Effects of Nerve Growth Factor Infusion on Behavioral Recovery and Graft Survival Following Intraventricular Adrenal Medulla Grafts in the Unilateral 6-Hydroxydopamine Lesioned Rat

(C)Freund Publishing House Ltd., 1994 Effects of Nerve Growth Factor Infusion on Behavioral Recovery and Graft Survival Following Intraventricular Adrenal Medulla Grafts in the Unilateral 6-Hydroxydopamine Lesioned Rat Valerie K. Bergdall and Jill B. Becker2 1Unitfor Laboratory Animal Medicine and 2Neuroscience Program, Department of Psychology, and Reproductive Sciences Program, The University ofMichigan, Ann Arbor, M1 48104, USA SUMMARY NGF was infused into the lateral ventricle of rats with unilateral 6-OHDA lesions of the substantia nigra along with adrenal medulla or control grafts. Treatment effectiveness, as measured by amphetamine-induced turning behavior, indicated that there were no significant differences between treated and control groups in spite of the survival of tyrosine hydroxylase (TH) immunoreactive grafts. Furthermore, adrenal medulla graft survival was not dependent on NGF infusion. These results indicate that TH-positive graft survival is not correlated with behavioral recovery as assessed by amphetamine-induced turning. These results differ from studies which utilized apomorphineinduced turning as a measure of behavioral recovery. We propose that adrenal medulla graft survival alone is not sufficient to promote behavioral recovery in the 6-OHDA lesioned rat. KEY WORDS NGF, adrenal medulla, Parkinson’s disease, tyrosine hydroxylase, transplantation, rat INTRODUCTION Transplantation of adrenal medulla tissue into the lateral ventricle of the hemiparkinsonian rat has been reported to result in decreased rotational behavior induced by apomorphine /5,10,14/ or amphetamine /1/. The decreased amphetamineinduced rotational behavior (but not apomorphineinduced turning) is correlated with enhanced striatal extracellular dopamine, decreased D dopamine receptor binding, and an impaired blood-brain barrier at the site of the graft /4/. The precise mechanisms mediating these effects, however, are not well understood, as it is not clear that restoration of extracellular dopamine is either necessary or sufficient to promote behavioral recovery of function. For example, there may be trophic effects of adrenal medulla grafts on the surrounding host tissue that produce behavioral recovery. Conversely, induction of an inflammatory response has been shown to produce a non-specific injury-induced trophic effect on the surrounding striatum/6/. Nerve growth factor (NGF) delivered to the lateral ventricle or striatum has been reported to enhance survival of grafted chromaffin cells/14/and promote behavioral recovery as assessed by apomorphine-induced rotational behavior/10/. The purpose of the study reported here was to determine whether continuous intraventricular infusion of NGF in rats with unilateral 6-hydroxydopamine (OHDA) lesions of the substantia nigra would enhance behavioral recovery in adrenal medulla grafted animals, and to determine whether this is correlated with tyrosine hydroxylase (TH) immunoreactivity in the graft and/or host brain. METHODS AND MATERIALS Reprint address: Dr. J.B. Becker 3074B Neuroscience Bldg. 1103 E. Huron Street Ann Arbor, MI 48109-1687, USA VOLUME 5, NO. 3, 1994 Animals Adult female Long-Evans rats (Charles River, Indianapolis, IN) were tested with D-amphetamine 163 164 V.K.BERGDALL AND J.B. BECKER sulfate (0.85 mg/kg, i.p.) for direction of rotational bias/12/. The substantia nigra and medial forebrain bundle on the side contralateral to the preferred direction of turning was then lesioned using 6OHDA.HBr at a concentration of 2 lag/gl in saline with 0.01% ascorbic acid. This was infused at a rate of 0.5 gl/min at the following coordinates (from bregma, skull flat): posterior 5.0 mm, lateral 2.0 mm, ventral 7.4 mm (8 min); and posterior 3.1 mm, lateral 1o 8 mm, and ventral 8.2 mm (4 min). Animals were allowed to recover for 2 weeks, then tested with amphetamine (0.85 mg/kg i.p., 60 min) and with apomorphine (0.25 mg/kg, s.c., 45 min). Test drugs were never given more than once every 48 hours. Rats which exhibited more than 50 turns per hour following amphetamine, and more than 100 turns per 45 min following apomorphine were continued in the study. This criterion results in the selection of animals with >95% depletion of dopamine in the lesioned striatum/4/. The animals then received daily injections of amphetamine (3 mg/kg, i.p.) every weekday for 2 weeks to sensitize animals to the psychomotor stimulant effects of amphetamine. The sensitization process helps to alleviate the increased rotation rate observed with repeated exposures to amphetamine/13/. Following the sensitization period, the rats were withdrawn from amphetamine for 2 weeks before undergoing pregraff behavioral testing. The behavioral testing consisted of four tests performed at least 48 hours apart. Each test consisted of a 15 minute habituation period followed by a 2 h period with amphetamine (3 mg/kg, i.p.) in an automated rotometer (flat-bottom, cylindrical). The mean number of 360 turns induced by amphetamine was then used as the baseline rate for each animal. Graft and cannulae placement Following the pregraft behavioral testing, the animals were anesthetized with sodium pentobarbital (30 mg/kg, i.p.) and received gratts of adrenal medulla or adrenal cortex from 1 month-old rat pups as described previously/3/at coordinates anterior 1.0 mm, lateral 1.2 mm, ventral-5.0 mm. Once the graft was in place, a brain infusion cannula (Alzet) was lowered at the same coordinates and secured in place with dental acrylic cement. The cannula was attached to a subcutaneous 200 l.tl osmotic minipump (Alzet #2002) filled with nerve growth factor (0.5 lag/lal, Harlan Biologics, Madison, Wisconsin)/8/or cytochrome c (CytoC) (0.5 gg/gl, Sigma) with an infusion rate of 0.5 gl/h. Animals were assigned to the following groups: adrenal medulla and NGF (n=10) (AMJNGF), adrenal medulla and CytoC (n=7) (AM/CytoC), adrenal cortex and NGF (n=9) (AC/NGF), and NGF only (n=7). The minipumps were changed every two weeks under metofane anesthesia for a total delivery time of 6 weeks. Behavioral testing Animals were tested in the automated rotometer at 4-5 and 8-9 weeks post graft as described for the pregraff period. The mean of each session was then used to calculate the percent change in rotational behavior [(postgraft-pregraff/pregraff) X 100%] at 4 and 8 weeks postgraff. Histologic analysis Half the animals were euthanized with an overdose of sodium pentobarbital and perfused with 4% paraformaldehyde at 6 weeks postgraff. The remaining animals were similarly euthanized at 10 weeks postgraff. Brains were immediately removed and placed in 4% paraformaldehyde overnight, then transferred into a 20% sucrose phosphate buffer for no more than 5 days. Thirty micron sagittal sections were cut on a freezing microtome and stored in 0.2% sodium azide buffer until stained. Every fourth section was mounted on chrom-alum-subbed slides and stained with Meyer’s hematoxylin and eosin. Imm (...truncated)