L III

Figure 17 Intracranial administration of NGF-loaded, PLGA-based microparticles in rats: (A) Microscopic picture of the septal area 2 weeks upon microparticle injection, NGF immunostaining (scale bar = 500 ^.m); (B) adjacent section stained with AchE histochemistry (* = microparticles). Note the apparently healthy neurons in contact with the microspheres (scale bar = 200 ^m); (C) percentage of surviving cholinergic neurons stained by AchE histochemistry (compared to the contralateral, intact side) after 2 and 6 weeks upon microparticle administration (drug-free or NGF-loaded systems as indicated in the figure; a control group without microparticle injection is included for reasons of comparison) Abbreviations: HDBV, horizontal limb of the diagonal band of Broca; MS, medial septal nucleus; VDBD, vertical limb of the diagonal band of Broca. Source: From Ref. 79.

specific astro- and micro-glial reaction around the microspheres, which was similar for drug-loaded and drug-free devices. Importantly, no neuronal toxicity was observed and healthy appearing neurons were visible that were in direct contact with the microparticles. In the non-treated control group, the percentage of axotomized surviving neurons (compared to the contralateral, intact side) was 31(±2)% and 27(±1)% at t = 2 and 6 weeks, respectively (Fig. 17C, white bars). Drug-free microparticles exhibited neither protective nor toxic effects for the neurons: The percentage of surviving neurons after 2 and 6 weeks was equal to 40(±9)% and 39(±6)%, respectively (gray bars in Fig. 17C). Importantly, the NGF-loaded, controlled release microparticles could significantly increase the survival rate of the neurons: up to 66(±9)% and 61(±5)% after 2 and 6 weeks, respectively (Fig. 17C, black bars). Thus, this type of biodegradable microparticles is able to release sufficient amounts of bioactive NGF in a time-controlled manner into the brain tissue to limit the lesion-induced disappearance of cholinergic neurons.

Recently, Jollivet et al. (83,84) proposed GDNF-loaded, PLGA-based microparticles for the treatment of Parkinson's Disease. The particles release the neurotrophic factor during at least 2 months in vivo and are well tolerated upon intracranial administration into rat brain. Importantly, they were able to stimulate the axonal regeneration of mesencephalic dopami-nergic neurons in "Parkinsonian rats" (the animals received two injections of 10 ^g 6-hydroxydopamine inducing a partial progressive and retrograde lesion of the nigrostriatal system). The administration of the GDNF-loaded microparticles led to sprouting of the preserved doperminergic fibers with synaptogenesis. As it can be seen in Figure 18, this neural regeneration was accompanied by a functional improvements of the rats. The amphetamine-induced rotational behavior was measured 1, 4, 6, 8, and 10 weeks after the lesion, the microparticles were administered 2 weeks after the lesion. For reasons of comparison, also placebo microparticles were administered and an untreated animal group included as control. Clearly, all animals in this study were sucessfully lesioned (results obtained after 1 week, Fig. 18). Importantly, the number of ipsiversive turns per minute (tpm) increased in the non-treated and placebo group: from 10.7(±2.2) to 15.4(±2.3) tpm and from 11.6(±2) to 20.4(±2.2) tpm, respectively (white and gray bars). In contrast, the number of ipsiversive tpm significantly decreased in the rats that received GDNF-loaded microparticles: from 15.4(±0.9) to 8.8(±2.1) tpm (Fig. 18, black bars).

A further promising approach to treat neurodegenerative diseases is to transplant living cells into the human brain which continuously produce biologically active agents, e.g. missing neurotransmitters (such as dopamine in the case of Parkinson's Disease) ("cell therapy"). Unfortunately, so far the success of this type of advanced treatment method is limited due to the: (1) low-survival rate of the transplanted cells within the brain tissue; and (2)

co 25

1=1 Blank microspheres

I GDNF-loaded microspheres

1=1 Blank microspheres

I GDNF-loaded microspheres

Lesion treatment (2 weeks)

Figure 18 Effects of GDNF-loaded, PLGA-based microparticles on the behavior of "Parkinsonian rats" (the animals received two injections of 10 ^g 6-hydroxydopamine inducing a partial progressive and retrograde lesion of the nigrostriatal system): Number of ipsiversive turns per minute (tpm) in the amphetamine-induced rotation test at 1, 4, 6, 8, and 10 weeks after the lesion. GDNF-loaded microparticles as well as placebo microparticles were injected 2 weeks after the lesion. Untreated animals were included for reasons of comparison. Source: From Ref. 83.

Lesion treatment (2 weeks)

Figure 18 Effects of GDNF-loaded, PLGA-based microparticles on the behavior of "Parkinsonian rats" (the animals received two injections of 10 ^g 6-hydroxydopamine inducing a partial progressive and retrograde lesion of the nigrostriatal system): Number of ipsiversive turns per minute (tpm) in the amphetamine-induced rotation test at 1, 4, 6, 8, and 10 weeks after the lesion. GDNF-loaded microparticles as well as placebo microparticles were injected 2 weeks after the lesion. Untreated animals were included for reasons of comparison. Source: From Ref. 83.

poor integration of the cells in their new environment. Generally, about 90% of the transplanted cells die within the first 2 weeks after administration. A promising approach aiming to overcome these restrictions is the combination of controlled release microparticles with cell transplantation (65). The microparticles can for instance release growth factors and/or cytokines in a pre-determined manner, helping to reduce cell death and to improve cell integration into the brain tissue. An even more sophisticated approach is to use the microparticles not only as controlled drug delivery systems, but also as microcarriers for the transplanted cells (65,86). These systems are also called "pharmacologically active microcarriers" (PAM). Figure 19A shows a schematic illustration of this concept. The microparticles are coated with cell adhesion or extracellular matrix molecules and release the biologically active agents at pre-determined rates. Figure 19B and C show optical and scanning electron micrographs of such devices carrying PC12 cells on their surfaces. The microparticles may also release drugs that are able to modify the microenvironment, for example,

Figure 19 PAM used to improve the efficiency of cell therapies: (A) Schematic illustration of concept of the approach; (B and C) optical and scanning electron micrographs of cells adhering onto PAM. Source: From Ref. 86.

favor angiogenesis or local immuno-depression. After complete micro-particle degradation, the cells can integrate the parenchyma. Recently, NGF-releasing PAM conveying PC12 cells were transplanted into "Parkinsonian rats" (65). When PC12 cells, which express TH, are exposed to NGF, they stop cell division, extend long neuritis, become excitable and after depolarization they can release significant amounts of dopamine (the neurotransmitter missing in Parkinson's Disease). First results showed that these NGF-releasing PAM can reduce cell death and improve the amphetamine-induced rotational behavior of the rats.

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