine storage and voiding function in conscious minipigs. Neurourology and Urodynamics, 23, 265–272. Danielsen, E. H., Cumming, P., Andersen, F., Bender, D., Brevig, T., Falborg, L., et al. (2000). The DaNEX study of embryonic mesencephalic, dopaminergic tissue grafted to a minipig model of Parkinson’s disease: Preliminary findings of effect of MPTP poisoning on striatal dopaminergic markers. Cell Transplantation, 9, 247–259. Danielsen, E. H., Smith, D. F., Poulsen, P. H., Østergaard, L., Gee, A., Ishizu, K., et al. (1998). Positron emission tomography of living brain in minipigs and domestic pigs. Scandinavian Journal of Laboratory Animal Science, 25(Suppl. 1), 127–135. Ettrup, K. S., Glud, A. N., Orlowski, D., Fitting, L. M., Meier, K., Sorensen, J. C., et al. (2011). Basic surgical techniques in the Göttingen minipig: Intubation, transurethral bladder catheterization, femoral vessel catheterization, and transcardial perfusion. Journal of Visualized Experiments, (www.jove.com/details.php?id=2652). Ettrup, K., Sørensen, J. C., & Bjarkam, C. R. (2010). The anatomy of the Göttingen minipig hypothalamus. Journal of Chemical Neuroanatomy, 39(3), 151–165. Fjord-Larsen, L., Kusk, P., Tornøe, J., Juliusson, B., Torp, M., Bjarkam, C. R., et al. (2010). Long-term delivery of nerve growth factor by encapsulated cell biodelivery in the Göttingen minipig basal forebrain. Molecular Therapy, 18 (12), 2164–2172. Glud, A. N., Hedegaard, C., Nielsen, M. S., Sørensen, J. C., Bendixen, C., Jensen, P. H., et al. (2010). Direct gene transfer in the Göttingen minipig CNS using stereotaxic lentiviral microinjections. Acta Neurobiologiae Experimentalis, 70, 1–8.
103 Goodman, S., & Check, E. (2002). The great primate debate. Nature, 417, 684–687. Jensen, K. N., Deding, D., Sørensen, J. C., & Bjarkam, C. R. (2009). Long-term implantation of deep brain stimulation electrodes in the pontine micturition centre of the Göttingen minipig. Acta Neurochirurgica, 151(7), 785–794. Lind, N. M., Moustgaard, A., Jelsing, J., Vajta, G., Cumming, P., & Hansen, A. K. (2007). The use of pigs in neuroscience: Modeling brain disorders. Neuroscience and Biobehavioral Reviews, 31(5), 728–751. Nielsen, M. S. (2010). Progressive Parkinson’s disease in the Göttingen minipig based on continuous MPTP intoxication. PhD thesis, Faculty of Health Sciences, Aarhus University, Denmark. Nielsen, M. S., Sørensen, J. C., & Bjarkam, C. R. (2009). The substantia nigra pars compacta of the Göttingen minipig: An anatomical and stereological study. Brain Structure & Function, 213(4–5), 481–488. Rosendal, F., Chakravarty, M. M., Sunde, N., Rodell, A., Jonsdottir, K. Y., Pedersen, M., et al. (2010). Defining the
intercommissural plane and stereotactic coordinates for the basal ganglia in the Göttingen minipig brain. Stereotactic and Functional Neurosurgery, 88(3), 138–146. Rosendal, F., Frandsen, J., Chakravarty, M. M., Bjarkam, C. R., Pedersen, M., Sangill, R., et al. (2009). New surgical technique reduces the susceptibility artefact at air-tissue interfaces on in vivo cerebral MRI in the Göttingen minipig. Brain Research Bulletin, 80(6), 403–407. Rosendal, F., Pedersen, M., Sangill, R., StødkildeJørgensen, H., Nielsen, M. S., Bjarkam, C. R., et al. (2009). MRI protocol for in vivo visualization of the Göttingen minipig brain improves targeting in experimental functional neurosurgery. Brain Research Bulletin, 79(1), 41–45. Watanabe, H., Andersen, F., Simonsen, C. Z., Evans, S. M., Gjedde, A., Cumming, P., et al. (2001). MR-based statistical atlas of the Göttingen minipig brain. NeuroImage, 14, 1089–1096.
CHAPTER 7
Development of neuromodulation treatments in a large animal model—Do neurosurgeons dream of electric pigs? J. C. Sørensen{,*, M. S. Nielsen{, F. Rosendal{, D. Deding{, K. S. Ettrup{, K. N. Jensen{, R. L. Jørgensen{, A. N. Glud{, K. Meier{, L. M. Fitting{, A. Møller{,}, A. K. O. Alstrup{, L. Østergaard} and C. R. Bjarkam{ {
Center for Experimental Neuroscience (CENSE), Department of Neurosurgery, Aarhus University Hospital, Århus C, Denmark { PET Center, Aarhus University Hospital, Århus C, Denmark } Center for Functionally Integrated Neuroscience (CFIN), Aarhus University Hospital, Århus C, Denmark
Abstract: The Göttingen minipig has been established as a translational research animal for neurological and neurosurgical disorders. This animal has a large gyrencephalic brain suited for examination at sufficient resolution with conventional clinical scanning modalities. The large brain, further, allows use of standard neurosurgical techniques and can accommodate clinical neuromodulatory devises such as deep brain stimulation (DBS) electrodes and encapsulated cell biodelivery devices making the animal ideal for basic scientific studies on neuromodulation mechanisms and preclinical tests of new neuromodulation technology for human use. The use of the Göttingen minipig is economical and does not have the concerns of the public associated with the experimental use of primates, cats, and dogs, thus providing a cost-effective research model for translation of rodent data before clinical trials are initiated. Keywords: neuromodulation; translational neuroscience; large animal models; minipig; mini swine; neurodegenerative disorders; deep brain stimulation; spinal cord stimulation; stem cells; encapsulated cells; stereotaxic surgery; high-tesla MRI.
*Corresponding author. Tel.: þ45-8949-3461; Fax: þ45-8949-3410 E-mail: [email protected] DOI: 10.1016/B978-0-444-53815-4.00014-5
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Introduction The aim of our translational research is to examine mechanisms of action and develop new treatment paradigms of neuromodulation in a large nonprimate animal model. Over the past 14 years, the CENSE group has worked to establish the Göttingen minipig as a research animal for neurological and neurosurgical disorders. The advantage of this animal is that it has a large gyrencephalic brain that can be examined at sufficient resolution using conventional clinical scanning modalities (Andersen et al., 2005; Cumming et al., 2001, 2003; Danielsen et al., 1998, 2000; Watanabe et al., 2001). The large brain, furthermore, enables the use of deep brain stimulation (DBS) electrodes and other neuromodulatory devices for human use, making the animal ideal for preclinical tests of new neuromodulation technology (Bjarkam et al., 2005, 2008, 2010; Dalmose et al., 2004; FjordLarsen et al., 2010; Glud et al., 2010; Jensen et al., 2009). The use of the Göttingen minipig is economical and without the concerns of the public, associated with the experimental use of primates, cats, and dogs (Goodman and Check, 2002). In a minipig model of DBS toward Parkinson’s disease, we have examined mechanisms of action of this treatment with positron emission tomography (PET) imaging (Nielsen, 2010). The model has been further developed with quantitative behavioral data and neurostereology (Nielsen, 2010; Nielsen et al., 2009). Minipig models of stem cell transplantation to the central nervous system (CNS) has been established (Cumming et al., 2001; Danielsen et al., 2000), to serve as a test platform for the in vivo characterization of stem cell lines developed toward neurodegenerative diseases and CNS cancer. The model also serves as a test system for new delivery systems for stem cell transfer to the CNS (Bjarkam et al., 2010). The minipig model is also being used for preclinical testing of encapsulated cell biodelivery
(ECB) for neurotrophic factor delivery to the brain (Fjord-Larsen et al., 2010). Studies concerning the possible role of Brodmann area 25 DBS for depression, hypothalamic DBS in the treatment of adiposity, and pontine DBS toward central bladder regulation dysfunction are ongoing (Dalmose et al., 2004; Ettrup et al., 2010). Finally, we use the minipig to address the mechanisms of action of spinal cord stimulation for pain. In conclusion, the minipig provides a cost-effective research model allowing further elucidation of rodent data on neuromodulation and safety testing of neuromodulation equipment for human use before clinical patient trials are initiated. The model also allows use and safety testing of neuromodulation equipment for human use. Finally, it provides a platform for the examination of mechanisms of action of neuromodulatory treatments.
The Göttingen minipig as a neurobiological research animal Pigs have been used for surgical training and as physiological nonprimate research model for several decades at our University center. It was therefore a logical step to use this animal as a translational platform to develop and test neuromodulation treatments intended for human use. As the landrace pigs grow to an inconvenient size (250–300 kg), we chose the Göttingen minipig (20–40 kg adult weight) as our experimental animal for chronic studies, whereas young Landrace animals can be used for acute studies (Bollen et al., 2010). This also lay to rest the public and economical concerns that have hampered the use of primates for neurobiological research (Goodman and Check, 2002). The Göttingen minipig was originally bred at the University of Göttingen (Germany), from a crossing between the Minnesota minipig and the Vietnamese potbelly swine (Lind et al., 2007).
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In 1992, a small colony of the white Göttingen minipig was brought to the Ellegaard farm in Denmark where it is now bread under specific pathogen free conditions. The Göttingen minipig used for our neuromodulation studies are standardized laboratory animals, with a welldefined genetic background and health status. They are mild-tempered, shy, and easy to handle (Bollen et al., 2010).
Neuroimaging in the Göttingen minipig The minipig has a gyrencephalic, and relatively large, brain, with a weight of 60–90 g (depending on age), and 6 5 4 cm in adult dimensions (Bjarkam et al., 2005). The cranium of the minipig is pneumatized with a large frontal sinus, which can prove a challenge in both imaging and surgery (Bjarkam et al., 2005). The minipigs are anesthetized, intubated, and maintained on artificial ventilation during all imaging procedures (Ettrup et al., 2011). Human-sized scanner beds fit the minipigs and magnetic resonance imaging (MRI) compatible head holders have been developed for minipig use (Bjarkam et al., 2004, 2005, 2009). At our center, we can use CT, MRI, and PET imaging for visualizing the minipig brain (Andersen et al., 2005; Cumming et al., 2001, 2003; Danielsen et al., 1998, 2000; Rosendal et al., 2009a,b, 2010; Watanabe et al., 2001). The scanning modality depending on the hypotheses and the experimental paradigm.
Neurosurgery in the Göttingen minipig Just as for the imaging procedures, the minipigs are anesthetized, intubated, and maintained on artificial ventilation during surgical procedures (Ettrup et al., 2011). To facilitate surgery and stereotaxic procedures, the head of the minipig is fixed in a head holder that also functions as an MRI compatible localizer box (Bjarkam et al., 2004, 2005, 2008, 2009). Access to the minipig
brain is achieved with standard neurosurgical technique and instruments, such as high-speed drills, fine tipped aspirators, bipolar coagulation, etc. The equipment used is the same as for human neurosurgical procedures. For stereotaxic, high-precision placement of DBS electrodes, intracerebral microinjections of stem cells, and encapsulated biodelivery devices, a stereotaxic MRI is performed prior to the surgery followed by calculation of the stereotaxic coordinates for the target site (Bjarkam et al., 2004, 2005, 2009). The calculation can be based on an implanted fiducial marker or by external fiducials in the side plates of the head holder. The latter allows import of the MR images into standard neuronavigation systems (Fig. 1f) and a precision in the stereotaxic targeting that equals that in routine clinical use (Bjarkam et al., 2009). The minipigs tolerate the anesthesia and surgery well and complications (bleeding, infections) are few.
Neural and stem cell transplantation in the Göttingen minipig In an MPTP minipig model of Parkinson’s disease, we have tested the feasibility of neural cell transplantation (Cumming et al., 2001; Danielsen et al., 2000), grafting neural tissue from the ventral mesencephalon of 28-week old pig embryos. The grafts were stereotaxically placed in the dopamine-depleted striatum of adult parkinsonian minipigs. PET imaging revealed increased fluorodopa uptake in the transplantation sites, and after 7 months, postmortem analysis revealed surviving grafts (Cumming et al., 2001; Danielsen et al., 2000). Subsequent experimental paradigms have included implantation of stem cells, using a newly developed intracerebral microinjection device (Bjarkam et al., 2010), in the minipig striatum, and stereotaxic implantation of lentiviruses, carrying an alpha-synuclein construct, into the minipig substantia nigra in order to overexpress alpha-synuclein locally in transfected nigral neurons (Glud et al., 2010).
100 (a)
(b)
(c)
(d)
(e)
(f)
Fig. 1. (a) Adult Göttingen minipig in its pen. (b) Intubated minipig in stereotaxic MRI compatible localizer box with fiducial side plates attached. (c) DBS electrode being stereotaxically inserted into the minipig hypothalamus by use of Frederic Hayer Company (FHC) micromanipulator (www.fh-co.com) attached to a Surgiplan compatible stereotaxic arch system. (d) Two burr holes with DBS electrodes fixed by titanium microplates to the scull. (e) Implantable impulse generator, with the distal ends of the DBS electrodes attached, being implanted into a subcutaneous pocket above the scapula in the neck region of the minipig. (f) Postoperative control MRI imported into the Surgiplan system showing calculated trajectories for the DBS electrodes and dark artifacts from the tips of the electrodes (seen just above the green target crosshair).
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In a series of good laboratory practice (GLP) monitored studies, we have, in collaboration with the biotech company NsGene A/S, shown that nerve growth factor (NGF) and glia derived nerve growth factor (GDNF) producing encapsulated cells survive implantation and expand in their ECB device and that the neurotrophic factors diffuse out into the brain parenchyma in therapeutic amounts (Fjord-Larsen et al., 2010). This has led to a preclinical test in Alzheimer patients.
syndromes, we have not clarified its mechanism of action. The treatment involves placement of an electrode in the epidural space over spinal cord segments innervating the dermatome inflicted by chronic pain. We have accordingly set out to map the dermatomes of the minipig, and with this neuroanatomical information, we intend to address the mechanism of action of the treatment by functional imaging modalities. Conclusion and perspectives
Deep brain stimulation in the Göttingen minipig In order to examine the mechanism of action of DBS for Parkinson’s disease, we implanted the DBS system for patients into an MPTP minipig model of Parkinson’s disease. The DBS was unilateral and resulted in improved motor performance on the side contralateral to the stimulation, leading to rotational behavior (Bjarkam et al., 2005). The PET studies revealed increased blood flow around the stimulation site in the subthalamic nucleus (STN) and increased oxygen uptake in the motor cortex leading us to the hypothesis that DBS for Parkinson’s disease results in a normalization of the neural signaling in the basal ganglia system. Newer paradigms have involved targeting the subgenual area (Brodmann area 25 analogue) in the minipig to establish a DBS treatment model of depression. We have, likewise, targeted the minipig ventral hypothalamus with DBS to induce satiety in the minipig as a potential treatment for obesity (Fig. 1). Finally, we have targeted the pontine micturition center and achieved central control of voiding in the minipig (Dalmose et al., 2004; Jensen et al., 2009). Spinal cord stimulation in the Göttingen minipig Although spinal cord stimulation is an established clinical treatment for severe chronic pain
Minipigs are increasingly recognized as useful animals for neuromodulation research. The characteristics that make them particularly useful for this purpose are: 1. Convenient body size for most clinical neuromodulatory and surgical experiments or trials involving repeated collection of blood samples, biopsies, etc. 2. Similarities with the biology of the human, in particular with respect to, for example, brain, immune mechanism, heart and blood vessels, lung, liver and kidney, and physiological stage of the newborn. 3. The ease of handling and housing under confined conditions. 4. The relatively low price at which they can be made available 5. The use of pigs in research is generally accepted in the public. 6. The pig model provides a unique translational platform to develop and test innovative therapeutic approaches whether pharmacologic or surgical. With the title reference to the novel of Philip K. Dick, we not only dream of electric minipigs but also want to emphasize the potential for translational research and preclinical testing of neuromodulatory treatments that we have uncovered by the use of this animal.
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Acknowledgments The authors gratefully acknowledge the collaboration and technical assistance of Ms. Dorete Jensen, Ms. Trine W Mikkelsen, Mr. Albert Meier, Mr. Mogens Koed, Mr. Graziano Cancian, the staff and researchers at the Institute of Clinical Medicine, Påskehøjgard, and at the animal research center Foulum, Aarhus University, Dr. Lars Wahlberg and the staff at NsGene A/S, Dr. Miles Cunningham, Mc Lean Hospital, Harvard University, Professor Jens Zimmer and the Staff at Institute of Biomedical Research, University of Southern Denmark. The studies have been supported financially by the Danish Research Council, The Lundbeck Foundation, and the Karen Elise Jensen Foundation. References Andersen, F., Watanabe, H., Bjarkam, C. R., Danielsen, E. H., & Cumming, P. The DaNeX Study Group. (2005). Pig brain stereotaxic standard space: Mapping of cerebral blood flow normative values and effect of MPTP-lesioning. Brain Research Bulletin, 66(1), 17–29. Bjarkam, C. R., Cancian, G., Glud, A. N., Ettrup, K. S., Østergaard, L., & Sørensen, J. C. (2009). Isocentric MRIguided stereotaxic procedures in pigs based on a stereotaxic localizer box fitted with an adapted Leksell frame and use of related clinical computer-planning software. Journal of Neuroscience Methods, 183(2), 119–126. Bjarkam, C. R., Cancian, G., Larsen, M., Rosendal, F., Ettrup, K. S., Zeidler, D., et al. (2004). A MRI-compatible stereotaxic localizer box enables high-precision stereotaxic procedures in quadrupeds. Journal of Neuroscience Methods, 139, 293–298. Bjarkam, C. R., Glud, A. N., Margolin, L., Reinhart, K., Franklin, R., Deding, D., et al. (2010). Safety and function of a new clinical intracerebral microinjection instrument (IMI) for stem cells and therapeutics examined in the Göttingen minipig. Stereotactic and Functional Neurosurgery, 88(1), 56–63. Bjarkam, C. R., Jorgensen, R. L., Jensen, K. N., Sunde, N. A., & Sørensen, J. C. (2008). Deep brain stimulation electrode anchoring using BioGlueÒ, a protective electrode covering, and a titanium microplate. Journal of Neuroscience Methods, 168, 151–155.
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