Primate Studies

The ability of cellularly delivered trophic factors to preserve neurons within the striatum in a rodent model of HD led to similar studies in nonhuman primates, a step that is crucial to the initiation of clinical trials. A paradigm similar to the one employed in the rodent studies was used in nonhuman primates (99). Polymer capsules containing CNTF-producing cells were grafted into the striatum of rhesus monkeys. One week later, a QA injection was placed into the putamen and caudate proximal to the capsule implants. As seen in the rodent studies, the volume of striatal damage was decreased, and both GABAergic and cholinergic neurons destined to degenerate were spared in CNTF-grafted animals. Although all animals had significant lesions, there was a 3- and 7-fold increase in GABAergic neurons in the caudate and putamen, respectively,

Figure 10 Sprouting of cholinergic fibers in young (a,b) and aged (c,d) NGF-treated monkeys. Low- (a,c) and high-power (b,d) photomicrographs of NGF receptor immunostained sections, illustrating a dense plexus of cholinergic fibers on the side of NGF treatment (arrows) adjacent to the lateral ventricle (LV). Scale bar in a is 1000 um for a and c; scale bar in d is 50 um for b and d.

Figure 10 Sprouting of cholinergic fibers in young (a,b) and aged (c,d) NGF-treated monkeys. Low- (a,c) and high-power (b,d) photomicrographs of NGF receptor immunostained sections, illustrating a dense plexus of cholinergic fibers on the side of NGF treatment (arrows) adjacent to the lateral ventricle (LV). Scale bar in a is 1000 um for a and c; scale bar in d is 50 um for b and d.

in CNTF-grafted animals relative to controls. Similarly, there was a 2.5- and 4-fold increase in cholinergic neurons in the caudate and putamen, respectively, in CNTF-grafted animals (Fig. 12).

The ability to preserve GABAergic neurons in animals models of HD is an important, although not entirely sufficient, step to develop a useful therapeutic. If the perikarya are preserved without sustaining their innervation, then the experimental therapeutic strategy under investigation is not likely to yield significant value. The striatum is a central station in series of loop circuits that receives inputs from all the neocor-tex, projecting to a number of subcortical sites, and then returns information flow to the cerebral cortex. One critical part of this circuitry is the GABA-ergic projections to the globus pallidus and substantia nigra pars reticulata, the parts of the direct and indirect basal ganglia loop circuits. One approach to examining the integrity of this circuit is to use an antibody that recognizes GABA-ergic terminals (DARPP-32) to determine if the preservation of GABA-ergic somata within the striatum also results in the preservation of the axons of these neurons to critical extrastriatal sites. Using quantitative morphological assessment of DARPP-32 optical density, it has been shown that monkeys receiving QA lesions have significant reductions in DARPP-32 immunoreactivity within the globus pallidus and substantia nigra (Fig. 13). The lesion-induced decrease in GABA-ergic innervation for both of these regions was prevented in CNTF-grafted monkeys demonstrating that this treatment strategy protected GABA-ergic neurons destined to die following excitotoxic lesion, as well as sustained the normal projection systems from this critical population of neurons (99).

The intrinsic striatal cytoarchitecture can be preserved in monkeys by CNTF grafts, and once exposed to these grafts, the cells apparently maintain their projections. But are afferents to the striatum, specifically from the cerebral cortex, also influenced by these grafts? This may be particularly critical if some of the more devastating nonmotor symptoms seen in HD result from cortical changes secondary to striatal degeneration. Because layer V neurons from motor cortex send a dense projection to the postcommissural putamen, a region that was severely impacted by the QA lesion, the effects of QA lesions and CNTF implants on the number and size of cortical neurons in this region were examined. Although the QA lesion did not effect the number of neurons in this cortical area, layer V neurons were significantly reduced in cross-sectional area on the side ipsilateral to the lesion in control-grafted monkeys (Fig. 14). This atrophy of cortical neurons was virtually completely reversed by CNTF grafts (99).

A recent set of studies using CNTF-producing cells in 3NP-treated monkeys have replicated and extended these results (100). Following 10 weeks of 3NP treatment, monkeys displayed pronounced chorea and severe deficits in frontal lobe cognitive performance as assessed by the object retrieval detour test. Following implantation of CNTF-producing cells, a progressive and significant recovery of motor and cognitive recovery occurred. Histological analysis demonstrated that

Figure 11 (a) Expression vector containing the CNTF gene. (b) CNTF levels, as determined by ELISA, in encapsulated BHK cells immediately prior to implantation (left) and immediately following retrieval from rodent lateral ventricle 70 days following implantation (right). (c) Implants of encapsulated CNTF producing cells reduce apomorphine rotations in rats after unilateral striatal injection of quinolinic acid.

Figure 11 (a) Expression vector containing the CNTF gene. (b) CNTF levels, as determined by ELISA, in encapsulated BHK cells immediately prior to implantation (left) and immediately following retrieval from rodent lateral ventricle 70 days following implantation (right). (c) Implants of encapsulated CNTF producing cells reduce apomorphine rotations in rats after unilateral striatal injection of quinolinic acid.

CNTF was neuroprotective and spared NeuN and calbindin-positive cells in the caudate and putamen.

Although the sparing of striatal neurons and maintanence of intrinsic circuitry is impressive, the magnitude of the effect is less than that seen in rodents. In primates, robust protection is limited to the area of the capsules. However, the area of the lesion remains extensive, and it is likely that diffusion of CNTF from the capsule may not be sufficient to protect more distant striatal regions undergoing degeneration. This concept is supported by a recent experiment that examined the effects of intraventricular grafts of encapsulated CNTF grafts in the nonhuman primate model of HD (101). In contrast to when the capsules were placed directly within brain parenchyma, intraventricular placements failed to engender neuroprotection for any striatal cell types; again suggesting that diffusion is a key factor in the efficacy of this experimental therapeutic strategy. The complete lack of neuroprotection provided by intraventricular implants in primates should be considered more carefully in the current clinical trials being conducted in which encapsulated cells are being placed into the lateral ventricles of HD patients (102,103). If human trials are to yield clinically relevant positive effects, the means of CNTF delivery used in these studies needs to be improved. Whether this entails grafting more capsules, enhancing the CNTF delivery from the cells by changing the vector system or cell type employed, or changing the characteristics of the polymer membrane remains to be determined.

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