H. Lee Vahlsing

A phase 1 clinical trial of nerve growth factor gene therapy for Alzheimer disease

Cholinergic neuron loss is a cardinal feature of Alzheimer disease. Nerve growth factor (NGF) stimulates cholinergic function, improves memory and prevents cholinergic degeneration in animal models of injury, amyloid overexpression and aging. We performed a phase 1 trial of ex vivo NGF gene delivery in eight individuals with mild Alzheimer disease, implanting autologous fibroblasts genetically modified to express human NGF into the forebrain. After mean follow-up of 22 months in six subjects, no long-term adverse effects of NGF occurred. Evaluation of the Mini-Mental Status Examination and Alzheimer Disease Assessment Scale-Cognitive subcomponent suggested improvement in the rate of cognitive decline. Serial PET scans showed significant (P < 0.05) increases in cortical 18-fluorodeoxyglucose after treatment. Brain autopsy from one subject suggested robust growth responses to NGF. Additional clinical trials of NGF for Alzheimer disease are warranted.

Delayed treatment with nerve growth factor reverses the apparent loss of cholinergic neurons after acute brain damage

Previous studies have shown that the loss after brain injury of adult rat septal cholinergic neurons whose axons are transected can be prevented by immediate intraventricular nerve growth factor (NGF) administration. This loss of axotomized neurons may be due to a reduction in detectability of neurotransmitter-related enzyme rather than to neuronal death. Here we report that NGF treatment, started after most of the neurons were no longer detectable (i.e., 1, 2, and 3 weeks), induced a dramatic reappearance of the apparently lost cholinergic neurons. These results may have important implications for potential trophic factor treatments of CNS trauma and neurodegenerative diseases, such as Alzheimers dementia, which are characterized by chronic and progressive losses in the function of specific sets of neurons.

Nerve growth factor (NGF) reverses axotomy-induced decreases in choline acetyltransferase, NGF receptor and size of medial septum cholinergic neurons

Intraventricular nerve growth factor (NGF) infusion in the adult rat can prevent and also, if delayed, reverse the disappearance of most of the axotomized medial septum cholinergic neurons immunostained for choline acetyltransferase (ChAT). We have utilized the delayed NGF treatment protocol to (i) extend to 3 months the delay time between axotomy and NGF treatment, (ii) define the time course of their recovery, (iii) determine that immunostaining for the (lower affinity) NGF receptor (NGFR) parallels loss and reversal of the ChAT marker, and (iv) evaluate changes in cholinergic somal size following axotomy and subsequent NGF treatment. While NGF treatments starting only 7 days after the fimbria-fornix transection (axotomy) almost entirely restored the number of both ChAT- and NGFR-positive medial septum neurons, longer delayed (2-3 weeks) treatment brought about recovery from the baseline of 20-25% to only about 70% of the control numbers. This limited recoverability, however, persisted even after a 95 day delay period. In all cases examined maximal recoveries were achieved within 3-7 days of NGF treatment. Neuronal size analyses provided evidence for an axotomy-induced atrophy. NGF treatments, started with 1 or 2 week delays, not only reversed fully the average somal size loss but also induced an actual hypertrophy of several of those neurons. These results provide additional evidence that at least half of the apparent loss of cholinergic medial septum neurons upon axotomy is due to a loss of markers such as the transmitter-related enzyme ChAT and NGFR rather than to actual neuronal cell death. These results also show that NGF exerts a genuine trophic influence by regulating the size of its target neurons as well as their content of several proteins.

A small-gauge cannula device for continuous infusion of exogenous agents into the brain

A method is described for the construction of an intraventricular or intraparenchymal cannula device, which when connected to an Alzet osmotic pump, can be used for the continuous infusion of experimental solutions into the brain. A 33-gauge, stainless-steel cannula is encased within a dental acrylic stabilization platform prior to stereotaxic implantation, and after implantation, the platform is glued to the animals skull using cyanoacrylate adhesive. This procedure provides for the long-term stability (at least 4 weeks) of the small-gauge cannula without the need for additional stabilization skull screws, thus minimizing damage to surrounding tissues by the cannula and postsurgical trauma to the animal. Using the stock model 2002 Alzet pump to infuse artificial cerebral spinal fluid at a flow rate equal to 0.5 microliter/h, an inflammatory tissue reaction around the cannula tip was consistently found after 2 weeks of continuous intraparenchymal infusion. However, the inflammatory reaction could be significantly reduced or eliminated by decreasing the flow rate to approximately 0.25 microliter/h, using a modified Alzet pump. Alternatively, the stock 0.5 microliter/h pump could be used without causing parenchymal damage if the cannula tip was implanted into the lateral ventricle.

Nerve growth factor promotes CNS cholinergic axonal regeneration into acellular peripheral nerve grafts

Peripheral nerve grafts promote vigorous regeneration of adult mammalian CNS axons. Elimination of nerve-associated cells by freeze-thawing abolishes this promoting quality, possibly by creating inhibitory cellular debris and/or destroying the production of stimulatory factors by living Schwann or other cells. Here, debris-free acellular peripheral nerve segments placed between the disconnected septum and the hippocampal formation acquired almost no cholinergic axons after 1 month. However, such acellular nerve grafts treated before implantation with purified beta-nerve growth factor (NGF) contained nearly as many longitudinally oriented cholinergic axons as did fresh cellular nerve grafts. These results suggest that (i) NGF is required for the regeneration of adult CNS cholinergic axons into nerve grafts and (ii) an important function of living cells within peripheral nerve may be the production of neuronotrophic factors such as NGF.

Septohippocampal cholinergic axonal regeneration through peripheral nerve bridges: Quantification and temporal development

Axons of the adult mammalian CNS have been shown to regrow vigorously into peripheral nerve grafts. Using a cholinergic septohippocampal model for adult CNS regeneration, involving complete denervation of the hippocampal formation from its basal forebrain cholinergic afferents, this study has established quantitative parameters and a temporal baseline of cholinergic fiber regeneration into the dorsal hippocampal tissue through a peripheral sciatic nerve graft. In nerve-implanted animals (i) the nerve grafts are maximally invaded by AChE-positive fibers between 2 weeks and 1 month postlesion, (ii) the fibers entering the hippocampal formation from the graft show a peak numerical increase and rate of elongation around the first month and/or in the proximal hippocampal region, (iii) an apparently normal innervation pattern and fiber density in the most rostral 1.5 mm of the dorsal hippocampal formation is reached by 6 months postlesion. The present study provides a basis for future quantitative comparisons of manipulations of different components of the system, e.g., the contributing neurons, the bridging material, and the receiving central nervous tissue. The temporal/spatial pattern of fiber regeneration suggests that the hippocampal CNS tissue can be a good axonal growth-promoting environment, albeit with temporal and/or spatial limitations, and is therefore not an immutably restrictive environment for axonal regeneration.

Retrograde transport in corticospinal neurons after spinal cord transection

Complete spinal cord transection at T-6/T-7 in rats caused a decrease in the number of surviving corticospinal neurons. Cell death began 5 and 10 weeks after cord injury. The number of surviving cells decreased progressively for at least 25 weeks after injury. Surviving cells were identified by their ability to transport horseradish peroxidase (HRP) retrograde from a T-1/T-2 insertion site to cortical cell somas. Therapy aimed at promoting corticospinal tract regeneration must be started early after spinal cord injury.

Labeled corticospinal neurons one year after spinal cord transection

One year after a T9 spinal cord transection, horseradish peroxidase was inserted into the spinal cord at T3-T4. Only about 7% of the number of corticospinal neurons labeled in control rats were labeled in exactly matched transected rats. This long-term loss of labeled neurons makes cell death the most likely explanation for the failure to identify corticospinal neurons in spinal-cord-transected rats.

A two-compartment modification of the silicone chamber model for nerve regeneration

In the nerve regeneration silicone chamber model, the regenerate which forms across a 10-mm gap between proximal and distal nerve stumps is a monofascicular structure with an outer perineurial-like cell sheath. Recent work has provided indications that the geometry of the regenerate within a silicone chamber can be altered by experimental modifications of the chamber matrix. In the present study we modified the standard silicone chamber into a two-compartment chamber by inserting a 6- or 10-mm-long siliconized nitrocellulose strip in order to obtain two separate regenerates. Light microscopy 16 days after implantation revealed that two separate nerve structures had formed, one on each side of the nitrocellulose partition and adjacent to it, and each with its own perineurial-like cell sheath. In chambers with 6-mm-long strips a monofascicular regenerate started from the proximal stump and divided into two separate structures as it approached the proximal end of the strip: the two fascicles joined again into a monofascicular structure in the distal portion of the chambers. The new two-compartment silicone chamber model appears suitable for future examinations of experimental fasciculation. In addition, the nitrocellulose partition should allow one to study specific effects of growth factors on axonal regeneration in vivo, as growth factors bind strongly to untreated nitrocellulose while retaining their biological activity.

Cell Death in Clarke's Column after Spinal Cord Transection

The death of embryonic central nervous system (CNS) neurons deprived of a target is well established. In adult rats, similar cell death of corticospinal and rubrospinal motor neurons occurs as a delayed response to spinal cord transection. We document the loss of neurons in Clarkes column, secondary ascending spino-cerebellar neurons in adult rats, after complete spinal cord transection at T-9. Twenty-five weeks after spinal cord transection, horseradish peroxidase (HRP) studies showed a dramatic loss of labeled cells in rats with transected spinal cords as compared to matched control rats. Cresyl echt violet-stained sections failed to support the hypothesis that unlabeled cells persist in a shrunken, inactive state; instead we found far fewer identifiable neurons in Clarkes column. Although we saw little gliosis in the area of cell loss, gliosis was evident in the adjacent corticospinal tract which was severed in the original surgical injury. Amputation of the right hind limb resulted in a paradoxical increase in labeled Clarkes column cells on the right. Total cells stained with cresyl echt violet in amputated animals were not different from right to left. The increase in labeled cells on the amputated side may have been caused by an increase in metabolic activity of these deafferentated neurons which resulted in more effective axoplasmic transport of the HRP label.