Quintero et al. 2006 - Medical University of South Carolina

Research Report
Behavioral and morphological effects of minocycline in the
6-hydroxydopamine rat model of Parkinson's disease
Elias Matthew Quintero a, ⁎, Lauren Willis a , Rachel Singleton a , Naida Harris b , Peng Huang c ,
Narayan Bhat a , Ann-Charlotte Granholm a
a
Department of Neurosciences, Medical University of South Carolina, 173 Ashley Avenue, Suite 403, Charleston, SC 29425, USA
b
Benedict College, Columbia, SC 29204, USA
c
Department of Biostatistics, Bioinformatics and Epidemiology, Medical University of South Carolina, Charleston, SC 29425, USA
ARTICLE INFO ABSTRACT
Article history:
Accepted 20 March 2006
Available online 18 May 2006
Keywords:
Parkinson's disease
6-Hydroxydopamine
Minocycline
Apomorphine
Inflammation
Neuroglia
1. Introduction
Neuroinflammation mediated by activated glial cells has
recently been investigated as a causatory factor for Parkin-
⁎ Corresponding author. Fax: +1 843 792 4423.
E-mail address: quintero@musc.edu (E.M. Quintero).
BRAIN RESEARCH 1093 (2006) 198– 207
available at www.sciencedirect.com
www.elsevier.com/locate/brainres
0006-8993/$ – see front matter © 2006 Elsevier B.V. All rights reserved.
doi:10.1016/j.brainres.2006.03.104
The neuropathology in many neurodegenerative diseases is mediated by inflammatory
cascades that influence neuronal dysfunction and death. Minocycline reduces the
neurodegeneration observed in various models of Parkinson's. We exploited the unilateral
6-hydroxydopamine (6-OHDA) lesion model to assess the effect of minocycline on related
neurodegeneration. Thirty Fisher 344 rats were divided into three daily treatment groups: (1)
after: 45 mg/kg of minocycline beginning 24 h after lesioning; (2) before: 45 mg/kg of
minocycline beginning 3 days before 6-OHDA lesioning; (3) control: corresponding salinetreated
controls. Animals were assessed for apomorphine-induced rotations for 4 weeks. A
longitudinal model for repeated measures showed that both after and before groups had
significantly lower rotations than controls (P < 0.001 for both comparisons). Pair-wise group
comparisons showed that the before animals rotated less compared to controls (mean
rotations: 164 ± 38 versus 386 ± 49, respectively, P = 0.001). After animals also rotated
significantly less then controls (mean rotations: 125 ± 41 versus 386 ± 49, respectively,
P < 0.001). Animals receiving minocycline displayed reduced tyrosine hydroxylase-positive
cell loss in the lesioned nigra versus contralateral nonlesioned nigra, compared to controls
(mean differences: 5065 for after, 3550 for before, and 6483 for controls; P = 0.158 for after
versus controls, P = 0.019 for before versus controls). The remaining lesioned nigral cells of
both minocycline-treated groups were larger than controls, with the most robust cell size
and fiber density observed in the after group. These data suggest that the therapeutic
potential of minocycline may depend on the time of drug administration relative to
neuropathogenic event.
© 2006 Elsevier B.V. All rights reserved.
son's disease (PD), an age-related neurodegenerative disease
(Barcia et al., 2003; Dawson and Dawson, 2003). Studies have
shown that activated microglial cells surround dopaminergic
neurons in patients with this disease and produce damaging

cytokines that may be directly responsible for the demise of
the dopamine (DA) neurons (Hirsch et al., 2003; Hunot and
Hirsch, 2003). Activated glia (astrocytes and microglia) also
contribute towards the rampant oxidative stress that is
associated with the neurodegenerative process via excessive
production of neurotoxic free radicals (Abramov et al., 2004;
Leblhuber et al., 2003). Although astrocytes normally protect
neurons through their production of growth and trophic
molecules, “aging” astrocytes, because of their expression of
pro-oxidant enzymes such as MAO-B and a high content of
free iron, can promote enzymatic and nonenzymatic oxidation
of DA, generating toxic levels of reactive oxygen species
(Johnstone et al., 1999; Hirsch et al., 2003).
Minocycline, a second generation tetracycline, has recently
fallen under close scrutiny as having possible therapeutic value
for treating several neurodegenerative disorders, including
Huntington's disease (Chen et al., 2000), amyotrophic lateral
sclerosis (Zhu et al., 2002), and autoimmune encephalomyelitis
(Popovic et al., 2001). Studies in models of PD, including the 1methyl-4-phenyl-1,2,3,6-tetrahydropyridine
(MPTP) model (Du
et al., 2001; Wu et al., 2002) and the 6-hydroxydopamine (6-
OHDA) model (He et al., 2001), have provided compelling
evidence that minocycline possesses vast potential for attenuation
of the neurodegenerative changes that occur in the brain.
In particular, He et al. (2001) compared the effects of
minocycline treatment to saline controls on nigral dopaminergic
neuronal and microglial status in male mice lesioned in the
right striatum with 6-OHDA. Tyrosine hydroxylase (TH) immunohistochemistry
revealed a significant sparing of dopamine
neurons in animals receiving minocycline treatment. In
addition, the number of activated microglia was significantly
decreased in those animals that received minocycline compared
to their saline counterparts. Lin et al. (2003) reported that
minocycline reduced the susceptibility of cultured rat cerebellar
granule neurons to 6-OHDA-induced death and prevented the
production of free radicals following 6-OHDA administration.
These two studies clearly demonstrate that minocycline
impacts the toxicity associated with 6-OHDA administration
and establish the possibility of manipulating the degenerative
cascade that follows upon introduction of this toxin. However,
6-OHDA studies in the rat model combined with minocycline
have been sparse. The allure of this phenomenon becomes
apparent when one considers that the biochemical and cellular
events associated with 6-OHDA toxicity may be, at least in part,
similar to the molecular neuroinflammatory cascades that are
engaged in the onset of neurodegenerative diseases such as
Parkinson's disease (Hirsch et al., 1998; Jenner and Olanow,
1998).
Since its first use by Ungerstedt in the late 1960s (Ungerstedt,
1968), the utilization of the catecholaminergic neurotoxin
6-hydroxydopamine (6-OHDA) to experimentally destroy
dopaminergic neurons in the brain has been well documented
and is regarded as the gold standard for studying mesencephalic
dopamine (DA)-containing neurons (Schwarting and
Huston, 1996a,b; Glinka et al., 1997; Blum et al., 2001; Deumens
et al., 2002). By stereotaxically introducing 6-OHDA into
individual components of the nigrostriatal pathway, we
have gained enormous insight into the neuroanatomical,
neurochemical, and electrophysiological parameters of DAcontaining
neurons and their interaction with other sys-
BRAIN RESEARCH 1093 (2006) 198– 207
tems. The most common application of this toxin is the
creation of a unilateral lesion in mesencephalic DA neurons
or their ascending fibers, which will result in a loss of target
striatal DA, or into the striatum directly. Consequently, the
efficacy of a 6-OHDA unilateral lesion can be demonstrated
by DAergic compounds, such as apomorphine or amphetamine
(Ungerstedt and Arbuthnott, 1970). The number of
apomorphine-induced turns per hour has been directly
correlated to the extent of DA loss when 6-OHDA is injected
into the medial forebrain bundle (Hudson et al., 1993). This
strategy has been proven to be an invaluable tool in
studying how diseases, such as PD, may encroach upon
the nigrostriatal pathway and how experimental manipulation
of this system may provide clues for possible clinical
intervention.
For this study, we hypothesize that minocycline will reduce
the number of lesioned nigral dopaminergic cells and will
reduce the number of apomorphine-induced rotations in rats
when administered prior to or after receiving a unilateral 6-
OHDA lesion. In order to test the preventive effects of this
drug, daily intraperitoneal injections of saline or minocycline
were initiated 3 days prior to neurotoxic lesioning and
continued for 4 weeks postlesioning. We also assessed the
rescue effects of minocycline by administering the drug (as
well as saline control counterparts) 24 h following 6-OHDA
lesioning and continuing for 4 weeks after surgery. In order to
obtain behavioral evidence of the neuroprotective properties
of this tetracycline, weekly apomorphine-induced rotational
behavior was assessed on all subjects, and histological
analysis of surviving dopaminergic neurons was performed
following sacrifice of all animals.
2. Results
2.1. Effects of minocycline on apomorphine-induced
rotations
199
Apomorphine-induced rotational analysis was performed in
order to examine the effect of minocycline treatment on the
survival of dopaminergic neurons exposed to 6-OHDA. All
animals were analyzed for apomorphine-induced rotations for
1 h, once a week, for 4 weeks following lesioning (previous
studies indicate that 6-OHDA requires 4 weeks before the full
rotational potential of each animal is realized (Ungerstedt and
Arbuthnott, 1970; Sauer and Oertel, 1994; Gerlach and
Riederer, 1996). Fig. 1 is a graphical representation of the
rotational assessments for the saline-treated animals (control
group), minocycline 3 days before lesioning (before group), and
minocycline 24 h after lesioning (after group) over the 4-week
testing period following 6-OHDA administration. The control
group consistently rotated the most number of times at each
weekly time point, compared to those animals that received
minocycline. At 1 week, the mean rotation rate for the controls
was 202 turns per hour compared to 140 turns in the before
and 34 turns in the after groups (n = 10). At 2 weeks postlesion,
the controls rotated 381 turns per hour, whereas the before
and after animals rotated 138 and 114 turns per hour,
respectively. Three weeks following toxin administration
resulted in 359 turns in the controls, 246 turns in the before

200 BRAIN RESEARCH 1093 (2006) 198– 207
Fig. 1 – Minocycline reduces the number of
apomorphine-induced rotations in the 6-OHDA-lesioned rat.
All animals that received minocycline rotated significantly
fewer times over the 4-week testing period compared to their
saline-treated counterparts (P < 0.001). At week 2, animals
treated before (Before) and after (After) 6-OHDA lesioning
rotated significantly fewer times than the saline-treated
(Control) animals (138, 114 and 381 turns, respectively.
P < 0.05). At week 4, the difference in rotations in the after
treated group approached significance (P = 0.07) compared to
the saline control group.
and 148 turns in the after. Finally, the fourth week of rotational
analysis revealed that the controls rotated 430 turns per hour,
while the before rotated 394 turns and the after rotated 149
turns.
A one-way ANOVA showed that there was a significant
difference among the 3 groups in overall turns across all
time points (P < 0.0001). Post hoc two-sample t tests to
compare the after group versus controls, before group versus
controls, and combined after and before versus controls all
yielded P values

minocycline treatment. Fig. 2 demonstrates the contrast
between lesioned and nonlesioned nigras of all three
groups. The dopaminergic neurons of the lesioned nigra
readily succumbed to 6-OHDA when treated with saline
only (Fig. 2A). These results were identical whether saline
treatment was initiated before or after 6-OHDA administration.
When minocycline was administered 3 days prior
to 6-OHDA administration and continuously thereafter,
there was a marked sparing of dopaminergic neurons in
the substantia nigra 4 weeks following toxin introduction
(Fig. 2B). There was also a similar demonstration of nigral
cell survival when minocycline was administered continuously
starting 24 h after 6-OHDA lesioning (Fig. 2C).
Stereological cell counts of TH-positive lesioned and
contralateral nonlesioned nigral cells in each animal was
performed to determine the number of surviving DA cells 4
weeks after lesioning. To analyze whether minocycline was
efficient in reducing the number of nigral cells that
succumbed to 6-OHDA exposure, we first computed the
difference in cell numbers by subtracting the nigral cell
count of the lesioned side from the nigral cell count of the
contralateral nonlesioned side in each animal (Fig. 4A). We
then compared the mean difference measures among the
three groups (after, before and control). A one-way analysis
of variance showed that the mean cell differences were
not the same among the three groups (P = 0.034). The
mean cell difference in the controls was 6483 ± 782. The
mean cell difference in all minocycline-treated animals
(after and before groups combined) was 4388 ± 446. A twosided
t test generated a P value of 0.047 (P < 0.05), implying
the effectiveness of the minocycline treatment. We next
examined whether early minocycline treatment (before
group) or late minocycline treatment (after group) gives
the best protection for cells against 6-OHDA exposure. We
BRAIN RESEARCH 1093 (2006) 198– 207
compared the mean cell differences of each minocycline
group (after or before) separately with the control group
values using a two-sided t test. The observed mean cell
differences were 3550 ± 628 and 5065 ± 476 in before and
after animals, respectively. The early minocycline (before)
treated group demonstrated a significant reduction in cell
count loss (P < 0.05) compared to the controls, while the
difference between the late (after) minocycline subjects
and controls was not significant (P > 0.1). These data imply
that fewer nigral cells succumbed to 6-OHDA-induced
toxicity in animals treated with minocycline, particularly
those that began the tetracycline three days prior to
lesioning.
Cell size measurements on randomly selected neurons
for each animal (>50 neurons/animal) revealed a significant
difference in cell size in the lesioned nigra between the
three groups (Fig. 4B). The before group had significantly
smaller neurons than the after group (746.87 ± 35.121
versus 897.07 ± 27.447 square pixels, respectively). The
smallest cell size measurements were obtained in the
controls (700.62 ± 185.240 square pixels). Significant cell
size differences were observed between the before and
after group (P < 0.001) as well as between the after and
controls (P < 0.0001). In addition, remaining nigral cells in
the after group appeared more robust with thick neuritic
processes, suggesting intact neuronal viability (Fig. 3C).
Thus, it appeared that the cell size, but not the cell
numbers, reflected the alterations seen in apomorphineinduced
behavior (see Fig. 3). Subsequent densitometric
measurements of TH-positive nigral fibers of all three
groups revealed that animals treated with minocycline
24 h after lesioning displayed more numerous thick
sprouting processes compared to those treated with
minocycline 3 days before or saline only (Fig. 4C). This
Fig. 3 – Minocycline increases TH-immunoreactive cell size and fiber density in lesioned nigras. Saline-treated animals display
typical degenerative profiles including small, weakly stained cell bodies (A) and sparse dendritic processes (D). However,
minocycline treatment before lesioning (B) and after lesioning (C) appears to enhance neuronal cell size and fiber density,
particularly in the after group. Panels (D), (E), and (F) illustrate lateral nigral fibers of saline-, minocycline before-, and minocycline
after-treated groups, respectively. Note the robust fiber sprouting and density in the minocycline-treated groups, particularly in
the after lesion group (F). (A, B, C: 60× magnification, scale bar = 20 μm; D, E, F: 40× magnification, scale bar = 15 μm).
201

202 BRAIN RESEARCH 1093 (2006) 198– 207
Fig. 4 – Minocycline reduces TH-positive cell loss and
increases the size and fiber density of remaining nigral cells.
The degree of cell loss is more attenuated in the minocycline
before group than the after group, compared to the saline
controls (A, *P < 0.05). However, close inspection reveals that
animals receiving minocycline after lesioning contained
larger, more healthy looking cells than either the minocycline
before or saline controls (B, *P < 0.001, **P < 0.0001). In
addition, the lesioned nigras of all minocycline-treated
animals contained more dense sprouting fibers compared to
controls, especially in the after group (C). See text for details.
was particularly evident in the lateral nigra of all animals
analyzed.
3. Discussion
Apomorphine-induced rotational results and TH immunohistochemistry
analyses support our hypothesis that minocycline
reduces the number of rotations and spares DA-containing
nigral neurons in 6-OHDA unilaterally lesioned rats. We
utilized apomorphine to evaluate the extent of DA depletion
because it serves as a more reliable predictor of lesion efficacy
following 6-OHDA administration and avoids the possibility of
sensitization associated with D-amphetamine (Hudson et al.,
1993; Carman et al., 1991; Casas et al., 1988). Animals receiving
daily minocycline beginning 3 days prior to lesioning rotated
almost half as many times as their saline-treated counterparts.
Surprisingly, those animals that began their regimen of
minocycline 24 h after lesioning displayed over a 50%
reduction in apomorphine-induced rotations compared to
their corresponding saline-treated lesioned counterparts,
suggesting that minocycline optimally spared DA depletion
when administered after surgical lesioning. Stereologic counts
and cell size measurements demonstrated a greater sparing of
neurons on the lesioned side of animals that received
minocycline before the lesion, although these cells displayed
a more atrophied profile. In contrast, fewer nigral cells
remained in animals receiving minocycline after the lesion.
Interestingly, the remaining nigral cells in animals treated
with minocycline after lesioning appeared much larger than
the lesioned nigral cells in the other two groups and contained
numerous thick, darkly stained neuronal fibers, particularly in
the lateral nigral area. It is possible that minocycline administered
24 h after lesioning promoted healthier surviving THpositive
nigral cells, compared to animals that began their
treatment three days prior to lesioning.
When 6-OHDA injections are administered into the MFB,
there is minimal variability, and there is >90% success in
obtaining animals with more than 90% reduction in striatal DA
levels using this method (Hudson et al., 1993; Bowenkamp et
al., 1995; Granholm et al., 1997). Thus, the technique is reliable
and reproducible. Extensive use of 6-OHDA in our laboratory
and from other investigators has clearly demonstrated that
sham-operated controls do not display neurodegenerative
characteristics typically present in toxin-treated subjects. In
recent studies in our laboratory utilizing minocycline, Hunter
et al. reported that the number of remaining cells in shamlesioned
animals treated with minocycline did not differ from
that in sham-lesioned animals receiving saline (Hunter et al.,
2004b), suggesting that minocycline did not alter the basal
histological profile observed in host brains. Based on the fact
that we found no effects of minocycline in control animals
after up to 3 months of treatment (Hunter et al., 2004b), we
opted to not include sham-lesioned controls in the present
study. In addition, inspection of the contralateral histological
data among all groups in this study corroborates the rationale
to exclude sham-lesioned animals in that there are no
observable effects of minocycline on the unlesioned side of
the brain.
In order to interpret the data presented here, one must
consider the time course of degeneration in nigral dopaminergic
neurons following a 6-OHDA lesion. Earlier studies
using 6-OHDA have determined that the behavioral phenotype
begins 1 week following toxin administration, when there
is a greater than 60–80% depletion of striatal DA; this
behavioral phenotype is fully evident at about 4 weeks
(Ungerstedt and Arbuthnott, 1970; Sauer and Oertel, 1994;
Gerlach and Riederer, 1996). In a previous study to determine
the temporal pattern of acute phenotypic and degenerative
alterations following 6-OHDA lesions in the MFB of rats, Zuch
et al. (Zuch et al., 2000) demonstrated that events leading to
dopaminergic neuronal loss begin almost immediately

following introduction of the toxin. FluoroJade staining, a
marker for cell death, was evident in the ipsilateral substantia
nigra pars compacta as early as 6 h postlesion, peaking at 48 h
following toxin administration. Tyrosine hydroxylase immunohistochemistry
of corresponding sections revealed that
degenerative alterations to DA neurons were evident at 6 h
postlesion and progressed to a dramatic loss of TH immunoreactivity
in the ventromedial substantia nigra pars compacta
by 96 h postlesion. These data clearly demonstrate that the
degenerative changes following 6-OHDA administration begin
within the first 24 h after exposure.
Early characterizations of the mechanisms of 6-OHDAinduced
degeneration have established that the toxin is
oxidized to reactive oxygen species, affects apoptosis (Choi
et al., 1999; Lotharius et al., 1999) and promotes the
production of proinflammatory cytokines (Mladenovic et al.,
2004; Mogi et al., 1999). There is well documented evidence
that minocycline imparts its anti-inflammatory properties by
inhibiting the activation of microglia and attenuating the p38
MAPK cascade with resultant reduction of inflammatory
cytokine synthesis (Yrjänheikki et al., 1998; Tikka et al.,
2001; Wu et al., 2002). These anti-inflammatory properties
were originally identified in studies of brain ischemia
(Yrjänheikki et al., 1998; 1999). In addition, previous reports
reveal that minocycline is capable of blocking components of
pro-apoptotic pathways, including a reduction in the formation
of caspase-3 and caspase-1 (Zhu et al., 2002; Wang et al.,
2003; Arvin et al., 2002; Chen et al., 2000). It is likely that these
events are being influenced by the minocycline administration
in our study. Therefore, this drug may be an effective tool
in deterring both caspase-dependent and -independent
components of neurodegenerative cascades.
It is possible that the differences in toxin susceptibility
observed in this study could be due to an interaction between
minocycline and 6-OHDA such that the full effect of the toxin
may not have been realized. For instance, it is evident that
the reduction in number of lesioned nigral cells by 6-OHDA
was more attenuated by minocycline in the before than the
after group. However, an interaction between minocycline
and 6-OHDA is unlikely. Tetracyclines such as minocycline
impart their bacteriostatic effects by binding to a single high
affinity site on the bacterial 30S ribosome within the cell,
effectively blocking the entry of aminoacyl transfer RNA into
the A site of the ribosome, preventing elongation of amino
acid residues into polypeptide chains, and inhibiting protein
synthesis (Zhanel et al., 2004). As for its anti-inflammatory
properties, the literature fully supports two mechanisms of
cell death prevention by tetracyclines: potent inhibition of
innate and adaptive immunity components and obstruction
of apoptotic cascades (Domercg and Matute, 2004). Finally, a
comprehensive consultation with the Medical University of
South Carolina Drug Information Center has revealed no
reported documentation or theoretical interaction between
minocycline (or other similar tetracyclines) and DA or DA
analogs, such as L-DOPA and hydroxydopamine, based on 12
major drug references, including many primary literature
sources (personal communication).
Current publications have proposed that the proinflammatory
response of microglial cells in the brain may consist of
both deleterious and beneficial responses (Vallat-Decouve-
BRAIN RESEARCH 1093 (2006) 198– 207
203
laere et al., 2003; Teismann et al., 2003). Most recently, a study
examining the effects of blueberry- and spirulina-enriched
diets on striatal dopamine recovery following 6-OHDA lesioning
revealed that an early phase of activated microglial
response is associated with successful dopaminergic protection
(Strömberg et al., 2005). When the activated microgliaassociated
OX-6 expression was suppressed using antisense to
TNFα, more severe dopamine depletion was observed. Alternatively,
others have reported that delayed inhibition of
cytokine release and microglial activation following 6-OHDA
lesioning will result in less severe dopamine destruction
(Gemma et al., 2003). Collectively, these data support the
possibility of an initial beneficial microglial response to
neuroinflammation and, if left unabated, subsequent detrimental
effect. If this phenomenon holds true, it may explain
why we observed a greater loss of TH-positive nigral cells but
better rotational phenotype in the minocycline after group,
compared to the minocycline before group. Because the early
beneficial wave was reduced in the before group but was
present in the after group, the remaining nigral cells may be
more healthy with enhanced fiber arborization in the latter,
suggesting continued nigral destruction but more viable
dopaminergic function because this first wave of glial
response is preserved. Indeed, upon examination of all
histological preparations for this study, we observed a marked
increase in size and fiber density of the remaining TH-positive
cells in animals treated with minocycline 24 h after 6-OHDA
lesioning, compared to the other two groups. Neuronal cell
size measurements revealed that, although stereology identified
more TH-positive cells in the lesioned nigra of animals
exposed to minocycline before neurotoxin exposure, these
cells displayed more classic apoptotic profiles (e.g., smaller,
ruptured cells with dense, clumped vesicular bodies in the
neuropil, etc.) than cells in the after group. Additionally, many
of the lesioned nigral neurons in the before group displayed
fewer or absent neuritic processes when compared to the after
group. We propose that the better outcome in apomorphine
rotational analyses in the after group may be due to fewer but
healthier TH-containing neurons remaining because the
initial beneficial wave of microglial response is preserved. A
more in-depth examination of the timing of minocycline
administration on neuronal viability relative to toxin exposure
is currently underway in our laboratory, using markers for cell
death and degeneration.
Previous work demonstrates that inflammation is a key
component of the degenerative cascade associated with
Parkinson's disease (McGeer et al., 2001; Cicchetti et al.,
2002). Microglial activation, in particular, has been previously
implicated as a key mediator of the inflammatory response in
the brain and has been observed in the brains of PD patients
(McGeer et al., 1988). Similar observations have been reported
in PD animal models, such as the 6-OHDA-induced Parkinsonian
rat (Akiyama and McGeer, 1989; He et al., 1999). Indeed,
we also observe this phenomenon in our 6-OHDA-lesioned
animals following CD11b immunohistochemistry for microglial
status (data not shown). Although the activation of
resting microglia is a normal event necessary for maintaining
neuronal homeostasis, unabated gliosis associated with a
pathological state such as PD may exacerbate the neurodegenerative
processes already established (Hirsch et al., 1998,

204 BRAIN RESEARCH 1093 (2006) 198– 207
2003; Stoll et al., 2002). Because activated microglia are
associated with phagocytosis and the liberation of cytotoxic
molecules (e.g., reactive oxygen species) and proinflammatory
cytokines (e.g., tumor necrosis factor α, interleukin-1β,
interferon-γ), it is probable that chronic microglial activation
will significantly contribute to the destruction of resident
cells. Because minocycline easily passes through the blood–
brain barrier, it may be indicated as a deterrent to the
inflammatory environment observed in diseases like Parkinson's,
Alzheimer's and others.
These data are concordant with previous literature that
supports the therapeutic potential of minocycline against
neurodegenerative events. Our findings suggest that the time
of minocycline delivery relative to neuropathogenic event
may determine the effectiveness of this drug for therapy.
However, recent studies suggest that minocycline treatment
may lead to adverse effects clinically, especially after longterm
use (Porter and Harrison, 2003; Wolthuis et al., 2003; Abe
et al., 2003; Bradfield et al., 2003). Although these reports
have been generated in the clinical setting, it is readily
apparent that further, comprehensive evaluations of longterm
minocycline use must be pursued before this tetracycline
can gain universal acceptance to treat neurodegenerative
diseases.
4. Experimental procedures
4.1. Minocycline protocol
Thirty male Fisher 344 rats (200 grams) were lesioned with 6-
OHDA (see details below) and randomly divided into three
groups (n = 10). One group received daily IP injections of
45 mg/kg of minocycline (ICN Biomedicals, Inc.) beginning 3
days prior to lesioning. Another group received daily IP
injections of 45 mg/kg of minocycline beginning 24 h after 6-
OHDA lesioning. The remaining control group received daily
IP saline injections either 3 days before or 24 h after lesioning.
All animals continued to receive daily injections of 45 mg/kg
of minocycline or saline for 1 week following surgical
procedures. After this period, all injections were reduced to
every other day for the remainder of the 4-week rotational
analysis phase. All experiments were carried out in accordance
with the National Institutes of Health Guide for the
Care and Use of Laboratory Animals (NIH Publications No. 80-
23; revised 1996).
4.2. 6-Hydroxydopamine lesioning
All animals were anesthetized with chloral hydrate (400 mg/kg
in 0.9% NaCl, IP) and stereotaxically lesioned in the right
medial forebrain bundle with 9 μg/4 μl of 6-OHDA in saline
containing 0.02% ascorbate (coordinates −4.4 mm AP, +1.3 mm
ML, and −7.8 mm DV from the dural surface; see Granholm et
al., 1997). The 6-OHDA neurotoxin was administered over
4 min followed by a 2-min period delay prior to needle
withdrawal to allow for toxin absorption into the brain
tissue. Animals were provided with acetaminophen in their
drinking water for 24 h to alleviate potential postsurgical
discomfort.
4.3. Rotational evaluation
All animals included in this study were evaluated for
apomorphine induced rotational behavior beginning 1 week
following 6-OHDA administration and tested once a week
thereafter for a total of 4 weeks. Briefly, animals were placed in
rotometer bowls (Hudson et al., 1993) and secured to the
counting sensor by a thoracic harness. Subjects were allowed
to acclimate to the environment for 10 min, then administered
a subcutaneous injection of apomorphine (0.05 mg/kg).
Subsequent rotational behavior was then recorded for 1 h.
Following rotational testing, animals were released from their
harnesses, removed from the rotometer bowls, and returned
to their cages.
4.4. Immunohistochemistry
After 4 weeks, animals were deeply anesthetized with
Halothane (Halocarbon Laboratories). Rats were then perfused
through the aorta with 0.9% saline followed by 4% paraformaldehyde
and transferred to 30% sucrose in phosphatebuffered
saline the next day. Sections (45 μm) were obtained
from the nigral region of each animal using a sliding
microtome (Microm). Sections were processed for free-floating
immunohistochemistry using antibodies against tyrosine
hydroxylase (1:1000, Pel-Freeze Biologicals). Briefly, the sections
were washed and incubated for 48 h with TH antibodies.
The sections were washed again in Tris-buffered saline (TBS)
and incubated with the secondary antibody reacted with the
ABC solution (Vectastain, Vector Laboratories) and diaminobenzidine
(Sigma) (DAB, 0.0300 g/100 ml imidazole-acetate
buffer solution, Fisher Scientific). Sections were mounted on
glass slides and cover slipped. Control sections were processed
without primary or secondary antibodies. Sections
from all groups were processed simultaneously in the same
bath, using mesh-bottom incubation wells, to avoid intersection
staining variability (for further details, see Granholm
et al., 2000).
4.5. Stereologic analysis
Estimated numbers of TH-positive cells in the substantia nigra
of all animals were obtained using the optical fractionator
stereological method (StereoInvestigator, MicroBrightfield,
Inc.) according to previously published protocols (Hunter et
al., 2004a). Briefly, the optical fractionator system consisted of
a computer-assisted image analysis system including a Nikon
Eclipse E-600 microscope hard coupled to a Prior H128
computer-controlled x–y–z motorized stage, an Olympus 750
video camera system, a Micron Pentium III computer and the
Stereoinvestigator stereological software (Microbrightfield
Inc.). The region of interest was outlined under low magnification
and the number of neurons in the outlined region
measured with a systematic random design of disector
counting frames. A 60× objective lens with a 1.4 numerical
aperture was used to count and measure individual cells
within the counting frames. The counting brick was approximately
20 μm thick, and upper and lower gard zones were
excluded from counting. Means and standard error of the
mean (SEM) of estimated cell counts were calculated from the

aw data using the stereology software system. The total cell
number for each lesioned nigra was compared to the cell
number of the contralateral nonlesioned nigra. These comparisons
were expressed as a percent decrease of cell number
in the lesioned nigra compared to the respective nonlesioned
nigra in the same animal.
4.6. Cell size measurements
In order to determine if the surviving neurons were healthy
in all groups or had undergone atrophic alterations, we
characterized the size of lesioned nigral neurons using Scion
Imaging software. Fifty neurons from the lesioned side of
each animal randomly picked from 5 sections for each
animal were measured. Only cells displaying a clearly
defined nucleus and/or at least two neuritic processes were
considered for measurement. The NIH Image software
system is written using Think Pascal from Symantec
Corporation. The perimeter of each neuron was measured
using a scale calibration feature. In addition, fiber density
measurements were made of the remaining lesioned nigral
neurons in all groups using the Scion Imaging software
package.
4.7. Statistical analysis
The apomorphine-induced rotation data were analyzed using
a one-way ANOVA for all 3 groups followed by several post hoc
two-sample t tests of the mean rotations for different group
pairs. We further analyzed time (week) effect and treatment
effects jointly by using a two-way ANOVA followed by a
several post hoc paired t tests to find which group demonstrated
significant increase in rotation from week 1 to week 4.
In addition, regression analysis was used to test for linear
trends in rotations over time for each group. For the cell count
results, the mean of the difference between lesioned and
nonlesioned nigras of each animal for each group was
analyzed using a two-sided t test. Descriptive statistics,
including the mean, median and mode, for each group's cell
size and fiber density measurements were obtained. The
analysis was done using SAS statistical software (SAS
Institute, Inc.) and Statview for Windows.
Acknowledgments
This work was made possible by a grant from the Murray
Center for Parkinson's Disease Research and Related Disorders.
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