Effect
of Nimodipine
and
Mortality
on Ischemia-Induced
in a Novel Artery
Hideaki
HARA,
Occlusion
Hiroshi and
Department Tohoku
Transient
rat
novel
was
used
Nimodipine MCA
and
ipsilateral
after that
transient
School
to
mortality
the
partly the rate
effect
left at
MCA 6
was has
Institute
March
and not
beneficial
occlusion 9 hr
of Brain Diseases,
of
artery
nimodipine immediately
45Ca
after
different effects
NAGASAWA
the
hr
after
the
in the
It has been widely assumed that disruption of calcium homeostasis is one of the detri mental factors leading to cell death after cerebral ischemia (1-4). Beneficial effects of calcium channel blockers after cerebral isch emia in animal and human studies have been reported (5-9). Nimodipine, a Ca2+ channel blocker of the dihydropyridine type, has also been shown to improve clinical out come and has been reported to be protective in animal models of ischemia (5, 10-13). Permanent unilateral MCA occlusion models in the rat have been commonly used to an alyze the protective roles of various drugs against ischemic brain damage (14, 15). In human ischemic strokes, however, recanaliza tion after MCA occlusion is not uncommon, particularly in the case of MCA occlusion caused by embolization. Thus, a recirculation model after MCA occlusion may be of great value for simulating focal ischemia in humans. We have developed a transient MCA occlu sion model in the rat, which was produced by inserting a piece of silicone-coated nylon thread into the internal carotid artery (16). The aim of the present report was to inves tigate the effect of nimodipine on post ischemic brain edema and on animal mor
(MCA)
occlusion
on
brain
after
3
the
hr
the
group. phase
of
in
of
survival These
the
reperfusion
the
mortality.
transient
unilateral tissue
water cortex
Nimodipine
although
early
of
and
parieto-temporal
reperfusion.
control
model
edema
increase
in
recirculation, from
980, Japan
post-ischemic
accumulation 3
Sendai
3, 1990
cerebral
significantly
attenuated
Haruo
of Medicine,
administered
attenuated
recirculation nimodipine
middle
evaluate
(30 ƒÊg/kg)
occlusion
content
the
to
Cerebral
Kyuya KOGURE
Accepted
Abstract-A
Middle
Edema
Model
ONODERA,
of Neurology,
University
Brain
decreased rate results
at
24
hr
suggest period
.
tality after reperfusion using a novel transient MCA occlusion model. The effect of hyper osmotics (glycerol) on brain edema was also studied. Materials and Methods 1. Animals: Male Wistar rats weighing 230-250 g were used. Animals were pur chased from Japan S.L.C., Ltd. and allowed free access to food and water. 2. MCA occlusion: Transient focal ischemia was induced in rats by means of left MCA occlusion (16, 17). With the animals under anesthesia (a mixture of 70% N20, 30% 02, and 1% halothane), a piece of nylon thread, one end of which was pre-coated with silicone resin, was inserted into the left com mon carotid artery in order to occlude the origin of MCA. 3. Brain edema and mortality: Three hours after reperfusion (6 hr after induction of ischemia), the animals were decapitated and tissue samples of both hemispheres were quickly dissected in a humidified chamber. The water content of the samples was deter mined by the dry-weight method. Brain areas used for analysis are presented in Fig. 1. The water content of animals killed 6 hr after
ministration of these compounds was started immediately after reperfusion using a pump (Terumo STC-521, Japan) at the rate of 0.2 ml/min for 30 min. 6. Statistics: For statistical analyses, Duncan's multiple range test, Dunnett's multiple range test and Fisher's exact prob ability test were used. Results
Fig. the
1.
Anatomic
striatum
temporal
regions
of rats. cortex,
1 and
3 and
of coronal 6: striatum, 4: frontal
section 2 and
through 5: parieto
cortex.
ischemia without reperfusion was also ex amined. The survival of animals treated with nimodipine or vehicle alone was compared at the time points of 3, 6, 9, 24 hr and 7 days after reperfusion following 3-hr ischemia. 4. 45Ca autoradiography and historogical study: 45Ca autoradiography was performed according to the method of Kato et al. (18). In brief, for the 45Ca autoradiographic study, the animals were injected intravenously with 45CaCl2 (Amersham; 300 ,uCi/animal, dis solved in 0.15 ml physiological saline) when the MCA was reperfused. Three hours after reperfusion following 3-hr ischemia, the brains were removed quickly and frozen in powdered dry-ice. Serial coronal sections, 20-ucm thick, were cut in a cryostat at -20°C, and they were exposed to Kodak NMC-1 film for 2 weeks. For histopathology, animals were perfusion-fixed (10% formalin, 10% acetate and 80% methanol) under deep pentobarbital anesthesia 3 hr after reperfusion, and paraffin sections, 5 /cm in thickness, were stained with Hematoxylin/Eosin and cresyl violet. Each group consisted of 3 experiments. 5. Test compounds: The doses of test com pounds were as follows: Nimodipine (30 iig/ kg, 6 ml/kg), nimodipine vehicle (6 ml/kg; it consisted of 200 g/I ethanol, 170 g/I macro gol 400, 2 g/I sodium citrate and 0.3 g/I citrate), 60% glycerol (4 g/kg, 5.3 ml/kg; Wako Co., Ltd., Japan), glycerol vehicle (5.3 ml/kg, physiological saline). Nimodipine and its vehicle were kindly donated by Bayer Co., Ltd. (FR Germany). The intravenous ad
1. Brain edema: Effect of nimodipine and glycerol on brain edema after transient focal ischemia is summarized in Table 1. The pre sent ischemic model caused a marked in crease in the water content in the areas per fused by MCA. In the brain areas ipsilateral to the left MCA occlusion, the water content in the striatum, parieto-temporal cortex, and frontal cortex (nimodipine vehicle group) of rats killed 3 hr after reperfusion (6 hr after induction of ischemia) increased by 6.3, 6.4 and 2.2%, respectively. By contrast, the in creases of water content in the striatum, parieto-temporal cortex, and frontal cortex ipsilateral to the left MCA occlusion in animals killed 6 hr after left MCA occlusion without reperfusion were 4.2, 3.8 and 1.2%, respec tively. Water content of the areas contralat eral to the left MCA occlusion was unaltered both in the reperfusion group and in the group with permanent left MCA occlusion. Brain edema during the 3-hr reperfusion that occurred after a 3-hr ischemia induced in our rat model was observed to be more severe than that which followed a 6-hr ischemia. Nimodipine significantly reduced the post ischemic increase of water content in the frontal and parieto-temporal cortex ipsilateral to the left MCA occlusion 3 hr after reper fusion (6 hr after left MCA occlusion). The increase in water content of the nimodipine group in the parieto-temporal cortex and frontal cortex ipsilateral to the left MCA oc clusion was reduced to 3.9 and 1%, respec tively. Glycerol significantly reduced the in crease of water content in the striatum, frontal cortex, and parieto-temporal cortex ipsilateral to the left MCA occlusion 3 hr after reperfusion. Interestingly, glycerol in duced a reduction of water content in the striatum and parieto-temporal cortex con tralateral to left MCA occlusion.
249
Table 2.
Effect of nimodipine on mortality after left MCA occlusion
2. Mortality: Effect of nimodipine on animal mortality after transient focal ischemia is sum marized in Table 2. At 3, 6, 9, 24 hr and 7 days after reperfusion, the mortality of the nimodi pine vehicle group was 7, 47, 87, 100 and 100%, respectively. A time-dependent in crease in mortality was observed following reperfusion. On the other hand the mortality of the nimodipine group was 0, 0, 27, 82 and 82% at 3, 6, 9, 24 hr and 7 days after reper fusion, respectively, with statistical signifi cance at 6 and 9 hr after reperfusion. 3. 45Ca autoradiography and histological study: Figure 2 shows typical 45Ca autoradio grams of each experimental group. After 3 hours of ischemia followed by 3-hr reper fusion, 45Ca accumulation and neuronal cell damage were high in the dorsolateral striatum, moderate in the MCA cortex, and slight in the anterior cerebral artery (ACA) cortex (Fig. 2, a, b, data of histological findings not shown). The contralateral areas and sham-operated rats showed no abnormal calcium accumula tion and neuronal cell damage (Fig. 2, g, h). The abnormal 45Ca accumulation distribution was in close accordance with the neuronal cell damage area in the histological findings. Nimodipine partly attenuated the abnormal 45Ca accumulation in the MCA and ACA cortex, not in the striatum, in two of three animals (Fig. 2, c, d). On the other hand, glycerol attenuated the abnormal 45Ca ac cumulation in all areas (Fig. 2, e, f). Discussion The
present
results
showed
that
the
in
(3 hr) and reperfusion in rats
crease in tissue water content was accentuat ed by reperfusion following ischemia, cor relating well with previous reports (19-21). calcium antagonists have been reported to be beneficial for the treatment of permanent MCA occlusion in rats (6, 14). In these models, the beneficial effects of drugs were obtained by influencing only the peripheral areas surrounding the ischemic core, where drugs could not reach. We observed that nimodipine administered immediately after recirculation attenuated the formation of brain edema in the areas perfused not only by MCA (parieto-temporal cortex) but also by ACA (frontal cortex), although water content in the striatum was not reduced by nimodipine treatment. Furthermore, nimodipine partly attenuated the 45Ca accumulation in the MCA and ACA areas. In contrast, glycerol, a hyperosmotic agent, was potent enough to ameliorate brain edema and water content in the striatum ipsilateral to the MCA occlusion. One reason why nimodipine failed to save the striatum seems to be that the damage to the striatum was greater than that to the cortex. Abe et al. (22) has reported that re covery from brain edema and of protein synthesis following a 1-hr ischemia in the same animal model used in our study differed between the cerebral cortex and the striatum, and that the inhibition of protein synthesis and the formation of brain edema began so oner in the striatum than in the cortex. Re cently, it has been generally accepted that an excitotoxic mechanism is involved in the neuronal damage that occurs after ischemia
Fig. 2. Representative 45Ca autoradiograms in the middle cerebral artery (MCA) occlusion model. a and b: 3-hr ischemia plus 3-hr reperfusion (1-3, R-3) control group. Marked calcium accumulation is noted in the dorsolateral striatum, the MCA cortex and the anterior cerebral artery (ACA) cortex. c and d: nimodipine-treated group. Abnormal calcium accumulation was attenuated partly in the MCA and ACA cortex. e and f: glycerol treated-group. Abnormal calcium accumulation was attenuated almost completely in the MCA cortex, ACA cortex and striatum. g and h: sham-operated group. No abnormal calcium accumulation is noted.
(23). The dorsolateral striatum has been ob served to be rich in receptors of excitatory neurotransmitters such as glutamate (24) and dopamine (25). Receptors of inhibitory neuro transmitters such as GABA and benzodiaze pine have been found to exist in smaller
amounts in the striatum than in the cortex (26). The release of glutamate and dopamine have been seen to greatly increase during ischemia and/or the early period of reper fusion (27, 28). Therefore, the damage to the striatum may be more severe than that to the
cortex because of the imbalance of excitatory and inhibitory innervation. Although the mechanisms of such bene ficial effects of nimodipine seen in the present study on MCA occlusion-induced brain edema in rats are by no means definitely established, there seem to be at least two possible explanations. The first one is an im provement of postischemic hypoperfusion. It has been suggested that the neurologic damage that occurs subsequent to ischemia is due at least in part to a delayed hypoper fusion state following reperfusion (29, 30). Steen et al. has reported previously that in fusion of the nimodipine, administered before or after ischemia (7, 8), improved both the postischemic cerebral blood flow and the neurologic outcome. Similar results with nimodipine have been reported by Hof fmeister et al. (31) and Kazda et al. (32). The second one is a reduction of Ca2+ overload in the neurons. Recently, it has been reported that a massive influx of calcium oc curs during the reperfusion period and may result in further damage to brain cells. This influx may occur as the result of other reper fusion injuries, such as lipid peroxidation and acidosis (2, 33). Furthermore, Uematsu et al. (34) has reported that nimodipine prevents an increase in cytosolic free calcium concen trations following cerebral ischemia in vivo. Therefore, nimodipine may prevent partly the formation of brain edema by a direct action (blocking the entry of calcium ions into the cell) on neurons. The effect of calcium antagonists on the histopathological outcome after cerebral ischemia is still unclear. Interestingly, studies that reported the beneficial effect of calcium antagonists analyzed the drug effects at earlier periods after ischemia (6, 14). The beneficial effect of nimodipine on animal mortality after transient left MCA occlusion was shown only at 6 and 9 hr after reperfusion but not at 24 hr and 7 days. Taken together, later administration of nimodipine may be less effective for the prevention of ischemic brain damage. However, since we administered nimodipine only once, repeated infusion of the drug might ameliorate animal survival. In conclusion, nimodipine treatment is beneficial in early phases of the recirculation
period. The present reversible MCA occlusion model provides a good system for estimating drug effects against ischemic brain damage and is of value for simulating human stroke. References 1
Siesjo,
B.K.:
lative
synthesis.
1, 2
3
155-185
J.
The
Ann.
Neurol.
R.P.,
and
Meldrum,
and
after
study
in
4,
Siesjo,
B.K.
Blockers
p.
135-150,
Gelmers,
A
Flow
specu Metab.
T.,
Mcevans,
rat.
350-361
the
and
electron
Cereb.
Wieloch,
T.:
selec
micro
Blood
Brain
homeostasis.
and
In
Tissue
Protection,
Vanhoutte,
P.M.
Raven H.J.:
in hippocampus
an J.
Swan,
Flow
(1984)
calcium
T.,
•@ J.H.,
overload of
ischemia:
brain
(1983)
Calcium
the
of
2-10
neurons
scopy Metab.
13,
B.:
during
Godfraind,
brain:
pathophysiology
Griffiths,
vulnerable
cellular
the Blood
(1981) M.E.:
Simon,
in
Cereb.
Raichle,
tively
5
damage
ischemia.
S.
4
Cell
Press, Nimodipine
New
ischemia Calcium
and York in
and Entry
Edited
by
Govoni,
S.,
(1985) acute
brain
336 (1984) 13 Kazda, S., Garthoff, B. and Luckhaus, G.: Calcium-antagonists prevent brain-damage in stroke-prone spontaneously hypertensive rats (SHR-Sp) independent of their effect on blood pressure: nimodipine versus nitrendipine. J. Cereb. Blood Flow Metab. 3, 526-527 (1983) 14 Nakayama, H., Ginsberg, M.D. and Dietrich, W.D.: (s)-Emopamil, a novel calcium channel blocker and serotonin S2 antagonist, markedly reduces infarct size following middle cerebral artery occlusion in the rat. Neurology 38, 1667 1673 (1988) 15 Tamura, A., Graham, D.I., McCulloch, J. and Teasdale, G.M.: Focal cerebral ischemia in the rat. 1. Description of technique and early neuro pathological consequences following middle cerebral artery occlusion. J. Cereb. Blood Flow Metab. 1, 53-60 (1981) 16 Nagasawa, H. and Kogure, K.: Correlation between cerebral blood flow and histologic changes in a new rat model of middle cerebral artery occlusion. Stroke 20, 1037-1043 (1989) 17 Koizumi, J., Yoshida, Y., Nakazawa, T. and Ooneda, G.: Experimental studies of ischemic brain edema. 1. A new experimental model of cerebral embolism in rats in which recirculation can be introduced in the ischemic area. Japan. J. Stroke 8, 1-7 (1986) (in Japanese) 18 Kato, H., Kogure, K., Sakamoto, N. and Wata nabe, T.: Greater disturbance of water and ion homeostasis in the periphery of experimental focal ischemia. Exp. Neurol. 96, 118-126 (1987) 19 Ito, U., Ohno, K., Nakamura, R., Suganuma, F. and Inaba, Y.: Brain edema during ischemia and after restoration of blood flow. Measurement of water, sodium, potassium content and plasma protein permeability. Stroke 10, 542-547 (1979) 20 Ito, U., Ohno, K., Suganuma, Y., Suzuki, K. and Inaba, Y.: Effect of steroid on ischemic brain edema: Analysis of cytotoxic and vasogenic edema occurring during ischemia and after restoration of blood flow. Stroke 11, 166-172 (1980) 21 Kogure, K., Busto, R. and Scheinberg, P.: The role of hydrostatic pressure in ischemic brain edema. Ann. Neurol. 9, 273-282 (1981) 22 Abe, K., Araki, T. and Kogure, K.: Recovery from edema and of protein synthesis differs between the cortex and caudate following transient focal cerebral ischemia in rats. J. Neurochem. 51, 1470-1476 (1988) 23 Rothman, S.M. and Olney, J.W.: Glutamate and
the pathophysiology of hypoxic-ischemic brain damage. Ann. Neurol. 19, 105-111 (1986) 24 Fagg, G.E. and Foster, A.C.: Amino acid neuro transmitters and their pathways in the mammalian central nervous system. Neuroscience 9, 701 719 (1983) 25 Onodera, H. and Kogure, K.: Autoradiographic localization of opioid and spirodecanone recep tors in the gerbil hippocampus as compared with the rat hippocampus. J. Cereb. Blood Flow Metab. 8, 568-574 (1988) 26 Onodera, H., Sato, G. and Kogure, K.: GABA and benzodiazepine receptors in the gerbil brain after transient ischemia: demonstration by quantitative receptor autoradiopgraphy. J. Cereb. Blood Flow Metab. 7, 82-88 (1987) 27 Benveniste, H., Drejer, J., Schousboe, A. and Dimer, N.H.: Elevation of the extracellular con centrations of glutamate and aspartate in rat hippocampus during transient cerebral ischemia monitored by intracerebral microdialysis. J. Neurochem. 43, 1369-1374 (1984) 28 Harik, S.I., Yoshida, S., Busto, R. and Ginsberg, M.D.: Monoamine neurotransmitters in diffuse reversible forebrain ischemia and early recircu lation: Increased dopaminergic activity. Neuro logy 36, 971-976 (1986) 29 Siesjo, B.K.: Brain Energy Metabolism. New York, John Wiley & Sons (1978) 30 White, B.C., Gadzinski, D.S., Hoehner, P.J., Krome, C., Hoehner, T., White, J.D. and Trombley, J.H., Jr.: Effect of flunarizine on canine cerebral cortical blood flow and vascular resistance post cardiac arrest. Ann. Emerg. Med. 11, 119-126 (1982) 31 Hoffmeister, F., Benz, U., Heise, A., Krause, H.P. and Neuser, V.: Behavioral effects of nimodipine in animals. Arzneimittelforschung 32, 347-360 (1982) 32 Kazda, S., Garthoff, B., Krause, H.P. and Schlossmann, K.: Cerebrovascular effects of the calcium antagonistic dihydropyridine derivative nimodipine in animal experimants. Arzneimit telforschung 32, 331-338 (1982) 33 Uematsu, D., Greenberg, J.H., Revich, M. and Karp, A.: In vivo measurement of cytosolic free calcium during cerebra! ischemia and reperfusion. Ann. Neurol. 24, 420-428 (1988) 34 Uematsu, D., Greenberg, J.H., Hickey, W.F. and Reivich, M.: Nimodipine attenuates both increase in cytosolic free calcium and histologic damage following focal cerebral ischemia and reperfusion in cats. Stroke 20, 1531-1537 (1989)