Chronic central administration of valproic acid

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Chronic central administration of valproic acid increased pro-survival phospho-proteins and growth cone associated proteins with no behavioral pathology
Bates, Ryan Christorpher ( author )
University of Colorado Denver
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Anticonvulsants ( lcsh )
Valproic acid ( lcsh )
Anticonvulsants ( fast )
Valproic acid ( fast )
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theses ( marcgt )
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Valproic acid (VPA) is the most widely prescribed antiepileptic drug due to its ability to treat a broad spectrum of seizure types. However, potential complications of this drug include anticonvulsant polytherapy metabolism, organ toxicity and teratogenicity which limit its use in a variety of epilepsy patients. Direct delivery of VPA intracerebroventricularly (ICV) could circumvent the toxic effects normally seen with the oral route of administration. An additional potential benefit would be significantly reduced dosing while achieving high brain concentrations. Epileptogenic tissue from patients with intractable seizures has shown significant cell death which may be mitigated by maximizing cerebral VPA exposure. Here we show ICV administration of VPA localized to the periventricular zone increased pro-survival phospho-proteins (pAktSer473, pAktThr308, pGSK3beta-Ser9, pErk1/2Thr202/Tyr204) and growth cone associated proteins (2G13p, GAP43) in a whole animal system. No significant changes in DCX, NeuN, synaptotagmin, and synaptophysin were detected. Assessment of possible behavioral alterations in rats receiving chronic central infusions of VPA was performed with the open field and elevated plus mazes. Neither paradigm revealed any detrimental effects of the drug infusion process.
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Integrative biology
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Department of Integrative Biology
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by Ryan Christopher Bates.

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CHRONIC CENTRAL ADMI NISTRATION OF VALPRO IC ACID: INCREASED PROSURVIVAL PHOSPHO PROTEINS AND GROWTH CONE ASSOCIATED PROTEINS WITH NO BEHAVIORAL PATHOLOGY by RYAN CHRISTOPHER BATES B.S ., Metropolitan State University of Denver, 2006 A thesis submitted to the Faculty of the Graduate School of the University of Colorado in partial fulfillment of the requirements for the degree of Master of Science Integrative Biology 2013


This thesis for the Master of Science degree by Ryan Christopher Bates has been approved for the Integrative Biology Program by Bradley J. Stith Chair Jefferson Knight Amanda Charlesworth July 9, 2013 ii


Bates, Ryan C hristopher (M. S., Integrative Biology) Chronic C entral A dministration of V alproic Acid: Increased Pro Survival PhosphoProteins and Growth Cone Associated Proteins with no Behavioral P athology Thesis Directed by Professor Bradley J. Stith ABSTRACT Valproic acid (VPA) is the most widely prescribed antiepileptic drug due to its ability to treat a broad spectrum of seizure types. However, potential complications of this drug include anticonvulsant polytherapy metabolism, organ toxicity and teratogenicity whic h limit its use in a variety of epilepsy patients. Direct delivery of VPA intracerebroventricularly (ICV) could circumvent the toxic effects normally seen with the oral route of administration. An additional potential benefit would be significantly reduc ed dosing while achieving high brain concentrations. Epileptogenic tissue from patients with intractable seizures has shown significant cell death which may be mitigated by maximizing cerebral VPA exposure. Here we show ICV administration of VPA localized to the periventricular zone increased pro survival phosphoproteins ( pAktSer473, pAktThr308, p GSK3 Ser9, pErk1/2Thr202/Tyr204) and growth cone associated proteins (2G13p, GAP43) in a whole animal system. No significant changes in DCX, NeuN, synaptotagmi n, and synaptophysin were detected. Assessment of possible behavioral alterations in rats receiving chronic central infusions of VPA was performed with the open field and elevated plus mazes. Neither paradigm revealed any detrimental effects of the drug infusion process. iii


The form and content of this abstract are approved I recommend its publication. Approved: Bradley J. Stith iv


DEDICATION I dedicate this thesis to my loving parents, Dale & Karen Bates, and to my little brother, Steven. v


ACKNOWLEDGEMENT I would like to thank Dr. Brad Stith and Dr. Doug Petcoff for their countless hours spent advising me about the complex world of bioscience and the games a scientist must play in order to win. The contents of this thesis were published on August 31st 2012: Bates RC, Stith BJ, Stevens KE. Chronic central administration of valproic acid: increased pro survival phosphoproteins and growth cone associated proteins with no behavioral pathology. Phar macology, Biochemistry and Behavior 2012;103:237244. DOI Link: Paper featured on Global Medical Discovery with additional data figure (Fig. 4.) on March 19th 2013: central administrationof valproic acid increased prosurvival phosphoproteins andgrowthcone associated proteins with nobehavioral pathology/ vi


TABLE OF CONTENTS CHAPTER I INTRODUCTION ............................................................................................... 1 II. MATERIALS AND METHODS ....................................................................... 3 Animals ............................................................................................................... 3 Implant Surgery .................................................................................................. 3 In Vivo Behavi oral Studies ................................................................................. 4 Tissue Preparations ............................................................................................. 5 Immunohist ochemistry ....................................................................................... 5 Sample Lysate Preparation ................................................................................. 6 Western Immunoblotting .................................................................................... 7 Statis tics .............................................................................................................. 8 III. RESULTS ......................................................................................................... 9 Behavior of ICV I mplanted R ats in Open Field and Elevated Plus M aze VPA, PhosphoProteins, and Growth Cone Associated Protein Localization in V entricle PhosphoProtein and Total Protein Levels in Regional Brain Tissue Lysates..28 IV DISCUSSION ................................................................................................. 18 V CONCLUSION ................................................................................................ 24 REFERENCES ..................................................................................................... 25 vii


LIST OF FIGURES FIGURE 1 Behavioral effects of ICV VPA administration ................................................. 11 2 Immunochemical results of ICV VPA administration ....................................... 14 3 Growth cone associated proteins after ICV VPA administration ...................... 16 4 Drug penetrance and cases of h ydrocephaly ...................................................... 17 viii


L IST OF TABLES TABLE 1 ANOVA results for Open Field and Elevated Plus Paradigms .......................... 10 2 ANOVA and Scheffe a posteriori results for Immunohistochemistry and Western blot studies ................................................................................................................... 12 ix


LIST OF ABBREVIATIONS Erk, extracellular signal regulated kinase GSK3 glycogen synthase kinase 3 beta GAP43, growth associated protein 43 ICV, intracerebroventricle VPA, valproic acid CSF, cerebrospinal fluid BBB, blood brain barrier EP, elevated plus maze OF, open field maze SUDEP, sudden unexpected death in epilepsy DCX, doublecortin NeuN, neuronal nuclei CC, corpus callosum LV, lateral ventricle TV, third ventricle H, hippocampus x


CHAPTER I INTRODUCTION Mortality in epilepsy patients, specifically the occurrence of sudden unexpected death (SUDEP), is 2440 times higher than those of the general population (Hitiris et al., 2007; Nobili et al., 2010) and seems to preferentially affect those with chronic int ractable epilepsy (Asadi Pooya and Sperling, 2009). Other negative outcomes are seen in chronic childhood epilepsy which is suggested to increase the likelihood of mental retardation and severely perturbed brain development (Choi et al., 2009; Wasterlain and Shirasaka, 1994). Brain tissue removed from patients with chronic intractable seizures displayed significant cell death among both neurons and astrocytes that was increased by seizure frequency (Choi et al., 2009). Surgical resection of an epileptoge nic zone is usually performed in a large portion of temporal lobe epilepsy patients who are refractory to pharmacological intervention at a tolerable antiepileptic dose range (Vreugdenhil et al., 1998). Resection is generally followed by initiation of tre atment with one of the most effective antiepileptic drugs, valproic acid (VPA) (Vreugdenhil et al., 1998), a drug shown to be effective in a significant proportion of treatment resistant patients who did not undergo surgical resection (Chayasirisobhon and Russell, 1983; Gal et al., 1988; Hassan et al., 1976; Redenbaugh et al., 1980). VPAs ability to help manage seizures is thought to be attributed to its multifaceted capability in affecting several targets. In rats, peripherally administered VPA increased the inhibitory neurotransmitter, aminobutyric acid (GABA) (Godin et al., 1969; Higuchi et al., 1986; Loscher, 1981, 1999), and within the timeframe of increasing GABA, rodents were found to have decreased susceptibility towards seizures 1


(Loscher, 1981; Schechter et al., 1978; Simler et al., 1973). Others report that VPA modulated voltage gated sodium channels (Cunningham et al., 2003; Vreugdenhil et al., 1998), calcium influx buffering during events of glutamate induced toxicity (Zhang et al., 2010) and lowered aspartate release (Loscher, 1999). Additionally, VPA exposure to neuron and glial cell cultures has shown increases in the pro cellular survival phosphoproteins, pAktSer473, pAktThr308, pGSK3 Ser9and pErk1/2Thr202/Tyr204 (Di Daniel et al., 2005; Lamarre and Desrosiers, 2008; Pan et al., 2005). While there are numerous benefits to the peripheral administration of VPA to patients, side effects such as hyperammonemia (DeWolfe et al., 2009) and a reduction in drug clearance (Faught et al., 1999) can lead to complications endangering or further lowering quality of life. In the present study, we record differential phosphoprotein and growth cone associated protein levels in brain tissue with ICV administration of VPA in a whole animal model. VPA deposition was visualized at the drug release site to help understand penetrance within the central infusion context. Behavioral paradigms were also run to help determine if there was any onset of overt route specific behavioral perturbations during the chronic exposure. 2


CHAPTER II MATERIALS AND METHODS Animals Adult male Sprague Dawley rats (HarlanTM Sprague Dawley; Indianapolis, IN) were housed in pairs prior to experiments under constant temperature (21 1 C) and 12hour lighting (lights on from 6 a.m. to 6 p.m.). Rats were given access to water and rodent chow (Harlan Teklad, Indianapolis, IN) ad libitum Procedures were approved by the Institutional Animal Care and Use Committee (IACUC, Veterans Affairs Medical Center, Eastern Colorado Healthcare System) and followed American Association for Laboratory Animal Science (AALAS) guidelines. Implant S urgery Rats (250280 g) were administered atropine sulfate (0.4 mg/kg, ip) as a preanesthetic 10 minutes pr ior to an injection of sodium pentobarbital (60 mg/kg, ip, Nembutal, Hospira, Inc.; Lake Forest, IL). They were stereotaxically implanted with a cannula (Brain Infusion Kit 1, ALZET, Durect, Cupertino, CA) aimed at one anterior lateral ventricle [ 1 mm posterior to bregma; 1.2 mm lateral to midline and; 4.0 mm ventral to skull (Paxinos and Watson, 1997)]. The cannula was attached by tubing to an osmotic minipump (0.25 l/hr for 28 days, ALZET, Durect, Cupertino, CA) loaded with saline, or valproic a cid (pH 7.4) 2 or 3 mM Great care was taken to guarantee no air bubbles were trapped in tubing (slow backfilling), our experience with this suggests that trapped air does affect pump rate. The cannula was secured to the skull with stainless steel screws and acrylic cement. The osmotic minipump was inserted into a subcutaneous pocket at shoulder level and the wound was closed with wound clips. A 3


prophylactic injection of enrofloxacin (20 mg/kg; Baytril; BAYER HealthCare Inc.; Leverkusen, Germany) was administered at the end of the surgical procedure. In Vivo Behavioral S tudies Implanted rats were allowed to recover for 14 days prior to behavioral testing. Behavioral testing was performed in a counterbalanced design to eliminate any potential order of testing effects. On the 15th day, half of the rats in each group were tested in the elevated plus (EP) maze (Carobrez and Bertoglio, 2005; Cruz et al., 1994; Doremus et al., 2006; Pellow et al., 1985) while the other half was tested in the open field (OF) maze (Ennaceur et al., 2006a, 2006b; Lipkind et al., 2004; Prut and Belzung, 2003; Stanford, 2007). On day 22, the groups were reversed. For the EP maze, a rat was placed in the open region of a b lack Plexiglas + shaped platform with 13 cm wide arms. Two of the arms were enclosed by 32 cm high walls while the remaining 2 arms were open. The maze was elevated 51 cm off the floor. Rat movements were computer tracked for 5 min; the percent time spent in each type of arm and distance traveled in each arm (open, closed or in the center square) were computer analyzed (TopScan; Clever Sys Inc.; Reston, VA). The open field consisted of a 104104 cm2 black Plexiglas floor with 39 cm high walls. The r at was placed near one side wall of the field and allowed to explore at will for 5 min during which its activity was computer monitored (TopScan; Clever Sys Inc.; Reston, VA). The field was divided into 3 regions (3 concentric squares), the outer region ( adjacent to the walls), a middle region and the center square. Time and distance of travel in each region were computer calculated. 4


Tissue P reparations A separate group of implanted rats was anesthetized with isoflurane inhalant anesthesia (3% in oxygen; Webster Veterinary; Sterling, MA) and perfused transcardially with a solution of 0.9% NaCl for 5 minutes followed by buffered 10% formaldehyde (Fisher Diagnostics; 23 245685; Kalamazoo, MI) for 15 minutes. Brains were then excised and postfixed in the sam e fixative for 2 days. 1 mm coronal sections through the whole brain were cut using a sectioning block (Alto, 1 mm; BS 6000C; Braintree Scientific, Inc.; Braintree, MA). Immunohistochemistry Rat brain sections were incubated in undiluted blocking buffer ( LI COR Biosciences; 92740000; Lincoln, NE) for 2 hours on a rotator (Stovall; The Belly Dancer Lab Shaker) at room temperature. The fixed tissue sections were then incubated with primary antibodies in undiluted blocking buffer for 12 hours at 48C. The specific antibodies employed in this study were: valproic acid pAb (rabbit; 1:1000; Abcam; ab37127; Cambridge, MA ), GSK3 Ser9 mAb (rabbit; 1:1000; Cell Signaling Technology; 9323; Boston, MA), 2G13 mAb (mouse; 1:200; Novus Biologicals; NB600 785; Littleton, CO), and AKTSer473 mAb (rabbit; 1:1000; Cell Signaling Technology; 4060; Boston, MA). Tissue slices were never allowed to dry; antibody incubations were unobstructed to prevent unequal tissue exposure (e.g. edge effect) in control and treated samples (True, 2008). Following two quick washes and two 30 minute washes, each with TBS containing 0.05% Tween20 (TBST), the tissue sections were incubated for 2.5 hours at room temperature with IRDye 800CW goat anti rabbit pAb antibody (1:5000; LI COR Biosciences; 926 32211; Lincoln, NE) or IRDye 5


680LT goat anti mouse pAb antibody (1:5000; LI COR Biosciences; 926 68020; Lincoln, NE). The tissue sections received two quick washes and then two 1.5 hour washes in TBST. After washing, the tissue slices were placed in PBS buffer (pH 7.4) until arranged on the Odyssey glass bed (LI COR Biosciences; Odyssey Infrared Imager System; Lincoln, NE). PBS was applied to slices and a silicone mat placed on top for equal laser strike between individual slices. Fluorescent immunocomplexes around the periventricular zone were detected with the LI COR Odyssey (700 nm and/or 800 included in data collection. Laser intensity was optimized for each sample group. Typical laser intensity settings ranged from 2.0 to 4.0 for VPA exposed samples. Control samples were hit with a higher intensity, 4.55.5, so the ventricles could be visualized for figure images. Integrated intensi ties were determined with the Odyssey system software (v 2.1). Sample Lysate P reparation A third group of implanted rats were anesthetized, perfuse fixed, and decapitated as described above. Brain tissue was removed, and 1 mm coronal sections were cut on a sectioning block as above, starting from the cannula insertion point and moving both anteriorly and posteriorly. The first two 1 mm sections from the insertion point were removed (anterior section/posterior section) and periventricular tissue regions c ollected. Dissected regions were placed in microfuge tubes with 50 l of 2% SDS, 200 mM DTT, 20 mM Tris HCl (pH 7), phosphatase inhibitors (Sigma Aldrich; P2850; P5726; St. Louis, MO), Protease Arrest (Cambiochem/EMD; 539124; Gibbstown, NJ) heated at 100o C for 20 minutes then lowered to 80o C for 2 hours (Addis et al., 2009; Becker et al., 6


2008; Faratian et al., 2011; Shi et al., 2006). Lithium dodecyl sulfate (LDS) buffer (Invitrogen; NP0008; Carlsbad, California) was added to samples, vortexed, and st ored at 20 C until used in Western blot studies. Western I mmunoblotting Samples were thawed at room temperature and loaded into an electrophoresis chamber (Invitrogen, Midi Cell Runner WR0100, Carlsbad, California) with NuPAGE Novex 412% Bis Tris Midi gels (Invitrogen, WG1403 Carlsbad, California) and transferred (Invitrogen, Novex Semi Dry Blotter SD1000 Carlsbad, California) to a PDVF membrane (Millipore, Immobilon FL IPFL07810 Billerica, MA). The membrane was then incubated in undiluted blocking buf fer (LICOR Biosciences; 927 40000; Lincoln, NE) for 1 hour on a rotator (Stovall; The Belly Dancer Lab Shaker) at room temperature. Antibodies were added to undiluted blocking buffer and poured over the blot, then allowed to rotate for 1 hour. The spec ific antibodies employed in this study were: GSK3 mAb (rabbit; 1:1000; Cell Signaling Technology; 9315; Boston, MA), GAPDH mAb (mouse; 1:1000; Novus Biologicals; NB300 221; Littleton, CO), GAP43 pAb (rabbit; 1:1000; Cell Signaling Technology; 5307; Bos ton, MA), NeuN mAb (mouse; 1:1000; Millipore; MAB377; Billerica, MA), Doublecortin pAb (rabbit; 1:1000; Cell Signaling Technology; 4604; Boston, MA), Synaptotagmin 1 (SYT1) pAb (rabbit; 1:1000; Cell Signaling Technology; 3347; Boston, MA), Synaptophysin mAb (rabbit; 1:1000; Cell Signaling Technology; 5461; Boston, MA), AKT mAb (rabbit; 1:1000; Cell Signaling Technology; 2938; Boston, MA), p44/42 MAPK (Erk1/2) mAb (rabbit; 1:1000; Cell Signaling Technology; 4695; Boston, MA), GSK3 Ser9 mAb (rabbit; 1:1 000; Cell Signaling Technology; 9323; Boston, MA), p44/42 MAPK 7


(Erk1/2)Thr202/Tyr204 mAb (rabbit; 1:1000; Cell Signaling Technology; 4370; Boston, MA), AKTSer473 mAb (rabbit; 1:1000; Cell Signaling Technology; 4060; Boston, MA), AKTThr308 mAb (rabbit; 1:1000; Cell Signaling Technology; 2965; Boston, MA). Following two quick washes and one 20minute wash, each with TBS containing 0.05% Tween 20 (TBST), the blot was then incubated for 1 hour at room temperature with IRDye 800CW goat anti rabb it pAb antibody (1:10,000; LI COR Biosciences ; 92632211; Lincoln, NE) and/or IRDye 680LT goat anti mouse pAb antibody (1:20,000; LI COR Biosciences ; 92668020; Lincoln, NE). The blot again received two quick washes and then one 20minute wash in TBST. After washing, it was placed in cold PBS buffer (pH 7.4) until arranged on the Odyssey glass bed (LI COR Biosciences ; Odyssey Infrared Imager System; Lincoln, NE). A silicone mat was placed on top for equal laser strike between locations. Fluorescen t immunocomplexes were detected with the LI COR Odyssey Channel sensitivity was optimized for each blot. Typical laser intensity settings ranged from 3.0 to 6.0 and integrated inten sities were determined with the Odyssey system software. The GAPDH intensity of each sample was used for normalization. Statistics One way analysis of variance (ANOVA), with Scheffe multiple comparison a posteriori analyses where appropriate, were perfor med using SSPS version 19 for Windows (IBM; Somers, NY). All graphs were made using SigmaPlot 12 (Systat Software; San Jose, CA). 8


CHAPTER III RESULTS Behavior of ICV I mplanted R ats in Open Field and Elevated Plus M aze Rats with chronic administration of either saline or VPA (2 or 3 mM) were tested in both the OF and EP mazes to determine if there were any significant behavioral alterations produced by the intervention ( n=8 each group; individual animals). Testing began after 14 days of post surgery to allow for any potential VPA toxicity to elicit effects before collecting data. ANOVA for the OF/EP studies, assessing percent time spent in specific regions or arms and total distance traveled, showed no significant changes (Fig. 1.; Table 1). These data demonstrate that chronic central administration of valproic acid did not produce any detrimental behavioral effects in these rats. 9


Table 1 ANOVA results for Open Field and Elevated Plus Paradigms Paradigm Variable Region F (2,21) p Open field Elevated plus Percent time in region Distance traveled in region Percent time in arm Distance traveled in arm Outer Middle Center Outer Middle Center Closed Open Center Closed Open Center 0.754 0.783 1.000 0.197 0.538 1.000 0.836 0.708 0.318 0.408 0.090 0.004 0.483 0.470 0.385 0.822 0.592 0.385 0.447 0.504 0.731 0.670 0.914 0.996 10


Figure 1 Behavioral effects of ICV VPA administration. Male Sprague Dawley rats were implanted with an osmotic minipump delivering saline (S), 2 mM (V2) or 3 mM (V3) valproic acid directly into the right lateral ventricle. In the open field and elevated plus maze, there were no significant differences observed in distance traveled over the 5 minutes of testing, suggesting that VPA did not cause sedation in these animals, nor was there any significant effect on anxiety. Data are mean SEM; n=8; individual animals. 11


Table 2 ANOVA and Scheffe a posteriori results for Immunohistochemistry and Western blot studies Figure Region sample F (2,15) Saline vs. 2 mM VPA p Saline vs. 3 mM VPA p 2 mM vs. 3 mM VPA p Fig.2.A. Valproic acid Fig.2.B. p Akt Ser473 Right vent Right vent 43.38 62.70 <0.001* <0.001* <0.001* < 0.001* 0.773 0.391 Fig.2.C. pGSK3 Ser9 Fig.2.D p Akt Ser473 Right vent Top right Right vent 200 117 19.26 <0.001* <0.001* <0.001* <0.001* <0.001* 0.001* 0.004* 0.616 0.532 Left vent 23.56 <0.001* <0.001* 0.878 Fig.2.E. pAktTh r308 Top right Right vent Left vent 13.18 22.30 10.38 0.006* <0.001* 0.002* 0.001* 0.001* 0.024* 0.597 0.231 0.434 Fig.2.F. pErk1/2 Thr202/Tyr204 Fig.2.G. pGSK3 Ser9 Top right Right vent Left vent Top right Right vent Left vent 65.59 7.72 4.57 37.57 36.25 59.89 <0.001* 0.024* 0.105 <0.001* <0.001* <0.001* <0.001* 0.009* 0.039* <0.001* <0.001* <0.001* 0.005* 0.865 0.855 0.886 0.623 0.081 Fig.3.A. 2G13 Fig.3.B. GAP43 Right vent Top right Right vent Left vent 45.50A 102 44.59 69.72 <0.001* <0.001* <0.001* <0.001* <0.001* <0.001* <0.001* <0.001* 0.006* 0.048* 0.130 0.511 Fig.3.B. Synaptophysin Fig.3.B. Synaptotagmin Top right Right vent Left vent Top right Right vent Left vent 0.385 0.497 0.532 0.224 0.360 0.285 0.865 0.981 0.537 0.802 0.836 0.762 0.692 0.649 0.908 0.940 0.720 0.967 0.948 0.759 0.788 0.951 0.977 0.890 12


Table 2 (cont) Fig.3.B. Doublecortin (DCX) Top right Right vent Left vent 0.318 0.397 0.242 0.752 0.679 0.993 0.844 0.909 0.868 0.984 0.903 0.811 Fig.3.B. NeuN Top right Right vent Left vent 0.166 0.421 0.710 0.858 0.925 0.516 0.921 0.665 0.760 0.989 0.875 0.913 AF(2,33): Due to the method, antibody multiplexing was possible and allowed for increased sample collection of this protein. (*p<0.05) 13


Figure 2 Immunochemical results of ICV VPA administration. Results of immunohistochemistry showing valproic acid, released intracerebroventricularly (ICV) for 28 days, localized primarily to the periventricular zone (A). Verifying VPA antibody specificity was don e by terminating the cannula end directly into brain tissue, adjacent to the lateral ventricle (LV) (A). Increases in phosphoprotein presence were also detected in tissue slices of this region (B, C). Infrared Western blotting results of VPA exposed brai n regions showed increases in pro survival protein phosphorylation states (D, E, F, G) 14


when compared to saline controls. Data are mean SEM; n=6 ; individual animals (*p<0.05, **p<0.01, ***p<0.001). VPA, Phospho Proteins, and Growth Cone Associated Protei n Localization in V entricle Fixed brain slices from rats treated with VPA showed a significant immunopositive presence for the drug itself versus saline control (Fig. 2. A.; Table 2; n=6; individual animals). With placement of the cannula within the ventricular space, penetrance of VPA appeared to primarily localize in the periventricular zone and cannula tract. An increase in 2G13p, a protein associated with axon growth cone formation, was detected in this same area (Fig. 3. A.; Table 2; n=12; indiv idual animals) along with pAktSer473 and p GSK3 Ser9 (Fig. 2. B, C.; Table 2; n=6; individual animals). Phospho P rotein and T otal P rotein L evels in R egional B rain T issue L ysates Pro survival phosphoproteins were assayed for in dissected tissue locations using infrared Western blot analysis. In rats with central administration of VPA there were significant increases in pAktSer473, pAktSer308 and p GSK3 Ser9 (Fig. 2. D, E, F.; Table 2; n=6; individual animals). p Erk1/2Thr202/Tyr204 had significant increases, except in saline vs. 2 mM (left vent) (Fig. 2. G.; Table 2; n=6; individual animals). GAP43, another growth cone associated protein, showed significant increases in saline vs. 2 mM/3 mM VPA within all three regions (Fig. 3. B.; Table 2; n=6; individual animals). Synaptophysin, synaptotagmin, NeuN and doublecortin (DCX) all had nonsignificant changes in the selected areas during exposure to valproic acid (Fig. 3. B.; Table 2; n=6; individual animals). 15


Figure 3 Growth cone associated proteins after ICV VPA administration. Tissue sections and immunoblots show increased presence of growth cone associated proteins, 2G13p and GAP43, after chronic ICV infusion of valproic acid (A, B). Results, however, did not displ ay any significant change in proteins associated with synaptic vesicle populations or mature/immature neurons (B). Data are mean SEM; (A) n=12; individual animals (B) n=6 ; individual animals (*p<0.05, **p<0.01, ***p<0.001). 16


Figure 4 Drug penetrance and cases of hydrocephaly. Fig. A. While testing cannula termination sites for VPA drug release, implantation into the corpus callosum displayed expanding increases of pErk1/2Thr202/Tyr204 and the growth cone protein (2G13p) within CC tissue (infused 28 days; 2 mM). Immunocomplexes for the two targets significantly increased as expected in the right CC release site, but also had expanded into the left hemisphere, suggested primarily through CC tissue. Fig. B. Several rats that were centrally administered 3 mM VPA within the right lateral ventricle (LV) displayed significant hydrocephaly. Arrows indicate some areas with markedly widened ventricles and the cannula insertion point is indicat ed between red lines. Abbreviations: VPA, valproic acid; CC, corpus callosum; LV, lateral ventricle; TV, third ventricle; H, hippocampus. 17


CHAPTER IV DISCUSSION The lack of behavioral changes in the EP/OF paradigms suggest s that, at least at the doses tested and for the two behavioral tests performed, continuous central administration of VPA has no overt behavioral effects on anxiety nor was there evidence of sedation or behavioral activation. These studies stand in contras t to work by Serralta and colleagues (Serralta et al., 2006) where central injections of VPA produced significant ataxia and sedation, albeit the concentrations used in present studies were significantly lower than the Serralta study (288 432 g/ml versus 400 mg/ml). While the present study did not assess seizure reduction with continuous central VPA administration, the Serralta study has shown significant reduction in seizure intensity, duration and after discharge duration with continuous ICV administrat ion of VPA using the kindled rat model (Serralta et al., 2006), again with the caveat of significantly lower concentrations in the present studies. In contrast to the lack of behavioral effects, there were significant changes observed with a number of biochemical markers. Tissue penetration of VPA, following chronic ICV release was localized primarily along the periventricular zone and the cannula track where there were increases in pro survival pAktSer473, pGSK3 Ser9 and growth cone 2G13p. In addition, analysis of the lysates of the three dissected regions of VPA treated rats showed elevated levels of pro survival pAktSer473, pAktThr308, p GSK3 Ser9, pErk1/2Thr202/Tyr204, and growth cone GAP43, while there were no significant changes in DCX, NeuN, synaptotagmin, and synaptophysin. These findings suggest that delivery of VPA directly into the lateral ventricle promotes a prosurvival and progrowth cone signaling environment in the periventricular region. 18


These profiles are present in tissue surrounding t he ventricular space which contains the cannula termination point and extend to other contiguous areas of cerebrospinal fluid (e.g. left ventricular space). Specific behavior, synaptic vesicle proteins, and neuron maturation markers appear to not be affected by the VPA infusion process. Low tissue penetrance may occur, which could limit drug efficacy and may explain the failed trials for Parkinsons disease (Nutt et al., 2003; Salvatore et al., 2006). VPA has been shown to elevate pAktSer473 in neural SH SY5Y cells (Pan et al., 2005) and p AktThr308 in glial U 87 cells (Lamarre and Desrosiers, 2008). The present study sought to determine if central administration of VPA would produce effects similar to those seen in the blastoma cell lines. Since phosphor ylation of Thr308 and Ser473 are both required for complete activation of Akt (Datta et al., 1999; Kandel and Hay, 1999; Scheid and Woodgett, 2001), our data suggests that the fully activated form is present when whole brain tissue is exposed to VPA. Othe r reports have shown variable conditions leading to differential Akt phosphoprotein states (Khor et al., 2004; Kitagawa et al., 2002; Kumari et al., 2001; Obara et al., 2002; Ouyang et al., 2000; Velling et al., 2008) along with downstream prosurvival eff ects. This suggests that both Akt sites are not needed for survival (Datta et al., 1999; Kandel and Hay, 1999; Scheid and Woodgett, 2001) and supports our measurement of both phosphorylation sites. An example of phosphospecific activity is reported by G oren et al (2008), who showed that phosphorylation at Thr308 of Akt1 allows for exclusion from the nucleus with insulin exposure in keratinocytes. Akt should induce an increase in the inhibitory phosphoform, GSK3 Ser9 (Chin et al., 2005; Cross et al., 1995; Grimes and Jope, 2001), and our results are in agreement with this effect. 19


Consistent with our results, oral administration of VPA in rats has been shown to increase pErk1/2Thr202/Tyr204 (Einat et al., 2003) which is also seen with exogenous exposure in SH SY5Y cells and rat primary cortical neurons (Di Daniel et al., 2005). The phosphorylation of Erk is required for Bad inhibition (Bonni et al., 1999), Bcl 2 activation and neurite growth induced by VPA in SH SY5Y cells (Yuan et al., 2001). GAP43 is also found to be elevated during neurite formation (Yuan et al., 2001) and is important in growth cone function (Aigner and Caroni, 1993; Biewenga et al., 1996). In the regions we dissected, pErk1/2Thr202/Tyr204 and GAP43 were both significantly increased in all three areas exposed to VPA. In addition, 2G13p, a protein found strictly in growth cones (Kim et al., 2011; Maier et al., 2008; Stettler et al., 1999), is increased in periventricular tissue with chronic ICV VPA administration. Doublecortin (DCX) levels are perturbed if exposure to certain agents alters immature neuron populations (Plane et al., 2008) or if there are asymmetrical distributions of neuroblasts caused by brain tumors (Bexell et al., 2007). We assayed for any abnormal changes in tota l DCX pools that could suggest tumorigenicity (Bexell et al., 2007) or lowering of immature neuron populations (Umka et al., 2010) but found no difference between samples. Detection of NeuN was used in the same manner, as a known mature neuron marker (Soylemezoglu et al., 2003; Wolf et al., 1996, 1997), and again there was no variance between treatment groups. Synaptophysin and synaptotagmin are key components of synaptic vesicles (Iwamoto et al., 2004). Assessment of these two proteins was used to look for any increase of synaptic vesicles since our results showed an increase of pErk1/2Thr202/Tyr204, 2G13p and GAP43. Our results did not show any change of the total vesicle proteins 20


after exposure to VPA. These data might reflect stalled growth cones, or possibly that the tissue block size was excessively large, which washed out any small change in synaptic vesicle pools. Three rats exposed to the highest VPA concentration (3 mM) in this study were not reported due to marked hydrocephaly (data not shown). The dilation of both lateral ventricles produced significant compression of the adjacent tissue making it unusable for tissue imaging and dissection. VPA at 3 mM is notably viscous and various studies that lowered ependymal ciliary movement induced hy drocephalus (Banizs et al., 2005; Del Bigio, 2010; Monkkonen et al., 2007; Nakamura and Sato, 1993; NybergHansen et al., 1975). In an inherited human disease, primary ciliary dyskinesia (PCD), in which cilia move abnormally or are stationary, leads to an increased likelihood of hydrocephalus (Lee, 2011). It is possible that the inhibition of ciliary movement due to high viscosity of the 3mM VPA led to the enlarged ventricles. In a manner similar to the work of Pan et al. (2005), who used SH SY5Y cells to determine neuroprotective dosages of VPA, our whole animal model allows for the collection of data to help determine biocompatible ICV dosages. Pan et al (2005) included concentrations of 5 mM and 10 mM VPA, however the present study would suggest that these levels could be potentially hazardous for ICV administration (e.g. hydrocephalus). VPA was localized to the periventricular zone suggesting low tissue penetrance, which may be related to the low concentrations used in the present study. Alternately a study by the Cornford group (Cornford et al., 1985) demonstrated that in a peripheral administration paradigm, the transport of VPA across the BBB was asymmetrical; the 21


efflux of brainto blood exceeded the influx from bloodto brain. A study by Shen et al (1992) analyzed cortical resections of patients who had been taking oral valproate and found large variances in plasmato brain concentrations (Godin et al., 1969; Shen et al., 1992), noting that cortical levels were significantly lower than unbound/ total plasma values; VPA showed the lowest brain to blood partitioning of antiepileptic medications (Shen et al., 1992). Patient concentrations of VPA have been reported from 18 M to 262 M in cerebrospinal fluid and 39 M to 185 M in brain tissue (Vajd a et al., 1981). Thus the VPA could have been rapidly carried out of the CNS before deep tissue penetration was obtained. This also suggests that rapid diffusion of VPA from CSF to plasma could recapitulate toxic side effects of oral administration of VP A (e.g. hepatotoxicity; Fisher et al., 1991; Jurima Romet et al., 1996; Loscher et al., 1984; Sidransky and Verney, 1996). The present studies coupled with previous work (Serralta et al., 2006) suggest that central administration may be a viable approach to the treatment of intractable seizures. However, some mechanism by which the VPA could be sequestered more effectively in the CNS would be of benefit in such a central administration paradigm. Attempts to retain peripherally administered drugs within the brain have generally used the single or multistep prodrug approach (Bundgaard, 1989; Stella, 1975). A key concept of the prodrug approach is administration of a nonactive form which is metabolized to the active form upon reaching the target site (Bu ndgaard, 1989). When a prodrug undergoes enzymatic processing, a component of the molecular structure would ensure retention at the site (i.e. brain) (Anderson, 1996; Bodor, 1994; Bodor and Buchwald, 1997). An example of this locked in approach would be a conjugated phosphate remaining after 22


metabolism to the active state. Presence of the conjugated phosphate would maintain an anionic charge, thus inhibiting passage back through the BBB and retention within the brain (Somogyi et al., 1998a, 1998b). Si nce valproic acid has an established efficacy for reducing the incidence of seizures in patients (Chayasirisobhon and Russell, 1983; Gal et al., 1988), applying lockin concepts to the VPA molecule with ICV administration could produce increased therapeu tic potential. While valproic acid has a known ability to help manage intractable seizures (Chayasirisobhon and Russell, 1983; Gal et al., 1988) there is an issue with variability in patient brain and blood plasma levels (Shen et al., 1992) possibly due t o the asymmetrical movement of VPA across the blood brain barrier (Cornford et al., 1985). Administering VPA intracerebrally in an altered form (i.e. PEGylated) to increase charge and size of the molecule could potentially lead to a more stable and increased level. With this method, a higher concentration within the CSF could possibly be achieved using less drug and yielding significantly decreased blood plasma levels thus reducing the potential for peripheral organ related side effects (DeWolfe et al., 2 009; Fisher et al., 1991; Jurima Romet et al., 1996; Loscher et al., 1984; Sidransky and Verney, 1996). Thus, there is potential for the use of a centrally administered redesigned VPA molecule to allow for improved seizure control in refractory patients a nd thus a better quality of life. 23


CHAPTER V CONCLUSION In an animal model, we have shown chronic, centrally administered VPA did not alter behavior, but increased levels of both cell survival phosphoproteins and proteins associated with growth cones. These data suggest that, contrary to the cell death often associated with seizures, central administration of VPA may increase cell survival. The data also suggest that central administration may be a viable route for exploration of efficacy in seizure reduction model to develop this route of administration for potential use in cases of intractable seizures. 24


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