Intrathecal catheter implantation decreases cerebrospinal fluid dynamics in cynomolgus monkeys.

A detailed understanding of the CSF dynamics is needed for design and optimization of intrathecal drug delivery devices, drugs, and protocols. Preclinical research using large-animal models is important to help define drug pharmacokinetics-pharmacodynamics and safety. In this study, we investigated the impact of catheter implantation in the sub-dural space on CSF flow dynamics in Cynomolgus monkeys. Magnetic resonance imaging (MRI) was performed before and after catheter implantation to quantify the differences based on catheter placement location in the cervical compared to the lumbar spine. Several geometric and hydrodynamic parameters were calculated based on the 3D segmentation and flow analysis. Hagen-Poiseuille equation was used to investigate the impact of catheter implantation on flow reduction and hydraulic resistance. A linear mixed-effects model was used in this study to investigate if there was a statistically significant difference between cervical and lumbar implantation, or between two MRI time points. Results showed that geometric parameters did not change statistically across MRI measurement time points and did not depend on catheter location. However, catheter insertion did have a significant impact on the hydrodynamic parameters and the effect was greater with cervical implantation compared to lumbar implantation. CSF flow rate decreased up to 55% with the catheter located in the cervical region. The maximum flow rate reduction in the lumbar implantation group was 21%. Overall, lumbar catheter implantation disrupted CSF dynamics to a lesser degree than cervical catheter implantation and this effect remained up to two weeks post-catheter implantation in Cynomolgus monkeys.

Personalized medicine: Vinpocetine to reverse effects of GABRB3 mutation.

OBJECTIVE: To screen a library of potential therapeutic compounds for a woman with Lennox-Gastaut syndrome due to a Y302C GABRB3 (c.905A>G) mutation. METHODS: We compared the electrophysiological properties of cells with wild-type or the pathogenic GABRB3 mutation. RESULTS: Among 1320 compounds, multiple candidates enhanced GABRB3 channel conductance in cell models. Vinpocetine, an alkaloid derived from the periwinkle plant with anti-inflammatory properties and the ability to modulate sodium and channel channels, was the lead candidate based on efficacy and safety profile. Vinpocetine was administered as a dietary supplement over 6 months, reaching a dosage of 20 mg three times per day, and resulted in a sustained, dose-dependent reduction in spike-wave discharge frequency on electroencephalograms. Improved language and behavior were reported by family, and improvements in global impression of change surveys were observed by therapists blinded to intervention. SIGNIFICANCE: Vinpocetine has potential efficacy in treating patients with this mutation and possibly other GABRB3 mutations or other forms of epilepsy. Additional studies on pharmacokinetics, potential drug interactions, and safety are needed.

Characterization of intrathecal cerebrospinal fluid geometry and dynamics in cynomolgus monkeys (macaca fascicularis) by magnetic resonance imaging.

Recent advancements have been made toward understanding the diagnostic and therapeutic potential of cerebrospinal fluid (CSF) and related hydrodynamics. Increased understanding of CSF dynamics may lead to improved detection of central nervous system (CNS) diseases and optimized delivery of CSF based CNS therapeutics, with many proposed therapeutics hoping to successfully treat or cure debilitating neurological conditions. Before significant strides can be made toward the research and development of interventions designed for human use, additional research must be carried out with representative subjects such as non-human primates (NHP). This study presents a geometric and hydrodynamic characterization of CSF in eight cynomolgus monkeys (Macaca fascicularis) at baseline and two-week follow-up. Results showed that CSF flow along the entire spine was laminar with a Reynolds number ranging up to 80 and average Womersley number ranging from 4.1-7.7. Maximum CSF flow rate occurred ~25 mm caudal to the foramen magnum. Peak CSF flow rate ranged from 0.3-0.6 ml/s at the C3-C4 level. Geometric analysis indicated that average intrathecal CSF volume below the foramen magnum was 7.4 ml. The average surface area of the spinal cord and dura was 44.7 and 66.7 cm2 respectively. Subarachnoid space cross-sectional area and hydraulic diameter ranged from 7-75 mm2 and 2-3.7 mm, respectively. Stroke volume had the greatest value of 0.14 ml at an axial location corresponding to C3-C4.

A comprehensive approach to identifying repurposed drugs to treat SCN8A epilepsy.

OBJECTIVE: Many previous studies of drug repurposing have relied on literature review followed by evaluation of a limited number of candidate compounds. Here, we demonstrate the feasibility of a more comprehensive approach using high-throughput screening to identify inhibitors of a gain-of-function mutation in the SCN8A gene associated with severe pediatric epilepsy. METHODS: We developed cellular models expressing wild-type or an R1872Q mutation in the Nav 1.6 sodium channel encoded by SCN8A. Voltage clamp experiments in HEK-293 cells expressing the SCN8A R1872Q mutation demonstrated a leftward shift in sodium channel activation as well as delayed inactivation; both changes are consistent with a gain-of-function mutation. We next developed a fluorescence-based, sodium flux assay and used it to assess an extensive library of approved drugs, including a panel of antiepileptic drugs, for inhibitory activity in the mutated cell line. Lead candidates were evaluated in follow-on studies to generate concentration-response curves for inhibiting sodium influx. Select compounds of clinical interest were evaluated by electrophysiology to further characterize drug effects on wild-type and mutant sodium channel functions. RESULTS: The screen identified 90 drugs that significantly inhibited sodium influx in the R1872Q cell line. Four drugs of potential clinical interest-amitriptyline, carvedilol, nilvadipine, and carbamazepine-were further investigated and demonstrated concentration-dependent inhibition of sodium channel currents. SIGNIFICANCE: A comprehensive drug repurposing screen identified potential new candidates for the treatment of epilepsy caused by the R1872Q mutation in the SCN8A gene.

Nonuniform Moving Boundary Method for Computational Fluid Dynamics Simulation of Intrathecal Cerebrospinal Flow Distribution in a Cynomolgus Monkey.

A detailed quantification and understanding of cerebrospinal fluid (CSF) dynamics may improve detection and treatment of central nervous system (CNS) diseases and help optimize CSF system-based delivery of CNS therapeutics. This study presents a computational fluid dynamics (CFD) model that utilizes a nonuniform moving boundary approach to accurately reproduce the nonuniform distribution of CSF flow along the spinal subarachnoid space (SAS) of a single cynomolgus monkey. A magnetic resonance imaging (MRI) protocol was developed and applied to quantify subject-specific CSF space geometry and flow and define the CFD domain and boundary conditions. An algorithm was implemented to reproduce the axial distribution of unsteady CSF flow by nonuniform deformation of the dura surface. Results showed that maximum difference between the MRI measurements and CFD simulation of CSF flow rates was <3.6%. CSF flow along the entire spine was laminar with a peak Reynolds number of ∼150 and average Womersley number of ∼5.4. Maximum CSF flow rate was present at the C4-C5 vertebral level. Deformation of the dura ranged up to a maximum of 134 µm. Geometric analysis indicated that total spinal CSF space volume was ∼8.7 ml. Average hydraulic diameter, wetted perimeter, and SAS area were 2.9 mm, 37.3 mm and 27.24 mm2, respectively. CSF pulse wave velocity (PWV) along the spine was quantified to be 1.2 m/s.

Prevalence and Incidence of Drug-Resistant Mesial Temporal Lobe Epilepsy in the United States.

OBJECTIVE: We reviewed data on the epidemiology of epilepsy in the United States and estimated the prevalence and incidence of drug-resistant temporal lobe epilepsy (TLE) due to hippocampal sclerosis (HS) based on extrapolation from the available data drawn from the literature. METHODS: We searched the electronic database PubMed on December 14, 2016, using the following search terms in the title: "epilepsy" OR "temporal lobe" OR "hippocampal sclerosis" AND "epidemiology" OR "prevalence" OR "incidence." Relevant original studies were included. We also reviewed several previous systematic reviews, meta-analyses, and their references. RESULTS: We concluded that the estimated current U.S. prevalence of drug-resistant HS-TLE is 0.51-0.66 cases per 1000 people, and the estimated U.S. incidence is 3.1-3.4 cases per 100,000 people per year. Based on a U.S. population of 324 million, we estimate that as many as 143,000-191,000 U.S. patients still suffer from drug-resistant HS-TLE and are in need of surgery or other therapeutic options. CONCLUSIONS: Although full scale epidemiologic studies on drug-resistant HS-TLE are needed, several observational studies allow adequate estimates of the range for the incidence and prevalence of this condition. Given the morbidity and mortality associated with poorly controlled seizures, this relatively large and growing patient population is a matter of concern. Considering the variety of treatment options that are available or in the pipeline to treat drug-resistant epilepsy, future efforts should focus on advocating for early referral of patients with drug-resistant HS-TLE for more comprehensive epilepsy management.

Onset Time and Durability of Huntingtin Suppression in Rhesus Putamen After Direct Infusion of Antihuntingtin siRNA.

One possible treatment for Huntington's disease involves direct infusion of a small, interfering RNA (siRNA) designed to reduce huntingtin expression into brain tissue from a chronically implanted programmable pump. Here, we studied the suppression of huntingtin mRNA achievable with short infusion times, and investigated how long suppression may persist after infusion ceases. Rhesus monkeys received 3 days of infusion of Magnevist into the putamen to confirm catheter patency and fluid distribution. After a 1-week washout period, monkeys received radiolabeled siRNA targeting huntingtin. After 1 or 3 days of siRNA delivery, monkeys were either terminated, or their pumps were shut off and they were terminated 10 or 24 days later. Results indicate that the onset of huntingtin mRNA suppression in the rhesus putamen occurs rapidly, achieving a plateau throughout the putamen within 4 days. Conversely, loss of huntingtin suppression progresses slowly, persisting an estimated 27-39 days in the putamen and surrounding white matter. These findings indicate the rapid onset and durability of siRNA-mediated target gene suppression observed in other organs also occurs in the brain, and support the use of episodic delivery of siRNA into the brain for treatment of Huntington's disease and possibly other neurodegenerative diseases.

Widespread suppression of huntingtin with convection-enhanced delivery of siRNA.

Huntington's disease is an autosomal dominant neurodegenerative disease caused by a toxic gain of function mutation in the huntingtin gene (Htt). Silencing of Htt with RNA interference using direct CNS delivery in rodent models of Huntington's disease has been shown to reduce pathology and promote neuronal recovery. A key translational step for this approach is extension to the larger non-human primate brain, achieving sufficient distribution of small interfering RNA targeting Htt (siHtt) and levels of Htt suppression that may have therapeutic benefit. We evaluated the potential for convection enhanced delivery (CED) of siHtt to provide widespread and robust suppression of Htt in nonhuman primates. siHtt was infused continuously for 7 or 28 days into the nonhuman primate putamen to analyze effects of infusion rate and drug concentration on the volume of effective suppression. Distribution of radiolabeled siHtt and Htt suppression were quantified by autoradiography and PCR, respectively, in tissue punches. Histopathology was evaluated and Htt suppression was also visualized in animals treated for 28 days. Seven days of CED led to widespread distribution of siHtt and significant Htt silencing throughout the nonhuman primate striatum in an infusion rate and dose dependent manner. Htt suppression at therapeutic dose levels was well tolerated by the brain. A model developed from these results predicts that continuous CED of siHtt can achieve significant coverage of the striatum of Huntington's disease patients. These findings suggest that this approach may provide an important therapeutic strategy for treating Huntington's disease.

Intracerebroventricular amyloid-beta antibodies reduce cerebral amyloid angiopathy and associated micro-hemorrhages in aged Tg2576 mice.

Although immunization against amyloid-beta (Abeta) holds promise as a disease-modifying therapy for Alzheimer disease (AD), it is associated with an undesirable accumulation of amyloid in the cerebrovasculature [i.e., cerebral amyloid angiopathy (CAA)] and a heightened risk of micro-hemorrhages. The central and peripheral mechanisms postulated to modulate amyloid with anti-Abeta immunotherapy remain largely elusive. Here, we compared the effects of prolonged intracerebroventricular (i.c.v.) versus systemic delivery of anti-Abeta antibodies on the behavioral and pathological changes in an aged Tg2576 mouse model of AD. Prolonged i.c.v. infusions of anti-Abeta antibodies dose-dependently reduced the parenchymal plaque burden, astrogliosis, and dystrophic neurites at doses 10- to 50-fold lower than used with systemic delivery of the same antibody. Both i.c.v. and systemic anti-Abeta antibodies reversed the behavioral impairment in contextual fear conditioning. More importantly, unlike systemically delivered anti-Abeta antibodies that aggravated vascular pathology, i.c.v.-infused antibodies globally reduced CAA and associated micro-hemorrhages. We present data suggesting that the divergent effects of i.c.v.-delivered anti-Abeta antibodies result from gradually engaging the local (i.e., central) mechanisms for amyloid clearance, distinct from the mechanisms engaged by high doses of anti-Abeta antibodies that circulate in the vasculature following systemic delivery. With robust efficacy in reversing AD-related pathology and an unexpected benefit in reducing CAA and associated micro-hemorrhages, i.c.v.-targeted passive immunotherapy offers a promising therapeutic approach for the long-term management of AD.

Neuropathology of the acid sphingomyelinase knockout mouse model of Niemann-Pick A disease including structure-function studies associated with cerebellar Purkinje cell degeneration.

Niemann-Pick A (NP-A) is an inherited metabolic (lysosomal storage) disease characterized by neurovisceral accumulation of sphingomyelin due to deficiency of acid sphingomyelinase (ASM). An ASM knockout (ASMKO) mouse model of NP-A is available through targeted disruption of the parent gene. This study presents the pattern and time course of lysosomal pathology and neurodegeneration in the ASMKO mouse nervous system. Cells throughout the nervous system developed the classic foamy appearance associated with lysosomal storage disorders. Despite this, neurons were capable of retrogradely transporting dyes within established brain pathways comparable to control animals. A silver degeneration staining method demonstrated widespread damage in the form of 'classic' impregnation of cells, fibers and synaptic terminals. Of particular interest was the degeneration of Purkinje cells (PC) within the cerebellum, beginning by 7 weeks of age in parasagittal bands and culminating with near complete degeneration of this cell type by 20 weeks. In parallel, ASMKO mice had progressively deteriorating motor performance on two versions of the rotating rod test (accelerating and rocking). ASMKO mice at 5-7 weeks of age performed similarly to controls on both rotating rod tests, but performance sharply deteriorated between 7 and 20 weeks of age. This study further characterized the neuropathology associated with ASM deficiency, and identifies quantitative histological and behavioral endpoints for evaluation of therapeutic intervention in this authentic NP-A mouse model.

A review of intrathecal fentanyl and sufentanil for the treatment of chronic pain.

Intrathecal infusion of morphine using implantable pumps is an accepted practice for long-term management of chronic pain. Despite clinical benefit, development of tolerance and side-effects associated with intrathecal morphine has prompted investigators to explore alternative opioids such as the potent anilinopiperidine analogs, fentanyl, and sufentanil. Relevant preclinical and clinical literature from the MEDLINE database was used primarily for this review. In vitro, both compounds are stable in solution, but studies have not been conducted using implantable pumps under simulated use conditions (e.g., long-term stability at body temperature). Preclinical studies of limited duration have demonstrated efficacy, but safety-toxicology studies have been limited to intermittent boluses of sufentanil only. Few clinical reports on the use of intrathecal sufentanil or fentanyl for chronic pain are available. Although results confirm potency and efficacy with intrathecal administration, further studies are needed to support the long-term use of either opioid in chronic pain management.

Intracranial delivery of CLN2 reduces brain pathology in a mouse model of classical late infantile neuronal ceroid lipofuscinosis.

Classical late infantile neuronal ceroid lipofuscinosis (cLINCL) is a lysosomal storage disorder caused by mutations in CLN2, which encodes lysosomal tripeptidyl peptidase I (TPP1). Lack of TPP1 results in accumulation of autofluorescent storage material and curvilinear bodies in cells throughout the CNS, leading to progressive neurodegeneration and death typically in childhood. In this study, we injected adeno-associated virus (AAV) vectors containing the human CLN2 cDNA into the brains of CLN2(-/-) mice to determine therapeutic efficacy. AAV2CUhCLN2 or AAV5CUhCLN2 were stereotaxically injected into the motor cortex, thalamus, and cerebellum of both hemispheres at 6 weeks of age, and mice were then killed at 13 weeks after injection. Mice treated with AAV2CUhCLN2 and AAV5CUhCLN2 contained TPP1 activity at each injection tract that was equivalent to 0.5- and 2-fold that of CLN2(+/+) control mice, respectively. Lysosome-associated membrane protein 1 immunostaining and confocal microscopy showed intracellular targeting of TPP1 to the lysosomal compartment. Compared with control animals, there was a marked reduction of autofluorescent storage in the AAV2CUhCLN2 and AAV5CUhCLN2 injected brain regions, as well as adjacent regions, including the striatum and hippocampus. Analysis by electron microscopy confirmed a significant decrease in pathological curvilinear bodies in cells. This study demonstrates that AAV-mediated TPP1 enzyme replacement corrects the hallmark cellular pathologies of cLINCL in the mouse model and raises the possibility of using AAV gene therapy to treat cLINCL patients.

Gene transfer of human acid sphingomyelinase corrects neuropathology and motor deficits in a mouse model of Niemann-Pick type A disease.

Niemann-Pick type A disease is a lysosomal storage disorder caused by a deficiency in acid sphingomyelinase (ASM) activity. Previously we showed that storage pathology in the ASM knockout (ASMKO) mouse brain can be corrected by adeno-associated virus serotype 2 (AAV2)-mediated gene transfer. The present experiment compared the relative therapeutic efficacy of different recombinant AAV serotype vectors (1, 2, 5, 7, and 8) using histological, biochemical, and behavioral endpoints. In addition, we evaluated the use of the deep cerebellar nuclei (DCN) as a site for injection to facilitate global distribution of the viral vector and enzyme. Seven-week-old ASM knockout mice were injected within the DCN with different AAV serotype vectors encoding human ASM (hASM) and then killed at either 14 or 20 weeks of age. Results showed that AAV1 was superior to serotypes 2, 5, 7, and 8 in its relative ability to express hASM, alleviate storage accumulation, and correct behavioral deficits. Expression of hASM was found not only within the DCN, but also throughout the cerebellum, brainstem, midbrain, and spinal cord. This finding demonstrates that targeting the DCN is an effective approach for achieving widespread enzyme distribution throughout the CNS. Our results support the continued development of AAV based vectors for gene therapy of the CNS manifestations in Niemann-Pick type A disease.

AAV vector-mediated correction of brain pathology in a mouse model of Niemann-Pick A disease.

Niemann-Pick A disease (NPA) is a fatal lysosomal storage disorder caused by a deficiency in acid sphingomyelinase (ASM) activity. The lack of functional ASM results in cellular accumulation of sphingomyelin and cholesterol within distended lysosomes throughout the brain. In this study, we investigated the potential of AAV-mediated expression of ASM to correct the brain pathology in an ASM knockout (ASMKO) mouse model of NPA. An AAV serotype 2 vector encoding human ASM (AAV2-hASM) was injected directly into the adult ASMKO hippocampus of one hemisphere. This resulted in expression of human ASM in all major cell layers of the ipsilateral hippocampus for at least 15 weeks postinjection. Transduced cells were also present in the entorhinal cortex, medial septum, and contralateral hippocampus in a pattern consistent with retrograde axonal transport of AAV2. There was a substantial reduction of distended lysosomes and an almost complete reversal of cholesterol accumulation in all areas of the brain that were targeted by AAV2-hASM. These findings show that the ASMKO brain is responsive to ASM replacement and that retrograde transport of AAV2 functions as a platform for widespread gene delivery and reversal of pathology in affected brain.

Enhanced delivery of octreotide to the brain via transnasal iontophoretic administration.

Transnasal drug delivery affords an opportunity to circumvent the blood-brain barrier and gain direct access to the brain. To date, this approach has used a relatively passive process relying on drug instillation high into the nasal cavity, formulation and gravity for drug delivery. The present study examined the use of an applied electrical field (transnasal iontophoresis or electrotransport) to actively drive a charged peptide, octreotide, into the rabbit brain. A simply designed electrode containing a reservoir of octreotide was placed deep into the nasal cavity on both sides. A return electrode was applied to the back of the head and a current strength of 3.0 mA was applied for 60 min. In control rabbits, electrodes were placed into the nasal cavity, but no current was applied (passive delivery). Additional control animals were given a bolus intra-arterial injection of octreotide. At the conclusion of drug delivery, animals were sacrificed and samples of brain, spinal cord, cerebrospinal fluid (CSF) and plasma were taken for measurement of octreotide levels by radioimmunoassay (RIA). In a second experiment, rabbits were exsanguinated prior to drug delivery to measure the ability of iontophoresis to transport octreotide into the brain in the absence of blood or CSF circulation. In both experiments, transnasal iontophoresis resulted in significantly elevated levels of octreotide in the brain, although results varied considerably due to electrode and tissue damage related to problems with electrode insertion into the rabbit's nasal cavity. Octreotide was present in samples extending from the olfactory bulb to the cerebellum with 2- to 13-fold increases in active compared to control/passive animals. High and sustained levels of octreotide were also present in the blood following transnasal delivery, but there were negligible amounts of octreotide in the brain following systemic administration indicating that the blood was not a significant route for drug redistribution. The results demonstrate that transnasal electrotransport is a unique, minimally invasive approach for enhancing drug delivery to the brain.

A mouse model of classical late-infantile neuronal ceroid lipofuscinosis based on targeted disruption of the CLN2 gene results in a loss of tripeptidyl-peptidase I activity and progressive neurodegeneration.

Mutations in the CLN2 gene, which encodes a lysosomal serine protease, tripeptidyl-peptidase I (TPP I), result in an autosomal recessive neurodegenerative disease of children, classical late-infantile neuronal ceroid lipofuscinosis (cLINCL). cLINCL is inevitably fatal, and there currently exists no cure or effective treatment. In this report, we provide the characterization of the first CLN2-targeted mouse model for cLINCL. CLN2-targeted mice were fertile and apparently healthy at birth despite an absence of detectable TPP I activity. At approximately 7 weeks of age, neurological deficiencies became evident with the onset of a tremor that became progressively more severe and was eventually accompanied by ataxia. Lifespan of the affected mice was greatly reduced (median survival, 138 d), and extensive neuronal pathology was observed including a prominent accumulation of cytoplasmic storage material within the lysosomal-endosomal compartment, a loss of cerebellar Purkinje cells, and widespread axonal degeneration. The CLN2-targeted mouse therefore recapitulates much of the pathology and clinical features of cLINCL and represents an animal model that should provide clues to the normal cellular function of TPP I and the pathogenic processes that underlie neuronal death in its absence. In addition, the CLN2-targeted mouse also represents a valuable model for the evaluation of different therapeutic strategies.

Mouse neural progenitor cells differentiate into oligodendrocytes in the brain of a knockout mouse model of Canavan disease.

Canavan disease (CD) is an autosomal recessive disorder that leads to spongy degeneration in the white matter of the brain. Aspartoacylase (ASPA) synthesizing cells, oligodendrocytes, are lost in CD. Transplantation of neural progenitor cells (NPCs) offers an interesting therapeutic approach for treating neurodegenerative diseases by replacing the lost cells. Therefore, the NPCs transplantation to the brain of the CD mouse was studied. Injection of mouse NPCs to the striatum and cerebellum of juvenile CD mouse showed numerous BrdU positive cells at 1 month after injection. The same result was also observed in the adult CD mouse brain after 5 weeks of post-transplantation period. The implanted cells differentiated into oligodendrocytes and fibrous astrocytes, as observed using glial cell marker. This is the first report to describe the survival, distribution and differentiation of NPCs within the brain of CD mouse and a first step toward the potential clinical use of cell therapy to treat CD.

The effects of transforming growth factor-beta2 on dopaminergic graft survival.

Dopaminergic cell transplantation is a promising therapeutic approach for the treatment of Parkinson's disease, the potential of which is limited due to poor survival and low dopamine content within engrafted tissue. In this study, the ability of transforming growth factor-beta2 (TGF-beta2) to influence transplant survival was evaluated. Cell suspensions containing fetal rat ventral mesencephalon (VM) cells were incubated prior to surgery with vehicle (DPBS), varying concentrations of TGF-beta2 (5-1000 ng/ml), or a pan-specific antibody against TGF-beta (1D11, 100 ng/ml). VM cell suspensions (200,000 cells) were unilaterally implanted into the striatum of adult Sprague-Dawley rats (n = 5-11 animals/group). Following a 3-week survival period, small but viable VM grafts containing tyrosine hydroxylase-positive (TH+) neurons and fibers were present in all animals. Addition of TGF-beta2 resulted in a steep, bell-shaped dose-response curve with a significant effect on TH+/dopamine cell survival. At 50 ng/ml TGF-beta2, the number of surviving dopamine neurons was increased twofold compared with controls. Addition of TGF-beta2 or 1D11 did not significantly influence graft volume. Further studies, possibly in combination with other neurotrophic factors, need to be performed to obtain a greater understanding of the effects of TGF-beta on dopamine neurons and fetal VM cell engraftment.

Comparison of fresh and cryopreserved porcine ventral mesencephalon cells transplanted in A rat model of Parkinson's disease.

To evaluate whether cryopreservation of porcine ventral mesencephalon cells influences graft survival and function in vivo, we have transplanted either freshly prepared or cryopreserved cells into the striatum of 6-hydroxydopamine-lesioned rats. A single cell suspension of porcine ventral mesencephalon cells from the same isolation either was stored at 4 degrees C and transplanted the next day or was cryopreserved for 4 weeks in liquid nitrogen vapor. The cryopreserved cells were then rapidly thawed, rinsed, and transplanted in the same manner as the fresh cells, with the same dose of viable cells. All animals received daily injections of cyclosporin A to prevent xenograft rejection. To monitor graft function, amphetamine-induced rotation was measured every 3 weeks between 6 and 15 weeks posttransplantation. After sacrifice at 15 weeks posttransplantation, histological methods were used to compare fresh cell and cryopreserved cell transplants with respect to graft survival, differentiation and integration, and host immune response. Cryopreserved cells were found to be either equivalent or in some cases superior to fresh cells with respect to rotational correction, graft survival, graft volume, numbers of graft-derived dopaminergic neurons, and host immune responses. In conclusion, the results indicate that it is feasible to cryopreserve porcine ventral mesencephalon cells for long-term storage of cells prior to transplantation in an animal model of Parkinson's disease.