MSU Udall Center Surgical Services
Director: Timothy Collier, Ph.D.
The MSU Udall Center lends its expertise as a resource to the Parkinson’s disease (PD) research community for implementation of surgical protocols in their programs. Specifically, MSU Udall Surgical Services provide consultation or collaboration to help investigators generate accurate, reproducible surgical PD rat models and neurosurgical therapeutic interventions including:
- Rat models of 6-hydroxydopamine (6-OHDA)-induced nigrostriatal degeneration to induce:
- Focal intrastriatal lesions
- Progressive nigral degeneration
- Complete unilateral nigrostriatal degeneration
- Rat models of alpha-synuclein (α-syn)-induced nigrostriatal degeneration using intranigral delivery of recombinant adenoassociated virus serotype 2/5 (rAAV2/5) to deliver human wildtype α-syn
- Implantation of stimulating electrodes into the subthalamic nucleus for the long-term rat model of deep brain stimulation
- Injection of AAV vectors for in vivo validation of transduction by AAV vectors
- Administration of experimental interventions including viral vectors for over-expression and silencing as dictated by experimental needs
The overall goal of the MSU Udall Surgical Services is to enrich local research opportunities through Center assets and broaden the research scope and opportunities for PD-related neurodegenerative disease research nationally. The direct participation of MSU Udall Center senior investigators in generation of animal models and experimental treatments brings a unique depth of expertise that provides strong oversight and uniformity in animal model generation. Further, we have considerable expertise in performing surgical interventions in aged rats with the overall goal of improved understanding of the influence the aged brain environment on the consequences of, and subsequent treatments for, PD.
Our workflow plan has been employed for over 15 years. The approach is team-based and functions as an assembly line. We believe strongly that the uniformity produced during the process of “batch” surgeries produces better experimental outcomes and is advantageous in experimental paradigms where timing is critical. We encourage adoption of the team surgery approach by our collaborators.
The MSU Udall Center uses a dedicated surgical suite within the Association for Assessment and Accreditation of Laboratory Animal Care (AAALAC) approved Van Andel Research Institute Vivarium. This surgical suite was custom built to accommodate our batch surgery workflow and includes 4 surgical stations, an animal prep and recovery area, an anesthesia induction station and a stereotaxic loading station. Each surgical station is permanently outfitted with a wall-mounted Zeiss OPMI-1FC surgical microscopes, Hamilton syringe pump, isoflurane delivery and scavenging units, circulating warming pad, glass bead sterilizer, dental drill with foot control and fiber optic light sources.
|Figure 1: Two of four surgical stations in use in the MSU Udall Center surgical suite.|
MSU Udall Surgical Services Participants
Timothy Collier, Ph.D., 30 years of experience in surgery on rodents and nonhuman primates.
Brian Daley, B.S.,1985, Biological Technology, SUNY Brockport. Mr. Daley has worked with Dr. Collier for 29 years, serving the role of surgical coordinator as part of his duties.
Christopher Kemp, M.S., 2003. B.S., 1996, Applied Biological Sciences, University of the West of England; M.S., 2003, Epidemiology and Biostatistics, University of Cincinnati. Mr. Kemp is the laboratory manager and surgical coordinator for Dr. Caryl Sortwell. He has twenty years experience working with small rodents.
All Udall Center Project Leaders and many Udall Center Co-Investigators have career-long experience in surgical techniques in animal models in excess of 10 years. MSU Udall Center investigators that routinely participate in surgical sessions include:
Core C Coordinating Committee (C4):
A team comprised of all surgeons and the surgical coordinators meet on a monthly basis to discuss issues related to scheduling, quality control, compliance with Animal Care and Use requirements, space allocation, maintenance of equipment, incorporation of new procedures and protocols and collaboration requests.
Models and Procedures
6-hydroxydopamine (6-OHDA) Models: Despite criticisms of toxin models in PD research, this paradigm does one thing very well: it evokes death of dopaminergic (DAergic) terminals in the striatum with corresponding striatal DA depletion, or DA neurons in the substantia nigra, or both, over a reproducible, well-characterized time period under our experimental conditions. This allows study of stages of neurodegeneration, beginning with axons, with a final endpoint of DA neuron death. We appreciate that DA neuron degeneration is not the only system affected in PD. However, it is a system central to the motor deficits of PD, and a pathological feature common to the varying forms of parkinsonism.
The MSU Udall Center consistently generates the three different 6-hydroxydopamine (6-OHDA)-induced models of nigrostriatal degeneration for use in Center projects:
- Focal intrastriatal lesions
- Progressive nigral degeneration
- Complete unilateral nigrostriatal degeneration
Focal Striatal Lesion: We have tailored the intrastriatal 6-OHDA lesion model to allow us to directly address axoprotection as an event distinct from neuroprotection. It has become clear that the earliest degenerative events in PD are associated with the axon. As degeneration progresses, neuronal cell bodies succumb. To provide a model system in which denervation within a circumscribed, reproducible region of the striatum is present, stable and available for study, over a time frame when degeneration of cell bodies is minimal, we have developed a focal, partial striatal DA denervation 6-OHDA lesion. This Focal Striatal Lesion paradigm is used to evaluate interventions capable of promoting re-innervation of the striatum. Our lesion parameters result in a zone of striatal tyrosine hydroxylase (TH) denervation that spans approximately 1.5 – 1.75 mm in diameter (Figure 2). No necrotic tissue is observed at the injection site however swollen tyrosine hydroxylase (THir) fibers often appear at the periphery of the depletion zone.
|Figure 2. Horizontal section illustrating the focal 6-OHDA striatal lesion. Section is stained for tyrosine-hydroxylase and shows the restricted zone of denervation in the left hemisphere bounded by a surround of intact innervation. At 28d post-lesion, no significant loss of THir neurons in substantia nigra has occurred (stereology). This model will be used to determine whether overexpression of genes of interest or STN DBS will modify the dimensions of the zone of denervation either as a pre-treatment or an inducer of re-growth in a stable lesion. Abbreviations: CC – corpus callosum, Str – striatum, Sep – septal area, Thal – thalamus.|
Progressive 6-OHDA-Induced Nigral Degeneration: To evaluate the ability of therapeutic interventions to protect nigral cell bodies or attenuate nigral loss we use intrastriatal 6-OHDA lesion parameters in which larger double site 6-OHDA injections result in a near immediate loss of all THir striatal terminals but a protracted loss of nigral cell bodies over a period of 6 weeks (Figure 3 and 4)[1, 2]. This pattern of degeneration appears to closely model the early striatal DAergic denervation in PD that precedes and exceeds degeneration of DA neuron cell bodies .
|Figure 3. Micrographs of THir neurons in the SN and THir terminals in the striatum at 2 (A), 4 (B) and 6 (C) weeks following unilateral (left) two site, larger volume intrastriatal injections of 6-OHDA. Progressive degeneration of nigral THir neurons is illustrated by comparing the number of THir neurons evident in the left hemisphere with the normal complement of THir nigral neurons in the contralateral mesencephalon.|
|Figure 4. Time course, magnitude and behavioral impact of nigrostriatal degeneration using our unilateral (left) two site, larger volume intrastriatal injections of 6-OHDA. A. Stereological counts of THir neurons in the SN reveal significantly fewer THir neurons in the lesioned SN (red bars) relative to the intact SN (black bars) at all time points examined (⁎p<0.001). B. Stereological counts of NeuNir neurons in the SN reveal significantly fewer NeuNir neurons in the lesioned SN relative to the intact SN at all time points examined (⁎p<0.001). C. THir neurite density in the striatum was significantly reduced at 2, 4 and 6 weeks following lesion (⁎p<0.0001) with no progression observed after 2 weeks. D. Contralateral forelimb akinesia in the cylinder task at 2, 4 and 6 weeks after intrastriatal vehicle injection (black triangles) or 6-OHDA injection (red circles). All rats receiving 6-OHDA exhibited a significant reduction in contralateral forepaw use at each time point examined compared to both pre-6-OHDA baseline contralateral forepaw use and vehicle injected controls (⁎p<0.002). E. Schematic illustrating the time course of degeneration of nigral THir neurons (red squares), nigral NeuNir neurons (red triangles), striatal THir neurites (white circles) and contralateral forelimb use (black circles).|
Complete Unilateral Nigrostriatal degeneration: We also use injections of 6-OHDA into the substantia nigra (SN) and medial forebrain bundle to produce a rapid > 95% unilateral lesion. This magnitude of lesion is required for the generation of levodopa-induced dyskinesias. We have used this lesion paradigm extensively in previous studies [4-8].
Nigrostriatal Alpha-Synuclein (α-syn) Overexpression Model: We believe it is important to determine whether our observations can be generalized to additional models of nigrostriatal system degeneration. A large body of evidence points to α-syn’s involvement in PD, including the fact that point mutations and multiplications of the SNCA gene have been linked to onset of familial forms of PD [9-11]. Subsequent discoveries of the presence of α-syn in the hallmark protein aggregations (Lewy Bodies) and dystrophic neurites of PD have linked α-syn to sporadic forms of the disease . In our laboratory, and in others, viral vector-mediated nigrostriatal overexpression of wildtype human α-syn results in α-syn aggregation, degeneration of SN THir neurons and striatal terminals and motor dysfunction (Figure 5) [13, 14]. We have characterized a degeneration model using viral vector mediated over-expression of human alpha-synuclein in the nigrostriatal system. This affords us the opportunity to compare our experimental findings in DA system degeneration induced by 6-OHDA to that produced by over-expression of alpha-synuclein.
Specifically, we utilize intranigral injections of recombinant adenoassociated virus serotype 2/5 (rAAV2/5) in which expression of the α-syn transgene is driven by the chicken beta actin/cytomegalovirus enhancer (CβA/CMV) promoter hybrid therefore resulting in the transduction of neurons .) This rAAV2/5 α-syn model produces: 1) transduction of the nigrostriatal system with human wildtype α-syn, 2) 60% nigral DA neuron loss and 40% reduction in striatal TH immunoreactivity 8 weeks after injection, and 3) significant impairment in contralateral forelimb use in the cylinder task at eight weeks post surgery (, Figure 5).
|Figure 5. Overexpression of α-synuclein in the Rat Nigrostriatal System Via rAAV2/5. A-C. Coexpression of human WT α-syn in THir neurons within the SNpc prior to degeneration. D. Degeneration of THir neurons of the SNpc at 8 weeks after rAAV2/5 α-syn injection compared to the uninjected contralateral SN (E). F. Stereological assessment reveals that at 4 weeks following α-syn vector injection there is ≈40% decrease in THir neurons in the SNpc that progresses to ≈60% at 8 weeks (*, p< 0.05 compared to GFP). G. Significant deficits in contralateral forelimb use are observed 8 weeks after rAAV2/5 injection. H. Partial dopaminergic striatal denervation ipsilateral to α-syn overexpression at 8 weeks visualized using near infrared immunofluorescence and quantified in I. (*,p< 0.05 compared to baseline).|
Rat Synucleinopathy Model: Our team has adapted the mouse model  of spread and aggregation of alpha-synuclein following intracerebral injection of pre-formed fibrils (PFFs) of alpha-synuclein to experiments in rats . Following injection of PFFs into the striatum, alpha-synuclein aggregates accumulate in DA neurons of the substantia nigra unilaterally, and cortical regions bilaterally, over time. At time points of 120 days post-injection and beyond, frank degeneration of DA neurons is observed with corresponding declines in striatal DA innervation and DA levels (Figure 6). This model provides a complimentary approach to the viral vector-mediated over-expression of alpha-synuclein, with the added features of synuclein seeding and transport associated with the pathogenic biology of this protein. Specifically, we use stereotaxic injection of 8µg of alpha-synuclein PFFs in a volume of 4µl into the striatum to produce progressive accumulation of alpha-synuclein aggregates in substantia nigra and cortex over a 180d time course.
Figure 6. Nigral & striatal pathology in rat PFF model. Following intrastriatal injection of PFFs, alpha-synuclein aggregates (labeled with antibody to pS129 synuclein) accumulate in substantia nigra early and are largely restricted to the ventral tier of neurons. Over time, dopamine innervation of the striatum declines.
Injection of AAV Vectors for Expression or Silencing of Genes of Interest: Our MSU Surgical Services also have developed surgical techniques specific for the efficiently deliver of AAV vectors into numerous sites (striatum, SN, M1 cortex) for gene or siRNA transfer. Precise targeting of AAV vectors requires a level of surgical accuracy above and beyond normal stereotaxic injections. The incorporation of hand-assembled, glass-pulled micropipette needles with an outer diameter of ≈100 μm into our injection strategy has elevated our consistency and minimized off-target vector spread. This method is used in delivery of rAAV2/5 α-syn (above) and is be used across all vector injection surgical sessions. Dr. Fredric Manfredsson, expert in viral vector construction (see MSU Vector Services) and delivery was instrumental in training all MSU Udall Center surgeons in this improved delivery system. Our group has published extensively using this methodology [1, 16-21].
Accurate Targeting and Long Term Implantation of Stimulating Electrodes in the Subthalamic Nucleus: The clinical use of subthalamic nucleus deep brain stimulation (STN DBS) as a treatment for PD has proceeded despite an incomplete understanding of the underlying biology. This certainly is justified by the dramatic benefits the treatment can provide. However, the investigators of our Center, led by Dr. Caryl Sortwell, believe that a better understanding of the underlying biology can provide information important for optimizing the treatment, patient selection, and timing of intervention. To this end, we have developed an accessible, cost-efficient model of chronic STN DBS in the rat.
Coordinates for implantation of STN electrodes were developed via systematic use of extracellular recording-guided placement, examination of current spread, and post-mortem histological verification [2, 22, 23].
|Figure 7. Extracellular recording-guided electrode placement within the STN A. Response of a neuron within the ventral posterior medial nucleus of the thalamus (VPM) to stimulation of the contralateral vibrissa, stimulation period indicated by horizontal lines beneath x axis. This VPM landmark is used to determine appropriate AP and ML electrode placement. The STN is located 0.5–1.0 mm ventral to this site and is readily distinguishable by a sudden increase of irregular spikes firing at a high rate. B. Dye infusion within STN recording site (arrow). After the coordinates of the STN were identified a bipolar concentric microelectrode was implanted with the electrode fixed in place using dental acrylic and bone screws. Scale bar=1000 μM.|
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Udall Projects & Services
Director: Kathy Steece-Collier, Ph.D.
Director: Caryl E. Sortwell, Ph.D.
Director: Jack W. Lipton, Ph.D.
Director: Timothy J. Collier, Ph.D
Director: Fredric P. Manfredsson, Ph.D.
Director: Timothy J. Collier, Ph.D