Eli Lilly & Co. Ltd., Lilly Research Centre, Windlesham, Surrey,
United Kingdom (A.B., C.A.H., G.M.W., M.A.W., M.F.O., M.J.O.); and Eli
Lilly & Co. Ltd., Lilly Corporate Center, Indianapolis, Indiana
(N.M.J., A.E.K., J.A.M., P.L.O., D.D.S., D.L.)
The mechanisms underlying the neuroprotective effects of the group II
metabotropic glutamate receptor (mGluR) agonist LY379268 were
investigated in a gerbil model of global ischemia. LY379268 (10 mg/kg
i.p.) 30 or 60 min after 5-min bilateral carotid artery occlusion
(BCAO) attenuated the ischemia-induced hyperactivity and provided
protection in the CA1 hippocampal cells. This neuroprotective effect
was maintained (P < .001) when histological
analysis was performed 14 and 28 days after BCAO. Furthermore, 24- or
48-h pretreatment with LY379268, 10 mg/kg i.p., before 5-min BCAO
markedly reduced (P < .001 and
P < .05, respectively) the damage to CA1 hippocampal neurons. This result is consistent with the induction of
neuroprotective factors or a very long brain half-life. To study the
possible induction of neuroprotective factors as contributing to this
action of LY379268, brains were examined for expression of neurotrophic
factors. Results indicated that LY379268 (10 mg/kg i.p.) failed to
alter the expression of transforming growth factor-
, brain-derived
neurotrophic factor, nerve growth factor, and basic fibroblast growth
factor in the hippocampal regions of brains taken from gerbils
sacrificed at 6, 24, 72, and 120 h postinjection. The new group II
mGlu antagonist, LY341495, administered 1 h before 5-min BCAO,
attenuated the neuroprotective effect of LY379268 administered 24 h before 5-min BCAO. Complementary pharmacokinetic studies showed that
a significant receptor-active concentration persisted in the brain
24 h after LY379268 10 mg/kg i.p. We conclude that group II mGluR
occupancy, rather than induction of neuroprotective factors, explains
the long-lasting neuroprotective effect of LY379268 in the gerbil model
of global ischemia.
 |
Introduction |
Glutamate
is the major excitatory neurotransmitter in the mammalian central
nervous system, activating ionotropic glutamate (iGlu) and metabotropic
glutamate (mGlu) receptors. It also is thought to play a central role
in acute neurodegeneration after cerebral ischemia (Meldrum and
Garthwaite, 1990
). However, for many years, investigators have focused
mainly on the roles of iGlu receptors and, in particular, of
N-methyl-D-aspartate (NMDA), 
-amino-3-hydroxy-5-methyl-isoxazole-4-propionic acid (AMPA), and
kainate receptors in ischemia (McCulloch, 1991; Gill and Lodge, 1997).
Because ionotropic receptors play such a critical role in fast synaptic
transmission, it has proved difficult to block these receptors without
side effects, and to date no such compounds have therapeutic use in
human stroke. Recent advances have provided selective ligands for
metabotropic glutamate receptor (mGluR) subtypes (Pin et al., 1999;
Schoepp et al., 1999), allowing further investigation of the proposed
role of mGluRs in aspects of neurodegeneration (Schoepp and Conn, 1993;
Buisson et al., 1996; Nicoletti et al., 1996).
To date, eight subtypes of the G protein-coupled mGluRs have been
cloned and classified into three groups according to their second-messenger association, sequence homology, and agonist
selectivity (Pin and Duvoisin, 1995; Pin et al., 1999). Group II (mGlu2
and -3) and group III (mGlu4, -6, -7, and -8) mGluRs are negatively coupled to adenyl cyclase and thought to act as presynaptic
autoreceptors, regulating glutamate transmission (Shigemoto et al.,
1995). Recent evidence indicates that mGlu3 receptors are also
expressed by astrocytes and glia (Petralia et al., 1996). Activation of
group II mGluRs is reported to protect neurons against excitotoxic
degeneration by the inhibition of glutamate release (Buisson and Choi,
1995; Buisson et al., 1996). In support of this idea, the selective group II mGluR agonist LY354740 (Schoepp et al., 1997) reduces veratradine-evoked striatal amino acid release (Battaglia et al., 1997)
and field excitatory postsynaptic potentials in rat hippocampal slices
(Kilbride et al., 1998).
Although reduction of glutamate release is an attractive hypothesis,
there is a body of evidence in vitro that suggests this may not always
account for the neuroprotective activity of group II mGluR agonists.
For example, Bruno et al. (1997) suggested that astrocytes, after
exposure to group II mGluR agonists, produce a heat-sensitive factor in
the culture medium, which itself has neuroprotective properties in
cortical cells. More recently, this group reported that transforming
growth factors-1 (TGF-1) and -2 were released from astrocytes
exposed to group II mGluR agonists and that antibodies that neutralized
the actions of TGF-1 or -2 prevented the neuroprotective effects
of DCG-IV and 4C3HPG in cultured cortical neurons (Bruno et al., 1998).
Group II mGlu agonists are also reported to be neuroprotective in
vivo (Chiamulera et al., 1996; Miyamoto et al., 1997), but these
earlier compounds were not very selective. A more selective group II
agonist, LY354740, has been shown to provide some neuroprotection in
gerbil global ischemia (Bond et al., 1998) but none in rat focal
ischemia (Lam et al., 1998). Recently, a more potent and highly
selective group II agonist, LY379268
[(1R,4R,5S,6R)-2-oxa-4-aminobicyclo[3.1.0]hexane-4,6-dicarboxylate], was discovered that has EC50 values of 2.69 ± 0.26 and 4.48 ± 0.04 nM at human mGluR2 and mGlu3 receptors,
respectively (Monn et al., 1999). LY379268 provided neuroprotection
against NMDA-mediated cell death in vitro (Kingston et al., 1999) and
almost complete protection against CA1 hippocampal damage after global
ischemia in gerbils but failed to show neuroprotection against focal
ischemia in the rat (Bond et al., 1999b). Therefore, the mechanism of
neuroprotection in the gerbil model remains to be elucidated.
In this study, we extended our original findings by investigating
neuroprotection in terms of both histological and functional behavioral
improvement in gerbils subjected to 5-min bilateral carotid artery
occlusion (BCAO). We have undertaken detailed time course studies to
evaluate the effects of LY379268 both during the induction of ischemic
brain injury and at later time points after occlusion. To understand
the mechanism of action of LY379268, we investigated the hypothesis
that group II mGlu agonists may induce neurotrophic factors with
neuroprotective properties, by examining its effects on the expression
of TGF-1, TGF-2, brain-derived neurotrophic factor (BDNF), nerve
growth factor (NGF), and basic fibroblast growth factor (bFGF) in brain
tissue using immunocytochemistry. We have also investigated the
interaction of LY379268 with the induction of "ischemic
tolerance" in the gerbil (Kirino et al., 1991). Finally, we
examined whether the protection afforded by 24-h pretreatment with
LY379268 is blocked by the new selective group II antagonist, LY341495
(Kingston et al., 1998) and made measurements of brain levels of
LY379268 at various time points after drug administration.
| |
Materials and Methods |
Animals and Surgery.
Male Mongolian gerbils (Bantin and
Kingman, Hull, UK) at least 3 months old and weighing in excess of
60 g were maintained in standard lighting conditions with food and
water available ad libitum. The animals were anesthetized with a 5%
halothane/oxygen mixture and maintained using 2% halothane delivered
with oxygen at 1 l/min via a face mask throughout the operation.
Through a midline cervical incision, both common carotid arteries were
exposed, freed from surrounding connective tissue, and clamped for 5 min, except for preconditioning experiments where clamps were applied for 2 min. At the end of the occlusion period, blood flow was reestablished. In sham-operated animals, the arteries were exposed but
not occluded. The wound was then sutured, and the animals were allowed
to recover. Throughout surgery, body temperature was maintained at
37°C using a K-TEMP temperature controller/heating pad (International
Market Supply, Cheshire, UK). After surgery, the animals were placed in
a four-compartment Thermacage (Beta Medical and Scientific, UK) that
maintained the environmental temperature at 28°C, and rectal
temperatures were measured for a 6-h period after occlusion.
General Histology.
Five days (or, in some experiments, 6, 24, 72, or 120 h or 14 or 28 days) after surgery, the animals were
perfused transcardially with 30 ml of 0.9% saline followed by 100 ml
of 10% buffered Formalin solution. The brains were removed and placed
in 10% Formalin for 3 days, processed, and embedded in paraffin wax.
Coronal sections (6 µm) were taken 1.5, 1.7, and 1.9 mm caudal to
bregma using a microtome (Leitz 1400 sledge microtome; Leica,
Milton-Keynes, UK). The slices were stained with H&E, and the neuronal
density in the CA1 subfield of the hippocampus was measured using a
microscope with grid lines (0.05 × 0.05 mm) as described
previously (O'Neill et al., 1998). The neuronal density was expressed
as number of viable cells per millimeter of CA1 hippocampus.
Statistical analysis of histological data was assessed using a
two-tailed unpaired Student's t test, with P
values of <.05 being considered statistically significant.
Locomotor Activity.
At 24 and 120 h postocclusion,
locomotor activity was measured in clear Perspex boxes (30 × 30 × 30 cm) with a metal base with a 2-cm covering of fine
sawdust. Each of the 16 cages had five, equally spaced horizontal
photocell beams 5.0 cm above the cage floor. Each beam break was
recorded as a photocell count. All the boxes were individually
connected to a Compaq PC, and the photocell interruptions were recorded
as the number of counts using software provided by Greenacre and Misac
Instruments (Ware, Hertfordshire, UK). All experiments were
videotaped for subsequent analysis. The animals were placed in
individual boxes for 60 min, and photocell interruptions were recorded
every minute.
Immunocytochemical Methods.
Immunohistochemical staining was
performed on paraffin-embedded coronal sections (6 µm) of
hippocampus. Hippocampal sections were deparaffinized in xylene and
rehydrated in ethanol and distilled water. Sections were incubated in
0.3% hydrogen peroxide
(H2O2) for 30 min to block
endogenous peroxidase activity, rinsed, incubated in pepsin/HCl for 30 min, rinsed in water, and then rinsed in PBS. Normal goat serum (1.5%
in PBS) was used as a preblock for 20 min, after which the sections
were incubated with the various primary antibodies: TGF-1 and -2
(Santa Cruz Biotechnology, Santa Cruz, CA) 1:100 for 2 h; bFGF
(Biogenex, San Ramon, CA) ready-to-use antibody for 2 h; and NGF
at 1:2000 and BDNF at 1:500 (Chemicon International, Temecula, CA),
both for 24 h at room temperature in a humidified immunostaining
chamber. Slides that were exposed to the bFGF antibody underwent a high
temperature antigen retrieval pretreatment with Citra solution
(Biogenex) immediately before the blocking step in 0.3%
H2O2. Briefly, sections were rinsed in water, placed in Citra solution, microwaved on high
until solution boiled, and then set to boil for 10 s every 30 s for a period of 15 min. Slides were allowed to cool for 30 min before
continuing the procedure.
After the incubation with primary antibodies, sections were
rinsed in PBS (3 × 5 min) and incubated in biotinylated secondary antibody (goat anti-rabbit Vector ABC Elite kit; 1:200 dilution in PBS
with 1.5% normal goat serum; Vector Laboratories, Peterborough, UK)
for 30 min at room temperature, after which they were again washed in
PBS (3 × 5 min). Subsequently, sections were incubated in
avidin-conjugated horseradish peroxidase (Vector ABC Elite kit, diluted
in PBS) for 30 min at room temperature and washed in PBS (3 × 5 min). Finally, sections were incubated with nickel-enhanced 3,3'-diaminobenzidine chromagen (Vector kit) until color developed and
then washed in tap water, dehydrated, coverslipped using DPX mountant, and examined under a light microscope. A detailed examination of the immunostaining product observed in the CA1 area of the hippocampus was performed, and digitized images were obtained using an
Optimus 5.2 image analysis system (Datacell, Finchampstead, England). The number of immunoreactive cells in the CA1 area was indicated by the use of the scoring system in which 
indicates no staining; +, little staining; ++, moderate staining; +++, intense staining; and ++++, most intense staining.
Bioavailability Studies.
Gerbils were dosed with LY379268 at
10 mg/kg i.p. and housed in incubator cages at 28°C for up to 8 h. At 15 and 30 min and 1, 2, 4, 6, 8, and 24 h after drug
administration, animals were sacrificed and brain levels of LY379268
were determined by HPLC/tandem mass spectrometry.
Ischemic Tolerance.
Gerbils were subjected to a 2-min
"preconditioning" ischemia and 48 h later were subjected to a
5-min "test" ischemia. Animals subjected to a 2-min ischemia before
the 5-min ischemia are protected with ischemic tolerance. The effects
of LY379268 (10 mg/kg i.p.) 30 min after the initial 2-min ischemia
were assessed.
| |
Results |
Neuroprotective Effects of LY379268: Locomotor Activity.
Locomotor activity was assessed 24 and 120 h postocclusion.
Results indicated that 5-min BCAO gerbils exhibited a large increase in
locomotor activity 24 h postocclusion (over a 60-min period) in
comparison with sham-operated animals (Fig.
1A). LY379268 produced a marked
attenuation (P < .001) in the ischemia-induced
hyperactivity when administered 30 or 60 min after occlusion (Fig. 1, A
and B).
View larger version (25K):
[in this window]
[in a new window]
|
Fig. 1.
The effects of LY379268 on ischemia-induced
hyperactivity 24 h postocclusion. Locomotor activity was measured
for a 60-min period and is illustrated as the number of counts
(mean ± S.E.; n = 8 animals/group) in 10-min
bins at 10, 20, 30, 40, 50, and 60 min (A) or total locomotor activity
counts for a 60-min period (B). Sham-operated animals habituated
rapidly to the apparatus and maintained low locomotor activity counts.
In contrast, 5 min of BCAO caused a large increase in locomotor
activity counts that was sustained for the 60-min period. LY379268 (10 mg/kg i.p.) commenced 30 or 60 min after the BCAO attenuated the
ischemia-induced hyperactivity (Student's t test).
***P < .001 versus sham control.
+++P < .001 versus BCAO control.
|
|
When tested at 120 h postocclusion, 5-min BCAO gerbils also
exhibited an increase in locomotor activity (over a 60-min period) in
comparison with sham-operated animals (Fig.
2A). This hyperactivity was much smaller
than that observed at 24 h (Fig. 2B). LY379268 attenuated
(P < .05) the ischemia-induced hyperactivity when
administered 30 or 60 min after occlusion (Fig. 2, A and B).
View larger version (22K):
[in this window]
[in a new window]
|
Fig. 2.
The effects of LY379268 on ischemia-induced
hyperactivity 120 h postocclusion. Locomotor activity was measured
for a 60-min period and is illustrated as the number of counts
(mean ± S.E.; n = 8 animals/group) in 10-min
bins at 10, 20, 30, 40, 50, and 60 min (A) or total locomotor activity
counts for a 60-min period (B). Sham-operated animals habituated
rapidly to the apparatus and maintained low locomotor activity counts.
In contrast, 5 min of BCAO caused a large increase in locomotor
activity counts that was sustained for the 60-min period. LY379268 (10 mg/kg i.p.) commenced 30 or 60 min after the BCAO attenuated the
ischemia-induced hyperactivity (Student's t test).
*P < .05, **P < .01, ***P < .001 versus sham control.
+P < .05 versus BCAO control.
|
|
Neuroprotective Effects of LY379268: Histological Assessment.
At the end of behavioral testing, the brains were removed for
histological analysis. Results indicated that there was a severe loss
of CA1 hippocampal cells 120 h postocclusion. LY379268 protected against this ischemia-induced cell death when administered 30 or 60 min
postocclusion. The protection was greatest when administration was
initiated 30 min after occlusion (85%, P < .001), but
significant protection was also observed when the compound was
administered 60 min postocclusion (47%, P < .05) as
shown in Fig. 3.
View larger version (40K):
[in this window]
[in a new window]
|
Fig. 3.
mGluR selectivity profile of LY379268 and the effects
of LY379268 (10 mg/kg i.p.) administered 30 or 60 min after occlusion
on the neuronal density at three stereotaxic levels in the CA1 region
of the hippocampus 5 days after surgery. Histological results are
expressed as mean ± S.E. viable cells per millimeter of CA1
hippocampal region (n = 8 animals/group). The 5-min
BCAO produced severe damage to the CA1 region (P < .001), whereas LY379268 produced protection when administration was
initiated 30 min after (P < .001) or 60 min after
(P < .05) occlusion (Student's t
test). ***P < .001 versus sham control.
+P < .05, +++P < .001 versus 5-min BCAO
control.
|
|
In separate studies, we examined the effects of LY379268 (10 mg/kg
i.p., 30 min postocclusion), on histological outcome 6, 24, 72, and
120 h and 14 and 28 days post-BCAO (5 min). Results indicated that
there was no cell damage in any of the treatment groups at 6 and
24 h postocclusion (Fig. 4).
However, we did observe a large loss in CA1 cells in 5-min BCAO animals
at 72 and 120 h and 14 and 28 days postocclusion
(P < .001). This form of "delayed neuronal death"
is well established in this gerbil model of transient global cerebral
ischemia (see Kirino, 1982). This 30-min postocclusion treatment with
LY379268 provided significant protection (P < .001) against this ischemia-induced cell death at all of the time points (Fig. 4). The results at 14 and 28 days indicate that LY379268 is not
delaying the onset of cell death but rather is producing a sustained
neuroprotection against BCAO-induced damage to CA1 cells.
View larger version (46K):
[in this window]
[in a new window]
|
Fig. 4.
The effects of LY379268 (10 mg/kg i.p.) administered
30 min after occlusion on the neuronal density in the CA1 region of the
hippocampus (1.7 mm caudal to bregma) at 6 h and 1, 3, 5, 14, and
28 days after surgery. Histological results are expressed as mean ± S.E. viable cells per millimeter of CA1 hippocampal region
(n = 8 animals/group). The 5-min BCAO produced
severe damage to the CA1 region (P < .001),
whereas LY379268 produced protection when administration was initiated
30 min after occlusion (P < .001) (Student's
t test). ***P < .001 versus sham
control. +++P < .001 versus 5-min BCAO
control.
|
|
Effect of Pretreatment with LY379268.
To investigate the
potential of LY379268 to induce expression of some neurotrophic
factors, LY379268 (10 mg/kg i.p.) was administered 24 or 48 h
before BCAO to allow time for new protein synthesis to occur. The
results clearly show that LY379268 at 10 mg/kg i.p. produced 93%
neuroprotection (P < .001) when dosed at 24 h and retained significant neuroprotective effects (29%; P < .05) when dosed 48 h before 5-min BCAO (Fig.
5).
View larger version (41K):
[in this window]
[in a new window]
|
Fig. 5.
The effects of LY379268 (10 mg/kg i.p.) administered
24 or 48 h before occlusion on the neuronal density at three
stereotaxic levels in the CA1 region of the hippocampus 5 days after
surgery. Histological results are expressed as mean ± S.E. viable
cells per millimeter of CA1 hippocampal region (n = 8 animals/group). The 5-min BCAO produced severe damage to the CA1
region (P < .001), whereas LY379268 produced
protection when administration was initiated 24 h before
(P < .001) or 48 h before
(P < .05) occlusion (Student's t
test). ***P < .001 versus sham control.
+P < .05, +++P < .001 versus 5-min BCAO
control.
|
|
Neuroprotective Effect of LY379268 on Induction of Ischemic
Tolerance.
This result raised the possibility that activation of
group II mGluRs may underlie the mechanism of ischemic tolerance
(Kirino et al., 1991) (i.e., glutamate released by the
"preconditioning ischemia" could activate group II receptors and
up-regulate some neurotrophic factor or factors). To investigate this,
we coadministered LY379268 with a 2-min preconditioning BCAO,
hypothesizing that if the above mechanism were true, they would act
additively. In fact, LY379268 (10 mg/kg i.p.) produced a marked
reduction (P < .001) in the induced tolerance to BCAO
tested 48 h later (Fig. 6).
View larger version (43K):
[in this window]
[in a new window]
|
Fig. 6.
Effect of LY379268 on induced ischemic tolerance in
the gerbil hippocampus. From left to right, mean ± S.E.
(n = 8) of the number of viable CA1 pyramidal cells
after 1) sham occlusion, 2) a preconditioning 2-min ischemia alone, 3)
a test 5-min ischemia alone, 4) a 2-min ischemia followed 2 days later
by a 5-min ischemia (tolerance), and 5) drug pretreatment before the
2-min preconditioning ischemia, followed 2 days later by a 5-min
ischemia (no drugs). LY379268 was dosed at 10 mg/kg i.p. and produced a
significant reduction (P < .01) in the induced
tolerance. **P < .01, ***P < .001.
|
|
Effects of LY379268 on Induction of Neuroprotective Growth
Factors.
To investigate directly whether there was any
up-regulation of the candidate factors bFGF, BDNF, NGF, TGF-1, and
TGF-2, we examined the effects of 10 mg/kg LY379268 i.p. on their
expression at 6, 24, 72, and 120 h in nonischemic gerbils. Results
show that LY379268 alone did not stimulate the expression of any of
these neurotrophic factors (Table 1).
Representative photomicrographs demonstrating immunostaining for bFGF,
BDNF, NGF, TGF-1, and TGF-2 in LY379268-treated and saline
control-treated gerbils are shown in Fig.
7. In additional experiments, we also
demonstrated that chronic administration (20 mg/kg i.p. for 4 days)
failed to alter the expression of neurotrophic factors in rat brain
(data not shown).
View this table:
[in this window]
[in a new window]
|
TABLE 1
A summary of immunohistochemical staining observed for antibodies to
BDNF, NGF, bFGF, TGF-1, and TGF-2 in tissue from saline and
LY379268 (10 mg/kg i.p.)-treated gerbils at 6, 24, 72, and 120 h
postinjection
The effect of LY379268 on immunohistochemical staining for these
neurotrophic factors was examined by light microscopy in the CA1 area
of the hippocampus of 6-µm sections using 5-min BCAO as a positive
control. Immunostaining product is described on a quantitative scale of
, no staining; +, little staining; ++, moderate staining; +++,
intense staining; and ++++, most intense staining.
|
|
View larger version (96K):
[in this window]
[in a new window]
|
Fig. 7.
Effects of saline or LY379268 (10 mg/kg i.p.) on
TGF-1, TGF-2, BDNF, NGF, and bFGF immunostaining in the
hippocampus 24 h after injection. The 5-min ischemic animal
results at 120 h are included as a positive control.
|
|
Duration of Action of LY379268.
These results suggest the
possibility that LY379268 remains in the brain for more than 24 h
at levels sufficient to activate group II receptors. To investigate
this possibility, we tested whether the new group II mGlu antagonist
LY341495 (Kingston et al., 1998) would prevent the protective effects
of the prior administration of LY379268. Dosing 1 h before 3-min
BCAO, LY341495 (5 mg/kg i.p.) alone did not cause a reduction or
exacerbation of the neuronal damage induced by a mild 3-min ischemia
(Fig. 8). Importantly, however, LY341495
at 5 mg/kg i.p. administered 24 h after LY379268 10 mg/kg i.p. and
60 min before 5-min BCAO reduced the level of neuroprotection from 97 to 20% (P < .001; Fig.
9).This ability of the competitive
antagonist LY341495 to reverse the effects of the agonist under these
conditions indicated that mGlu2/3 receptors were still occupied by the
agonist 24 h after LY379268 dosing.
View larger version (37K):
[in this window]
[in a new window]
|
Fig. 8.
Structure and selectivity of the mGluR antagonist
LY341495 and the effects of LY341495 (5 mg/kg i.p.) administered 1 h before 3-min BCAO on neuronal density at three stereotaxic levels in
the CA1 region of the hippocampus 5 days after surgery. Histological
results are expressed as mean ± S.E. viable cells per millimeter
of CA1 hippocampal region (n = 8 animals/group).
The 3-min BCAO produced damage to the CA1 region (P < .01). LY341495 did not provide any potentiation (or protection)
against this damage (Student's t test).
|
|
View larger version (38K):
[in this window]
[in a new window]
|
Fig. 9.
The effects of LY379268 (10 mg/kg i.p.) administered
24 h before occlusion alone or when dosed in combination with
LY341495 (5 mg/kg i.p., 1 h before 5-min BCAO) on the neuronal
density at three stereotaxic levels in the CA1 region of the
hippocampus 5 days after surgery. Histological results are expressed as
mean ± S.E. viable cells per millimeter of CA1 hippocampal region
(n = 8 animals/group). The 5-min BCAO produced
severe damage to the CA1 region (P < .001).
LY379268 produced almost complete protection when administration was
initiated 24 h before occlusion (P < .001).
This protection provided with LY379268 was prevented by LY341495
(P < .001) administered 1 h before occlusion
(Student's t test). ***P < .001.
|
|
Pharmacokinetic studies also demonstrated that at 24 h after a 10 mg/kg i.p. dose of LY379268, a significant receptor-active concentration of the compound was present in brain tissue (Fig. 10).
View larger version (8K):
[in this window]
[in a new window]
|
Fig. 10.
Line graph showing the brain concentrations of
LY379268 after a single dose of 10 mg/kg i.p. Ordinate, concentration
of LY379268 (ng/g of brain tissue); abscissa, time (h).
|
|
| |
Discussion |
mGluRs are increasingly being considered as targets for the
therapeutic intervention into neurodegenerative disorders, because their activation affects intracellular events that contribute to both
the induction and progression of neuronal damage (Schoepp and Conn,
1993; Buisson et al., 1996; Nicoletti et al., 1996; Bruno et al.,
1998). The lack of selective agents has made it difficult to clarify
the exact contribution of the various mGluRs to neurodegenerative
diseases. However, recently, several new ligands for mGluRs have been
synthesized that have allowed the role of mGluRs in ischemia to be
studied in detail (Pin et al., 1999; Schoepp et al., 1999). It has
recently been demonstrated that the newer group II mGluR agonists
LY354740 and LY379268 are systemically active (Bond et al., 1998,
1999b). Here, we have provided further insight into possible mechanisms
whereby the selective group II mGluR agonist LY379268 has
neuroprotective actions in a model of global ischemia in vivo.
Acute Neuroprotection.
In this study, we extended our previous
finding that LY379268 is neuroprotective by demonstrating that LY379268
provides not only histological protection but also functional
improvement when dosed after ischemia. A large increase in locomotor
activity was observed in control animals after BCAO, as noted
previously by several groups (e.g., Mileson and Schwartz, 1991), and it
has been suggested that this hyperactivity correlates with loss of hippocampal neurons (Anderson et al., 1997). In agreement with this,
both the hyperactivity and the cell loss were considerably attenuated
by LY379268, administered 30 or 60 min after BCAO (Fig. 1-3). We also
demonstrated that the good neuroprotection of CA1 neurons previously
observed with LY379268 (Bond et al., 1999b) is long-lasting. This
maintained neuroprotection is in contrast to the reported short-lasting
effects of ionotropic antagonists (e.g., NBQX; Nurse and Corbett,
1996). Because in our model of global ischemia cell death occurs by 3 days and LY379268 maintains protection up to 28 days (Fig. 4), it
appears that LY379268 is blocking, rather than merely delaying, the
process of cell death. The slow death of cells in our model is in
accordance with the delayed neuronal death that has been described in
this model (Kirino, 1982). Interestingly, the level of protection
observed with LY379268 in this model is much greater than that we
previously observed using the same protocol with calcium channel
blockers (NCC 09-0026, CNS1237; O'Neill et al., 1997), NMDA
antagonists (MK-801, ACEA1021, GV150526A; Hicks et al., 1999), and AMPA
antagonists (GYKI52466, LY300164; Lodge et al., 1996) and similar to
what we have seen with the iGluR5 antagonist (LY377770; O'Neill
et al., 1998).
Other studies have shown that group II mGluR agonists are
neuroprotective in vitro and in vivo. In mouse cortical neurons, DCG-IV, 4C3HPG, and L-CCG-1 were shown to protect against NMDA-induced excitotoxicity (Bruno et al., 1995), and the in vivo administration of
DCG-IV directly into the cerebral ventricles can block kainate-induced degeneration in rats (Shinozaki, 1994). However, these compounds are
not selective for group II mGluRs and have additional pharmacology that
could account for their neuroprotective effects. LY379268, being more
selective, confirms the involvement of group II mGluRs. Interestingly,
neither LY354740 nor LY379268 is protective in focal ischemia (Lam et
al., 1998; Behrens et al., 1999; Bond et al., 1999b). The exact reason
for this is not clear. One possible explanation may be that the
mechanisms of cell death are different in these models. Other recent in
vitro studies have reported that both LY354740 and LY379268 are more
potent against apoptotic indices of neuronal injury in vitro at
concentrations that are more commensurate with their actions at group
II mGluRs than those required to reduce lactate dehydrogenase release,
which occurs as a result of both apoptotic as well as necrotic cell
death (Kingston et al., 1999). In another recent study, LY354740 failed
to attenuate acute (24 h) excitotoxicity of NMDA or oxygen-glucose
deprivation in vitro or of intrastriatal NMDA injections in vivo
(Behrens et al., 1999). Taken together, these results indicate that
selective group II mGlu agonists appear to have little or no effect in
conditions where there is rapid development of neuronal cell loss such
as acute excitotoxicity and focal ischemia, but they appear to be most
effective in circumstances of slowly developing neuronal cell death
(i.e., global ischemia). In this study, we have demonstrated further
convincing evidence for the group II mGluR agonist LY379268 being
neuroprotective in global ischemia using various dosing protocols.
Mechanism of Action of LY379268.
The neuroprotective activity
of group II mGluR agonists can be ascribed to their presynaptic actions
at mGlu2 receptors and the prevention of glutamate release (Pin and
Duvoisin, 1995). However, this mechanism of action does not account for
neuroprotection seen with this class of compound against apoptosis
induced by oxygen-glucose deprivation and -amyloid peptide (Copani
et al., 1995), nor for the demonstration that DCG-IV can protect
cultured neurons even when applied after a toxic pulse of NMDA, when
large amounts of endogenous glutamate have already accumulated in the extracellular medium (Nicoletti et al., 1996). Furthermore, protein synthesis inhibitors can block the neuroprotective actions of group II
mGluR agonists in vitro against NMDA-induced toxicity in cortical
neurons (Bruno et al., 1997; Kingston et al., 1999). A recent report
demonstrated that transient exposure of astrocytes to DCG-IV or 4C3HPG
led to an increase in TGF- and that neutralizing antibodies to
TGF-1 or -2 prevented the neuroprotection afforded by DCG-IV and
4C3HPG in mixed cortical cultures. This then suggested that activation
of mGlu3 receptors in astrocytes led to the production of TGF-,
which protects cells (Bruno et al., 1998).
In this study, we sought to investigate this latter possibility. First,
we demonstrated that LY379268 was neuroprotective when dosed 24 h
(P < .001) and 48 h (P < .01)
before a 5-min period of BCAO (Fig. 5), a result consistent with
up-regulation of a neurotrophic factor, such as TGF- (Bruno et al.,
1998). However, LY379268 did not induce expression of the specific
neuroprotective factors TGF-1, TGF-2, bFGF, BDNF, and NGF.
The prolonged activity of LY379268 after 24- and 48-h pretreatment
raises the possibility that it shares a common mechanism with that of
ischemic tolerance (Kirino et al., 1991), whereby glutamate released by
a preconditioning ischemia may activate group II mGluRs and induce a
neuroprotective factor. We argued that if LY379268 induced a
neuroprotective factor, it should act additively with the
preconditioning ischemia. Our results indicate that LY379268 almost
blocked the degree of tolerance induced by a preconditioning ischemia.
It has previously been demonstrated that NMDA antagonists (Kato et al.,
1992; Bond et al., 1999a), but not AMPA antagonists, attenuated the
induction of tolerance in this model (Bond et al., 1999a). These
results suggest that LY379268 is acting via mGlu2 presynaptic
autoreceptors to reduce glutamate release, and hence NMDA receptor
activation, to interfere with the development of tolerance.
These results leave open the possibility that even at 24 and 48 h,
sufficient LY379268 remains in the brain to reduce the degree of damage
after BCAO in the gerbil. We investigated this in two ways. First, we
took advantage of the newly available group II mGlu antagonist LY341495
(Kingston et al., 1998), which given alone was without effect on the
outcome of a submaximal global ischemia (Fig. 8). Given 24 h after
10 mg/kg LY379268 and 1 h before a 5-min BCAO, 5 mg/kg LY341495
virtually prevented the neuroprotective effect of LY379268. Second, we
assayed the levels of LY379268 in the brain at time points up to
24 h postadministration (Fig. 10). Although there was a rapid
decline in central nervous system levels between 0.5 and 8 h, they
plateaued at a level above that required for group II mGluR activation.
Taken together, these results support a mechanism that is directly
dependent on receptor activation by LY379268 rather than through a
mechanism that involves a long-lasting induction of other factors that
would be farther downstream of receptor activation and not affected by
the actions of an mGlu antagonist such as LY341495.
In conclusion, we have shown that LY379268 provides marked long-lasting
neuroprotection against ischemia-induced damage in the gerbil. In this
model of global ischemia, the mechanism of action of LY379268 does not
appear to be via induction of neurotrophic factors such as NGF, BDNF,
or TGF but most likely occurs via a mechanism that is closely coupled
with receptor activation. Such a mechanism may involve inhibition of
glutamate release and/or activation of biochemical pathways that
inhibit programmed cell death. However, these findings do not rule out
the possibility that other mechanisms may also contribute to the quite
remarkable neuroprotection seen with LY379268 and encourage further
investigation of the use of group II mGlu agonists in chronic rather
than acute neurodegenerative disorders.
iGlu, ionotropic glutamate;
mGlu, metabotropic
glutamate;
mGluR, mGlu receptor;
BCAO, bilateral carotid artery
occlusion;
TGF-1, transforming growth factor-1;
TGF-2, transforming growth factor-2;
BDNF, brain-derived neurotrophic
factor;
NGF, nerve growth factor;
bFGF, basic fibroblast growth
factor;
NMDA, N-methyl-D-aspartate;
AMPA, -amino-3-hydroxy-5-methyl-isoxazole-4-propionic acid;
DCG-IV, (2S,1'R,2'R,3'R)-2-(2,3-dicarboxycyclopropyl)glycine;
4C3HPG, (S)-4-carboxy-3-hydroxyphenylglycine;
L-CCG-1, (2S,1S,2S)-2(carboxycyclopropyl)
glycine;
LY354740, (+)-2-aminobicyclo[3.1.0]hexane-2,6-dicarboxylic acid;
LY379268, (1R,4R,5S,6R)-2-oxa-4-aminobicyclo[3.1.0.]hexane-4,6-dicarboxylate;
LY341495, 2S-2-amino-2-(1S,2S-2-caroxycyclopropy-1-yl)-30(xanth-9-yl)propionic
acid;
NBQX, 2,3-dihydroxy-6-nitro-7-sulfamoyl-benzo[f]quinoxaline;
MK-801, (5R,10S)-(+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]
cyclohepten-5,10-imine;
ACEA1021, 5-nitro-6,7-dichloro-2,3-quinoxalinedione;
GV150526A, [(E)-3[(phenylcarbamoyl)
ethenyl]-4,6-dichloroindole-2-carboxylic acid;
GYKI52466, 1-(aminophenyl)-4-methyl-7,8-methylenedioxy-5H-2,3-benzodiazepine;
LY300164, (+)-3-N-acetyl-1(aminophenyl)-4-methyl-7,8-methylenedioxy-5H-2,3-benzodiazepine.
![[Feedback]](/icons/banner/feedbackACT.gif){kind=icon}
![[For Subscribers]](/icons/banner/subscriberACT.gif){kind=icon}
![[Archive]](/icons/banner/archiveACT.gif){kind=icon}
![[Search]](/icons/banner/searchACT.gif){kind=icon}
![[Contents]](/icons/banner/tocACT.gif){kind=icon}
Top
Abstract
Introduction
