Intraparenchymal nerve growth factor improves behavioral deficits while minimizing the adverse effects of intracerebroventricular delivery.

Nerve growth factor (NGF) delivered via intracerebroventricular (ICV) infusion restores behavioral and biochemical deficits in animal models of cholinergic hypofunction. However, ICV infusion of NGF induces an array of adverse events including weight loss, thermal hyperalgesia, and Schwann cell hyperplasia. We compared ICV administration with three different doses of intraparenchymally delivered NGF with cytochrome C infusion serving as a control. The goal of the study was to determine whether direct infusion of NGF would result in a more restricted topographical distribution of NGF leading to a reduction or elimination of the adverse events while still augmenting cholinergic functioning sufficiently to restore spatial mnemonic processing. Subsequent to bilateral ibotenic acid lesions of the nucleus basalis magnocellularis (NBM), NGF was delivered into the lateral ventricle or adjacent to the NBM for 11 weeks. Ibotenic acid lesions resulted in reductions in choline acetyltransferase (ChAT) activity in the cortex. The highest and medium dose of NGF led to significant restoration in ChAT activity on par with ICV infusion. The lowest dose was ineffective in altering ChAT activity in any region assayed. Similarly, the two highest doses did not alter weight gain, but ICV-NGF led to a significant weight loss. Intraparenchymal infusion resulted in a dose-dependent attenuation of the development of thermal hyperalgesia. However, the highest dose of intraparenchymal NGF induced Schwann cell hyperplasia at the level of the medulla and upper cervical spinal cord. ICV-NGF was able to completely restore spatial learning and memory as predicted while only the highest intraparenchymal dose was able to able to restore the mnemonic deficits. These data suggest that intraparenchymal infusion of growth factors may provide a viable delivery method in clinical trials using this mode of drug delivery once an optimal dose has been established.

Intraparenchymal infusions of 192 IgG-saporin: development of a method for selective and discrete lesioning of cholinergic basal forebrain nuclei.

The immunotoxin 192 IgG-saporin has a high degree of selectivity for cholinergic neurons within the basal forebrain (CBF). Intracerebroventricular delivery of 192 IgG-saporin results in a diffuse and massive depletion of choline acetyltransferase (ChAT) activity in projections of the CBF, and non-selective loss of Purkinje cells. To dissociate the basal-cortical and septo-hippocampal cholinergic systems and to minimize non-specific effects, we developed intraparenchymal parameters to deliver 192 IgG-saporin discretely to either the nucleus basalis magnocellularis (NBM) or the medial septum (MS). Intraparenchymal administration of the immunotoxin into the NBM or MS resulted in a dose-dependent depletion of ChAT activity in the corresponding projection areas and a concomitant loss of ChAT immunoreactive neurons in both nuclei. Both lesions were regionally restricted, having a minimal diffusion into adjacent CBF nuclei. Control infusions did not result in non-specific parenchymal damage. In addition, immunotoxic infusions had no effect on monoamine neurotransmitter systems. By optimizing the dosages for both CBF nuclei, we maximized ChAT depletion while minimizing diffusion into the adjacent CBF nuclei. This study delineated injection parameters enabling a selective dissociation of two cholinergic subpopulations in the basal forebrain for further functional characterization.

Role of the cholinergic system in the regulation of neurotrophin synthesis.

Nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF) and neurotrophin-3 (NT-3) are members of the family of neurotrophins that are highly expressed in the adult hippocampus, and to a lesser extent, in the cerebral cortex and olfactory bulb. Since neuronal expression of neutrophins is controlled by some neurotransmitters and there is a topographical correlation between neurotrophin expression and cholinergic terminal distribution from the cholinergic basal forebrain (CBF) neurons in these areas, the question arises as to whether the cholinergic system can also regulate neurotrophin gene expression in the CNS. When CBF neurons were selectively and completely destroyed by intraventricular injection of 192 IgG-saporin, resulting in a cholinergic deafferentation of the hippocampus, cortex, and olfactory bulb, there were no significant changes in NGF, BDNF and/or NT-3 mRNA levels in these areas from 1 week to 5 months after the lesion. These results suggest that afferents from CBF neurons may not play a significant role in maintaining basal levels of neurotrophin gene expression in the adult rat brain under physiological conditions. However, potential cholinergic regulation of brain neurontrophin expression may occur under other circumstances.

NGF-producing transfected 3T3 cells: behavioral and histological assessment of transplants in nigral lesioned rats.

The rodent fibroblast clonal cell line, 3T3, was retrovirally transfected with the rat nerve growth factor (NGF) gene and selected for NGF synthesis. This study tested the hypothesis that transplanted 3T3 cells, transfected to secrete nerve growth factor (3T3NGF+), change motor behavioral indices created by striatal denervation in a dose-dependent fashion. 3T3NGF+ cells were transplanted into the lateral ventricle of rats following ipsilateral lesions of the substantia nigra pars compacta by stereotaxic injections of 6-hydroxydopamine (10 micrograms), an established lesion model. Control groups included vehicle injections and transplanted untransfected cells. The extent of the lesions was measured by determining rotational behavior before and two weeks after transplantation. Immediately prior to transplantation, cells were incubated with the fluorescent dye marker, Dil. To assess cell viability, whole brains were cryosectioned and examined for Dil-labeled 3T3 cells using fluorescent microscopy. The number of Dil-labeled profiles in five animals per group were counted in at least five noncontiguous sections per animal. From these data a statistically derived estimate of viable, transplanted 3T3 cells was obtained. The number of surviving transplanted cells correlated with the behavioral changes measured. The 3T3NGF+ transplants reduced rotational behavior, while control 3T3 transplants exacerbated rotational behavior. Thus, while NGF delivery was found to be beneficial, it was apparent that naive 3T3 had detrimental effects. These results underscore the importance of making dose-response measurements when attempting transplant-based modifications of CNS behavior.

Migration and differentiation of PC12 cells transplanted into the rat spinal cord.

To test the hypothesis that transplanted neuronal or neuronal-like cell lines, grown in vitro, might survive and differentiate in the mammalian spinal grey matter, adult male Sprague-Dawley rats (N = 5) were injected with a suspension of between 3 x 10(5) and 1.0 x 10(6) DiI labeled, undifferentiated rat pheochromocytoma (PC12) cells in sterile phosphate buffered saline. The PC12 cell line was chosen since, in certain in vitro conditions, this cell line serves as a model of neuronal differentiation, which includes the ability to conduct action potentials and form functional synapses. After a survival time of 7 or 8 days, the spinal cords were removed, cryosectioned longitudinally and examined for detection of DiI labeled PC12 cells using fluorescent microscopy. The number of DiI labeled profiles and the proportions of the DiI cells which were differentiated were counted per section in at least five non-contiguous sections per animal. Differentiation was defined as cells with neurite-like extension which exceeded twice the soma diameter. Results demonstrated the following: (1) from 2 to 15% of the transplanted PC12 cells survived; (2) migration within the spinal grey matter occurred since PC12 cells were found as much as 510 microns away from the injection site; (3) of the surviving PC12 cell population, a proportion of between 60 and 80% were differentiated, many with two or more neurite-like processes, in all of the rats; (4) no mitotic profiles were observed in DiI labelled cells; (5) undifferentiated PC12 cells were juxtaposed to the lumens of small blood vessels or within the lesion cavity. Although the specific factors remain to be elucidated, the observed PC12 migration and differentiation within the host spinal grey matter appears to be controlled by factors in the microenvironment. These data support the use of a homogeneous in vitro population of neuronal or neuronal-like cells, which are readily accessible to transfection with the appropriate genes, as transplant sources for the injured spinal cord.

Effects of nerve growth factor and acetyl-L-carnitine arginyl amide on the human neuronal line HCN-1A.

The HCN-1A clonal cell line, derived from the cortical tissue of a patient with unilateral megencephaly, was shown to differentiate into a mature neuronal-like state in the presence of the nerve growth factor, dibutyryl cyclic adenosine, 3',5'-monophosphate and either 1-isobutyl-3-methylxanthine or forskolin. Differentiation was assessed by measuring the percentage of cells that displayed branched, varicose processes that stained for synaptophysin. Treatment of cultures with a cocktail containing forskolin increased immunocytochemical staining for gamma aminobutyric (GABA), neurofilament protein and the nerve growth factor receptor species p75NGFR. Treatment with acetyl-L-carnitine alone had some effects on the cell morphology while acetyl-L-carnitine arginyl amide and nerve growth factor together increased the GABA content. Positive staining levels for the neurotransmitters gamma aminobutyric acid, glutamate, somatostatin, cholecystokinin and vasoactive intestinal polypeptide were measured quantitatively for HCN-1A under basal conditions.