- Email: [email protected]
The fate of biodegradable microspheres injected into rat brain
Neuroscience Letters 323 (2002) 85–88 www.elsevier.com/locate/neulet
The fate of biodegradable microspheres injected into rat brain Anthony P. Nicholas a,*, Carey McInnis a, Kiran B. Gupta a, William W. Snow a, Darryl F. Love b, David W. Mason b, Teresa M. Ferrell b, Jay K. Staas b, Thomas R. Tice b a
Department of Neurology, University of Alabama at Birmingham and the Birmingham Veterans Administration Medical Center, 619 19th Street South, Birmingham, AL 35249-7340, USA b Pharmaceutical Formulations Department, Southern Research Institute, Birmingham, AL, USA Received 28 September 2001; received in revised form 27 November 2001; accepted 27 November 2001
Abstract Biodegradable microspheres made with poly-[d,l-lactide-co-glycolide] represent an evolving technology for drug delivery into the central nervous system. Even though these microspheres have been shown to be engulfed by astrocytes in vitro, the purpose of the present study was to track the fate of biodegradable microspheres in vivo. This was accomplished using microspheres containing the fluorescent dye coumarin-6 followed 1 day, 1 week and 1 month after intracerebral injections of this material were made into the rat brain. Using dual color immunohistochemistry and antisera against glial fibrillary acidic protein for astrocytes versus phosphotyrosine for microglia, results demonstrate that phagocytosis of small coumarin-containing microspheres ,7.5 mm in diameter was primarily by microglia in vivo during the first week post-injection. In contrast, only a small minority of these microspheres appeared to be engulfed by astrocytes. q 2002 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Astrocytes; Glia; Huntington disease; Parkinson disease; Poly-[d,l-lactide-co-glycolide] microspheres; Microglia
The use of biodegradable microspheres represents an evolving technology for eventual treatment of human brain disease [13]. In animal models of Parkinson and Huntington diseases, biodegradable microspheres containing various substances have produced lasting beneficial effects. For example, in a rodent animal model of Huntington disease, striatal lesion sizes caused by quinolinic acid injections were significantly reduced in rats pretreated with nerve growth factor-containing striatal microspheres [4,15]. In a rodent model of Parkinson disease utilizing unilateral destruction of the nigro-striatal pathway [1,2,19], abnormal rotational behavior was reversed after dopamine-containing biodegradable microspheres were injected into the injured striatum [8,11]. In fact, these beneficial effects were present for months past injection [8,11] and also seemed to have a trophic effect with evidence of new dopaminergic terminals sprouting in the damaged striatum [9,11]. However, the fate of biodegradable microspheres injected into the brain has not been sufficiently studied. It has been suggested that intracerebrally-injected microspheres may be engulfed by astrocytes that slowly release
* Corresponding author. Tel.: 11-205-975-8509; fax: 11-205934-0928. E-mail address: [email protected] (A.P. Nicholas).
their contents into the surrounding neuropil, because phagocytosis of microspheres by astrocytes was demonstrated in cell culture [10]. Previous in vivo studies have demonstrated no widespread phagocytosis by any cell type in the brain, but most of these experiments used microspheres of different sizes, many of which were as large or larger than average glial somata [8,12,14,15]. The purpose of the present study was to determine if glial phagocytosis of microspheres took place in vivo, using different microsphere sizes and injection survival times. Biodegradable coumarin-containing microspheres were prepared by an emulsion-based, solvent extraction process. Briefly, this procedure involved first preparing a solution containing the polymer poly-[d,l-lactide-co-glycolide] and the fluorescent dye coumarin-6 dissolved in ethyl acetate. This polymer solution was then added to a stirring aqueous process medium containing the surfactants carboxymethylcellulose and polyvinyl alcohol. An emulsion was then formed as the polymer/coumarin solution was mixed with the aqueous process medium. Once an emulsion containing appropriately-sized polymer/coumarin droplets was formed, the emulsion was then transferred to a large water bath to extract the ethyl acetate from the polymer/coumarin droplets forming the microspheres. The microspheres were then washed, centrifuged and finally collected by lyophili-
0304-3940/02/$ - see front matter q 2002 Elsevier Science Ireland Ltd. All rights reserved. PII: S03 04 - 394 0( 0 1) 02 53 4- 4
86
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zation. Properly sized coumarin-containing microspheres were ensured by passing them through 40, 20 or 7.5 mm screens, respectively. A total of eight male Sprague–Dawley rats were used for these experiments and each received unilateral or bilateral injections of coumarin-containing microspheres into the striatum. Half of the animals were injected with primarily large microspheres (20 or 40 mm diameter) while the other half were injected with smaller microspheres (,7.5 mm). A total of 12 intracerebral injections were made between both groups and representatives of each were sacrificed 1 day, 1 week or 1 month post-injection. Prior to injection, each animal was anesthetized with intraperitoneal injections of ketamine (120 mg/kg) plus xylazine (20 mg xylazine per ml of 100 mg/ml ketamine) and placed in a stereotaxic apparatus (Kopf). The scalp was then excised and retracted. Using a dental drill, a burr hole was then placed in the skull 3 mm lateral to and 0.4 mm rostral to the bregma. A modified syringe assembly [7] was then used to deliver a 2 mm dry pellet of coumarin-containing microspheres packed into the bottom of a 1.5 ml Wiretrol II glass microcapillary pipette (Drummond), and injected with a steel wire plunger into the striatum, unilaterally or bilaterally using stereotaxic co-ordinates [16]. After the injection, the micropipette was removed, the scalp was sutured and the animals were returned to their cages for 24 h, 1 week or 1 month with food and water ad lib. All animals showed no ill effects from their microsphere injections and were treated humanely in accordance with standard protocols at the University animal facilities. On the date of sacrifice, each animal was re-anesthetized as before and perfused transcardially with normal saline at 37 8C, followed by ice cold 4% paraformaldehyde in 0.1 M phosphate-buffered saline (PBS; pH 7.4). The brains were then removed and blocks containing the injection sites were post-fixed in 4% paraformaldehyde overnight, cryoprotected in 20% sucrose for 2 days, immersed in TissueTek w embedding compound (Miles, Inc.) and then frozen using dry ice vapor. Sections of this material were cut (12– 14 mm) using a Microm HM 505E cryostat, melted onto glass slides coated with 1% gelatin and 0.1% chromealum and prepared for immunohistochemical analysis. All immunohistochemical incubations were performed in humidified chambers. Sections containing the injection sites were first blocked with 3% bovine serum albumin plus 0.3% Triton X-100 in PBS for 30 min at room temperature, followed by three rinses in PBS for 10 min each. These sections were then incubated overnight at 4 8C in rabbit primary antisera diluted in PBS with 0.3% Triton X-100 either against phosphotyrosine (PT; Zymed; 1:500) to stain microglia [17,18] or glial acidic fibrillary protein (GFAP; Chemicon; 1:30) to stain astrocytes. After similar washes in PBS as before, the sections were then incubated for 1 h at 37 8C with rhodamine-conjugated donkey anti-rabbit IgG (Jackson ImmunoResearch) secondary antiserum diluted 1:50 in PBS and Triton X. The sections were then rinsed in
PBS as before, coverslipped with VECTASHIELD w (Vector), and examined using epi-fluorescence with the Nikon E-800 microscope. Green coumarin-fluorescent microspheres were seen with a Nikon B-2E/C filter cube, while red rhodamine-fluorescent structures were visualized with a Nikon G-2E/C filter cube along with an additional Nikon EF4-4 multiband barrier filter to avoid ‘bleed through’ of fluorescence. Images were transferred to a G3 Power Macintosh OS 8.1 computer using a supercooled SPOTe color video camera and green and red images were saved and merged using Adobe Photoshop 5.5 software. The present study demonstrated that injection sites in animals that received deposits of large microspheres (20 or 40 mm diameter) into the striatum showed no evidence of phagocytosis by PT-immunoreactive microglia or GFAPimmunoreactive astrocytes after 1 day, 1 week or 1 month survival times. The large microspheres remained in the injection site without much appreciable difference during these three time periods. Similar results were seen with the small microspheres (,7.5 mm), 1 day post-injection, but phagocytosis of these microspheres by microglia was evident at 1 week (Fig. 1) and 1 month post-injection. In these specimens, discrete cell clusters containing small fluorescent microspheres (Fig. 1A,B) were seen anywhere from 600 to 900 mm ventrally from the injection sites. Using dual color immunohistochemistry, these microspheres were demonstrated in some, but not all, PT-immunoreactive microglia (Fig. 1A,B) and usually filled up the cytoplasm of these cells when visualized with merged dual color immunofluorescence (Fig. 1C). In contrast, GFAP-positive astrocytic fibers were primarily demonstrated adjacent to the injection site as well as around cells that contained the microspheres (Fig. 2). While the cytoplasm of PT-positive microglia in these cell clusters adjacent to the injection site seemed to be filled with coumarin-containing microspheres (Fig. 1C), only few examples of apparent co-localization of microspheres within GFAP-positive astrocytes could be seen in these same areas (Fig. 2). Biodegradable microspheres containing neurotransmitters [8–11] or trophic substances [4,15] can be safely injected into the brains of animals with little trauma and provided clinical benefit in animal models of neurodegenerative diseases with evidence of beneficial effects lasting many months. It has been theorized that these long-term effects may be due to slow diffusion of neuroactive substances from within the microspheres into the surrounding neuropil that would continue until the microspheres eventually degrade, as determined by the microsphere composition [3,5]. However, another explanation for extended clinical benefits may be due to controlled release by glia after phagocytosis of drug-containing microspheres into these cells. Indeed, some neurotransmitters such as dopamine can be reabsorbed into glia for reuse [6]. In vitro studies have demonstrated phagocytosis of microspheres by astrocytes [10]; however, widespread phagocytosis of microspheres by astrocytes was not seen in vivo
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using GFAP-immunofluorescence staining in the present study. Instead, most of this phagocytosis was done by microglia. However, the amount of microsphere phagocytosis by astrocytes in vivo may be somewhat underestimated, because GFAP-staining labels astrocyte processes much better than cell soma, and thus the detection of some of these microspheres in astrocytes may be below the detection of our two-color immunofluorescence technique. In agreement with the present study, most previous experiments demonstrated a non-specific astrocytic proliferation and microglia reaction along the injection track [11,14,15] that reportedly disappeared 2 months post-injection [14]. Reactive immunohistochemical staining for astrocytes and microglia in these cases was demonstrated among
Fig. 1. Dual color immunofluorescence of intracerebrallyinjected, coumarin-containing microspheres (A,C) and PT-positive microglia (B,C). On low magnification, clusters of green cells containing coumarin microspheres (A) showed examples of colocalization with many (large arrows) but not all (small arrows) reactive microglia (red) positive for PT (B). On high magnification (C), merged green and red images demonstrated a high degree of co-localization (yellow) of coumarin fluorescence and PT-immunoreactivity representing microglia containing large amounts of microspheres filling their cytoplasm. Bars, 50 mm.
Fig. 2. Dual color immunofluorescence of intracerebrallyinjected, coumarin-containing microspheres (green) and GFAPpositive astrocytes (red). On high magnification, merged images demonstrated that most GFAP-positive astrocyte processes were seen primarily adjacent to coumarin microsphere-containing cells. However, there were a few examples (arrows in A ¼ B– D) of small numbers of microspheres (yellow) contained within some of these GFAP-positive structures. Bar, 50 mm.
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and around the microspheres without widespread phagocytosis from either cell class [11,14,15], except for one report of a microsphere appearing to be engulfed by a histiocytic cell, 9 months post-injection [14]. These previous experiments used microspheres that varied in maximum diameters of 20–35 mm [12,14,15] to as large as 40 mm [8]. In contrast, the present study showed, not only that small microspheres with diameters ,7.5 mm were engulfed primarily by microglia in vivo, but also that the first evidence of phagocytosis happened after 24 h and within the first week post-injection. Due to their small size, it was difficult to determine if other cell types engulfed any individual microspheres, but there was no evidence of widespread transport of these particles to other parts of the brain, including the substantia nigra. In one study utilizing microspheres of different sizes injected in the periphery, an increased immune response was seen when microsphere diameters were ,10 mm [3]. As a result, further studies are warranted to determine the significance of phagocytosis of these microspheres by glia on the clinical efficacy of drugs contained in different sized microspheres to treat neurological disease. Further studies are also needed to determine if astrocytes are more involved with the phagocytosis of microspheres in vivo under different conditions, such as with more variable survival times, microsphere sizes or composition, or with different techniques, such as GFAP-immunohistochemical examination of injection sites with electron microscopy. The use of intracerebrally-injected biodegradable microspheres to treat neurological diseases will require understanding of the fate of these particles within the brains of those who receive them. The present study is the first to demonstrate that, at least for small microspheres ,7.5 mm in diameter, many of these particles are engulfed primarily by microglia in vivo within the first week post-injection. Since most past experiments using brain microsphere injections have been a mixture of different size particles, future studies should focus on the clinical significance of phagocytosis of small versus large microspheres on the efficacy of this drug delivery system.
[4]
[5]
[6]
[7]
[8]
[9]
[10]
[11]
[12]
[13]
[14]
This work was supported by grants from the Parkinson Association of Alabama, The Strain Family Foundation and a MREP Award from the Department of Veterans Affairs. Many thanks to Dee Parsons and Dr Lindy Harrell for the use of their stereotaxic apparatus and Dr Yancey Gillespie for the use of his cryostat.
[15]
[16] [1] Anden, N.-E., Dahlstrom, A., Fuxe, K. and Larsson, K., Functional role of the nigro-neostriatal dopamine neurons, Acta Pharmacol. Toxicol., 24 (1966) 263–274. [2] Costall, B., Marsden, C.D., Naylor, R.J. and Pycock, C.J., The relationship between striatal and mesolimbic dopamine dysfunction and the nature of circling responses following 6-hydroxydopamine and electrolytic lesions of the ascending dopamine systems of rat brain, Brain Res., 118 (1976) 87–113. [3] Eldridge, J.H., Staas, J.K., Tice, T.R. and Gilley, R.M., Biode-
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gradable poly(dl-lactide-co-glycolide) microspheres, Res. Immunol., 143 (1992) 557–563. Gouhier, C., Chalon, S., Venier-Julienne, M.-C., Bodard, S., Benoit, J.-P., Besnard, J.-C. and Guilloteau, D., Neuroprotection of nerve growth factor-loaded microspheres on the D2 dopaminergic receptor positive-striatal neurones in quinolinic acid-lesioned rats: a quantitative autoradiographic assessment with iodobenzamide, Neurosci. Lett., 288 (2000) 71–75. Hausberger, A.G. and DeLuca, P.P., Characterization of biodegradable poly(d,l-lactide-co-glycolide) polymers and microspheres, J. Pharm. Biomed. Anal., 13 (1995) 747–760. Henn, F.A. and Hamberger, A., Glial cell functions: uptake of transmitter substances, Proc. Natl. Acad. Sci. USA, 68 (1971) 2686–2690. Kratskin, I.L., Yu, X. and Doty, R.L., An easily constructed pipette for pressure microinjections into the brain, Brain Res. Bull., 44 (1997) 199–203. McRae, A., Hjorth, S., Dillon, L. and Tice, T., Implantable microencapsulated dopamine (DA): prolonged functional release of DA in denervated striatal tissue, J. Neural Transm., 29 (Suppl.) (1990) 207–215. McRae, A., Hjorth, S., Dahlstrom, A., Dillon, L., Mason, D. and Tice, T., Dopamine fiber growth induction by implantation of synthetic dopamine-containing microspheres in rats with experimental hemi-Parkinsonism, Mol. Chem. Neuropathol., 16 (1992) 123–141. McRae, A., Ling, E.A., Hjorth, S., Dahlstrom, A., Mason, D. and Tice, T., Catecholamine-containing biodegradable microsphere implants as a novel approach in the treatment of CNS neurodegenerative disease. A review of experimental studies in DA-lesioned rats, Mol. Neurobiol., 9 (1994) 191–205. McRae, A., Dahlstrom, A., Hjorth, S., Ling, E.A., Mason, D. and Tice, T., Catecholamine-containing biodegradable microsphere implants: an overview of experimental studies in dopamine-lesioned rats, In I. Hanin (Ed.), Alzheimer’s and Parkinson’s Diseases, Plenum Press, New York, 1995, pp. 421–427. Menei, P., Daniel, V., Montero-Menei, C., Brouillard, M., Pouplard-Barthelaix, A. and Benoit, J.-P., Biodegradation and brain tissue reaction to poly(d,l-lactide-co-glycolide) microspheres, Biomaterials, 14 (1993) 470–478. Menei, P., Benoit, J.-P., Boisdron-Celle, M., Fournier, D., Mercier, P. and Guy, G., Drug targeting into the central nervous system by stereotactic implantation of biodegradable microspheres, Neurosurgery, 34 (1994) 1058–1064. Menei, P., Croue, A., Daniel, V., Pouplard-Barthelaix, A. and Benoit, J.-P., Fate and biocompatibility of three types of microspheres implanted into the brain, J. Biomed. Mater. Res., 28 (1994) 1079–1085. Menei, P., Pean, J.M., Nerriere-Daguin, V., Jollivet, C., Brachet, P. and Benoit, J.-P., Intracerebral implantation of NGF-releasing biodegradable microspheres protects striatum against excitotoxic damage, Exp. Neurol., 161 (2000) 259–272. Paxinos, G. and Watson, C., The Rat Brain in Stereotaxic Coordinates, Academic Press, New York, 1986. Tillotson, M.L. and Wood, J.G., Phosphotyrosine antibodies specifically label ameboid microglia in vitro and ramified microglia in vivo, Glia, 2 (1989) 412–419. Tillotson, M.L. and Wood, J.G., Tyrosine phosphorylation in the postnatal rat brain: a developmental, immunohistochemical study, J. Comp. Neurol., 282 (1989) 133–141. Tolosa, E., Marti’, M.J., Valldeoriola, F. and Molinueyo, J.L., History of levodopa and dopamine agonists in Parkinson’s disease treatment, Neurology, 50 (Suppl. 6) (1998) S2–S10.
The fate of biodegradable microspheres injected into rat brain Anthony P. Nicholas a,*, Carey McInnis a, Kiran B. Gupta a, William W. Snow a, Darryl F. Love b, David W. Mason b, Teresa M. Ferrell b, Jay K. Staas b, Thomas R. Tice b a
Department of Neurology, University of Alabama at Birmingham and the Birmingham Veterans Administration Medical Center, 619 19th Street South, Birmingham, AL 35249-7340, USA b Pharmaceutical Formulations Department, Southern Research Institute, Birmingham, AL, USA Received 28 September 2001; received in revised form 27 November 2001; accepted 27 November 2001
Abstract Biodegradable microspheres made with poly-[d,l-lactide-co-glycolide] represent an evolving technology for drug delivery into the central nervous system. Even though these microspheres have been shown to be engulfed by astrocytes in vitro, the purpose of the present study was to track the fate of biodegradable microspheres in vivo. This was accomplished using microspheres containing the fluorescent dye coumarin-6 followed 1 day, 1 week and 1 month after intracerebral injections of this material were made into the rat brain. Using dual color immunohistochemistry and antisera against glial fibrillary acidic protein for astrocytes versus phosphotyrosine for microglia, results demonstrate that phagocytosis of small coumarin-containing microspheres ,7.5 mm in diameter was primarily by microglia in vivo during the first week post-injection. In contrast, only a small minority of these microspheres appeared to be engulfed by astrocytes. q 2002 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Astrocytes; Glia; Huntington disease; Parkinson disease; Poly-[d,l-lactide-co-glycolide] microspheres; Microglia
The use of biodegradable microspheres represents an evolving technology for eventual treatment of human brain disease [13]. In animal models of Parkinson and Huntington diseases, biodegradable microspheres containing various substances have produced lasting beneficial effects. For example, in a rodent animal model of Huntington disease, striatal lesion sizes caused by quinolinic acid injections were significantly reduced in rats pretreated with nerve growth factor-containing striatal microspheres [4,15]. In a rodent model of Parkinson disease utilizing unilateral destruction of the nigro-striatal pathway [1,2,19], abnormal rotational behavior was reversed after dopamine-containing biodegradable microspheres were injected into the injured striatum [8,11]. In fact, these beneficial effects were present for months past injection [8,11] and also seemed to have a trophic effect with evidence of new dopaminergic terminals sprouting in the damaged striatum [9,11]. However, the fate of biodegradable microspheres injected into the brain has not been sufficiently studied. It has been suggested that intracerebrally-injected microspheres may be engulfed by astrocytes that slowly release
* Corresponding author. Tel.: 11-205-975-8509; fax: 11-205934-0928. E-mail address: [email protected] (A.P. Nicholas).
their contents into the surrounding neuropil, because phagocytosis of microspheres by astrocytes was demonstrated in cell culture [10]. Previous in vivo studies have demonstrated no widespread phagocytosis by any cell type in the brain, but most of these experiments used microspheres of different sizes, many of which were as large or larger than average glial somata [8,12,14,15]. The purpose of the present study was to determine if glial phagocytosis of microspheres took place in vivo, using different microsphere sizes and injection survival times. Biodegradable coumarin-containing microspheres were prepared by an emulsion-based, solvent extraction process. Briefly, this procedure involved first preparing a solution containing the polymer poly-[d,l-lactide-co-glycolide] and the fluorescent dye coumarin-6 dissolved in ethyl acetate. This polymer solution was then added to a stirring aqueous process medium containing the surfactants carboxymethylcellulose and polyvinyl alcohol. An emulsion was then formed as the polymer/coumarin solution was mixed with the aqueous process medium. Once an emulsion containing appropriately-sized polymer/coumarin droplets was formed, the emulsion was then transferred to a large water bath to extract the ethyl acetate from the polymer/coumarin droplets forming the microspheres. The microspheres were then washed, centrifuged and finally collected by lyophili-
0304-3940/02/$ - see front matter q 2002 Elsevier Science Ireland Ltd. All rights reserved. PII: S03 04 - 394 0( 0 1) 02 53 4- 4
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zation. Properly sized coumarin-containing microspheres were ensured by passing them through 40, 20 or 7.5 mm screens, respectively. A total of eight male Sprague–Dawley rats were used for these experiments and each received unilateral or bilateral injections of coumarin-containing microspheres into the striatum. Half of the animals were injected with primarily large microspheres (20 or 40 mm diameter) while the other half were injected with smaller microspheres (,7.5 mm). A total of 12 intracerebral injections were made between both groups and representatives of each were sacrificed 1 day, 1 week or 1 month post-injection. Prior to injection, each animal was anesthetized with intraperitoneal injections of ketamine (120 mg/kg) plus xylazine (20 mg xylazine per ml of 100 mg/ml ketamine) and placed in a stereotaxic apparatus (Kopf). The scalp was then excised and retracted. Using a dental drill, a burr hole was then placed in the skull 3 mm lateral to and 0.4 mm rostral to the bregma. A modified syringe assembly [7] was then used to deliver a 2 mm dry pellet of coumarin-containing microspheres packed into the bottom of a 1.5 ml Wiretrol II glass microcapillary pipette (Drummond), and injected with a steel wire plunger into the striatum, unilaterally or bilaterally using stereotaxic co-ordinates [16]. After the injection, the micropipette was removed, the scalp was sutured and the animals were returned to their cages for 24 h, 1 week or 1 month with food and water ad lib. All animals showed no ill effects from their microsphere injections and were treated humanely in accordance with standard protocols at the University animal facilities. On the date of sacrifice, each animal was re-anesthetized as before and perfused transcardially with normal saline at 37 8C, followed by ice cold 4% paraformaldehyde in 0.1 M phosphate-buffered saline (PBS; pH 7.4). The brains were then removed and blocks containing the injection sites were post-fixed in 4% paraformaldehyde overnight, cryoprotected in 20% sucrose for 2 days, immersed in TissueTek w embedding compound (Miles, Inc.) and then frozen using dry ice vapor. Sections of this material were cut (12– 14 mm) using a Microm HM 505E cryostat, melted onto glass slides coated with 1% gelatin and 0.1% chromealum and prepared for immunohistochemical analysis. All immunohistochemical incubations were performed in humidified chambers. Sections containing the injection sites were first blocked with 3% bovine serum albumin plus 0.3% Triton X-100 in PBS for 30 min at room temperature, followed by three rinses in PBS for 10 min each. These sections were then incubated overnight at 4 8C in rabbit primary antisera diluted in PBS with 0.3% Triton X-100 either against phosphotyrosine (PT; Zymed; 1:500) to stain microglia [17,18] or glial acidic fibrillary protein (GFAP; Chemicon; 1:30) to stain astrocytes. After similar washes in PBS as before, the sections were then incubated for 1 h at 37 8C with rhodamine-conjugated donkey anti-rabbit IgG (Jackson ImmunoResearch) secondary antiserum diluted 1:50 in PBS and Triton X. The sections were then rinsed in
PBS as before, coverslipped with VECTASHIELD w (Vector), and examined using epi-fluorescence with the Nikon E-800 microscope. Green coumarin-fluorescent microspheres were seen with a Nikon B-2E/C filter cube, while red rhodamine-fluorescent structures were visualized with a Nikon G-2E/C filter cube along with an additional Nikon EF4-4 multiband barrier filter to avoid ‘bleed through’ of fluorescence. Images were transferred to a G3 Power Macintosh OS 8.1 computer using a supercooled SPOTe color video camera and green and red images were saved and merged using Adobe Photoshop 5.5 software. The present study demonstrated that injection sites in animals that received deposits of large microspheres (20 or 40 mm diameter) into the striatum showed no evidence of phagocytosis by PT-immunoreactive microglia or GFAPimmunoreactive astrocytes after 1 day, 1 week or 1 month survival times. The large microspheres remained in the injection site without much appreciable difference during these three time periods. Similar results were seen with the small microspheres (,7.5 mm), 1 day post-injection, but phagocytosis of these microspheres by microglia was evident at 1 week (Fig. 1) and 1 month post-injection. In these specimens, discrete cell clusters containing small fluorescent microspheres (Fig. 1A,B) were seen anywhere from 600 to 900 mm ventrally from the injection sites. Using dual color immunohistochemistry, these microspheres were demonstrated in some, but not all, PT-immunoreactive microglia (Fig. 1A,B) and usually filled up the cytoplasm of these cells when visualized with merged dual color immunofluorescence (Fig. 1C). In contrast, GFAP-positive astrocytic fibers were primarily demonstrated adjacent to the injection site as well as around cells that contained the microspheres (Fig. 2). While the cytoplasm of PT-positive microglia in these cell clusters adjacent to the injection site seemed to be filled with coumarin-containing microspheres (Fig. 1C), only few examples of apparent co-localization of microspheres within GFAP-positive astrocytes could be seen in these same areas (Fig. 2). Biodegradable microspheres containing neurotransmitters [8–11] or trophic substances [4,15] can be safely injected into the brains of animals with little trauma and provided clinical benefit in animal models of neurodegenerative diseases with evidence of beneficial effects lasting many months. It has been theorized that these long-term effects may be due to slow diffusion of neuroactive substances from within the microspheres into the surrounding neuropil that would continue until the microspheres eventually degrade, as determined by the microsphere composition [3,5]. However, another explanation for extended clinical benefits may be due to controlled release by glia after phagocytosis of drug-containing microspheres into these cells. Indeed, some neurotransmitters such as dopamine can be reabsorbed into glia for reuse [6]. In vitro studies have demonstrated phagocytosis of microspheres by astrocytes [10]; however, widespread phagocytosis of microspheres by astrocytes was not seen in vivo
A.P. Nicholas et al. / Neuroscience Letters 323 (2002) 85–88
87
using GFAP-immunofluorescence staining in the present study. Instead, most of this phagocytosis was done by microglia. However, the amount of microsphere phagocytosis by astrocytes in vivo may be somewhat underestimated, because GFAP-staining labels astrocyte processes much better than cell soma, and thus the detection of some of these microspheres in astrocytes may be below the detection of our two-color immunofluorescence technique. In agreement with the present study, most previous experiments demonstrated a non-specific astrocytic proliferation and microglia reaction along the injection track [11,14,15] that reportedly disappeared 2 months post-injection [14]. Reactive immunohistochemical staining for astrocytes and microglia in these cases was demonstrated among
Fig. 1. Dual color immunofluorescence of intracerebrallyinjected, coumarin-containing microspheres (A,C) and PT-positive microglia (B,C). On low magnification, clusters of green cells containing coumarin microspheres (A) showed examples of colocalization with many (large arrows) but not all (small arrows) reactive microglia (red) positive for PT (B). On high magnification (C), merged green and red images demonstrated a high degree of co-localization (yellow) of coumarin fluorescence and PT-immunoreactivity representing microglia containing large amounts of microspheres filling their cytoplasm. Bars, 50 mm.
Fig. 2. Dual color immunofluorescence of intracerebrallyinjected, coumarin-containing microspheres (green) and GFAPpositive astrocytes (red). On high magnification, merged images demonstrated that most GFAP-positive astrocyte processes were seen primarily adjacent to coumarin microsphere-containing cells. However, there were a few examples (arrows in A ¼ B– D) of small numbers of microspheres (yellow) contained within some of these GFAP-positive structures. Bar, 50 mm.
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and around the microspheres without widespread phagocytosis from either cell class [11,14,15], except for one report of a microsphere appearing to be engulfed by a histiocytic cell, 9 months post-injection [14]. These previous experiments used microspheres that varied in maximum diameters of 20–35 mm [12,14,15] to as large as 40 mm [8]. In contrast, the present study showed, not only that small microspheres with diameters ,7.5 mm were engulfed primarily by microglia in vivo, but also that the first evidence of phagocytosis happened after 24 h and within the first week post-injection. Due to their small size, it was difficult to determine if other cell types engulfed any individual microspheres, but there was no evidence of widespread transport of these particles to other parts of the brain, including the substantia nigra. In one study utilizing microspheres of different sizes injected in the periphery, an increased immune response was seen when microsphere diameters were ,10 mm [3]. As a result, further studies are warranted to determine the significance of phagocytosis of these microspheres by glia on the clinical efficacy of drugs contained in different sized microspheres to treat neurological disease. Further studies are also needed to determine if astrocytes are more involved with the phagocytosis of microspheres in vivo under different conditions, such as with more variable survival times, microsphere sizes or composition, or with different techniques, such as GFAP-immunohistochemical examination of injection sites with electron microscopy. The use of intracerebrally-injected biodegradable microspheres to treat neurological diseases will require understanding of the fate of these particles within the brains of those who receive them. The present study is the first to demonstrate that, at least for small microspheres ,7.5 mm in diameter, many of these particles are engulfed primarily by microglia in vivo within the first week post-injection. Since most past experiments using brain microsphere injections have been a mixture of different size particles, future studies should focus on the clinical significance of phagocytosis of small versus large microspheres on the efficacy of this drug delivery system.
[4]
[5]
[6]
[7]
[8]
[9]
[10]
[11]
[12]
[13]
[14]
This work was supported by grants from the Parkinson Association of Alabama, The Strain Family Foundation and a MREP Award from the Department of Veterans Affairs. Many thanks to Dee Parsons and Dr Lindy Harrell for the use of their stereotaxic apparatus and Dr Yancey Gillespie for the use of his cryostat.
[15]
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