Possible involvement of interleukin-1 in ischemic brain edema formation

Neuroscience Letters, 142 (1992) 45 47 c 1992 Elsevier Scientific Publishers Ireland Ltd. All rights reserved 0304-3940/92,$ 05.00

45

NSL 08789

Possible involvement of interleukin-1 in ischemic brain edema formation Y a s u n d o Y a m a s a k i ~', T a k a s h i S u z u k i b, H i d e t o s h i Y a m a y a b, N a o s u k e M a t s u u r a b, H i r o s h i O n o d e r a " a n d K y u y a K o g u r e ~' "DcT~artnwnt q/'Neurology, b~stitute olBrain Disease, Tohoku University School o/Medicine. Sendal, f.lapan ) and ~'Hanno Research ('enter. Taiho Pharmaceutical Co. Lid.. Sailama ~J~q~an )

(Received 9 March 1992: Revised version received 24 April 1992: Accepted 24 April 19921 Key wordsv Transient I\~cal ischemia: Brain edema: lnterleukin 1

To determine the contribution of interleukin 1 (IL-I) on ischemic brain edema formation, the effect of recombinant human intcrleukin Ifl (rhIL-lfl), or zinc protoporphyrin (ZnPP) as an IL-1 blocker, on brain edema was studied in rats. The animals were subjected to 60 min of ischemia in a middle cerebral artery occlusion model. Immediately after reperfusion, rhl L-lfl at a dose of 10 ng/2,ul, or ZnPP at doses of 1 and 10/,tg/2ul were topically applied into lateral cerebroventricle. In rhlL-lfl-treated rats, ischemic brain edema formation was significantly increased in the dorsal and ventral areas of the caudate putamen 24 h after reperfusion, compared to that of vehicle-treated control rats. Furthermore, in ZnPP-treated rats, brain edema was decrcased in both caudate-putamen areas. This suggests that IL-1 plays an important role in pathogenesis for post-ischemic brain edema,

Interleukin 1 (IL-I), a cytokine originally detected in macrophages, has biological effects when injected into the brain. Injection o f IL-1 into the brain induces fever [3], and slow-wave sleep [1 1], suppression o f food intake [17], and activates the hypothalamic-pituitary axis by corticotropin-releasing factor and adrenocorticotropic h o r m o n e [1]. Moreover, IL-I is p r o d u c e d by astrocytes and microglia in vitro [5] and IL-1 has been shown to stimulate astrocytes growth in vitro and in vivo [6]. These biological lines o f evidence suggest that IL-I participates in the m o d u l a t i o n o f the C N S physiology and behavior in a m a n n e r characteristic o f n e u r o m o d u l a t o r or neuroendocrine hormones. Furthermore, IL-1 has been reported to be present in the ventricular fluid o f patients with traumatic brain injury [13], and in the traumatic lesions o f rats even from I to 4 days after brain d a m a g e [6]. These reports indicate that IL-I might be responsible at least in part for the formation o f post-traumatic and ischemic brain edema. However, few studies have focused on the role o f IL-I to post-ischemic brain edema in rats, or the effectiveness o f I k - I blocker on brain damage. In the present study, we assessed the contribution o f IL-I to ischemic brain edema formation by application of exogenous recombinant h u m a n IL-lfl (rhlL-lfl), or selective IL- 1 blocker. ('orrespomh'nce. Y. Yamasaki, Hanno Research Center, Taiho Pharmaceutical ('o. Ltd., 216-1 Nakayashita, Yaoroshi, Hanno-city, Saitama 357. Japan.

Adult male Wistar rats, weighing 270 320 g, were used. Animals were anesthetized with a gas mixture o f N~O/O= (70:30) containing 2% halothane. After a median incision o f the neck skin, the right external carotid artery (ECA) was carefully dissected and an 18 m m long 4-0 nylon thread (coated by silicon) was inserted from the E C A to the right internal carotid artery to occlude the origin o f the right middle cerebral artery ( M C A ) by a modification o f the m e t h o d o f L o n g a et al. [12]. Atier 60 rain o f M C A occlusion, the thread was removed to allow complete reperfusion o f the ischemic area via the right c o m m o n carotid artery. Neurologic deficits characterized by severe left-sided hemiparesis and right H o m e r ' s s y n d r o m e were used as criteria for complete ischemic insults. Ischemic insults can be induced without the occurrence o f seizure activity in this model. One day after reperfusion, animals were decapitated and the brain was dissected into the cerebral cortex perfused by M C A ( M C A area), the dorsal area of the caudate p u t a m e n (DCP) and the ventral area o f the caudate putamen (VCP) in a humidified glove-box. To assess brain water, samples were dried in an oven at I10°C for 24 h and water contents o f these samples were then measured by the wet and dry weight m e t h o d [10] as described below. Water contents (%)={wet wt. (mg) - dry wt. (mg)} / wet wt. (ng) x 100 To verify the contribution o f IL-I on ischemic brain

r

I

i [ -i

! [

r

1

Zrfii ~ , ! .

/~llPl ] 0

lJ{[

85

g ,2, 80

MCA

DCP

VCP

75

Fig. 1. Water contents on occluded side 24 h after 60 min of ischemia. rhIL-lfl at a dose of 10 ng/brain was injected into the lateral cerebroventricle immediately after reperfusion. Results are the mean _+ S.E.M. of 5 rats. Note the enhanced brain edema formation by application of exogenous rhIL-lfl. *P<0.05, ***P<0.001 compared to each vehicletreated group (Dunnett's multiple analysis).

edema formation, rhIL-lfl (kindly donated by Otsuka Pharmaceutical Co. Ltd.) diluted with 1% bovine serum albumin (BSA), or zinc protoporphyrin disodium salt that was purchased from Aldrich (USA) and converted to disodium salt with usual methods (ZnPP, MW 780 Da) dissolved in saline as a potent IL-1 blocker, was applied topically unto the lateral cerebroventricle unilaterally over 4 rain, and the needle was left for 2 min. Coordinates were as follows: 0.8 mm posterior to the bregma, 1.5 mm lateral to the midline, and 4.5 mm deep to the dural surface, according to the atlas of Paxinos and Watson [15]. The assay system for IL-1 blocking activity was prepared by the method of Conlon et al. [2] with minor modification. Briefly, murine thymocytes (collected from C3H/HeJ mice, non-responsive to peanut agglutinin) were cultured in RPMI 1640 medium including 10% fetal calf serum, and cells (2 x 106 cells/well) were treated with 10 U/ml rhIL-la (Genzyme, USA) and with each concentration of ZnPP. Cultures were incubated for 72 h at 37°C in 5% CO2. Eighteen hours before harvest, wells were pulsed with 0.5 ktCi [3H]thymidine (NEN) and then subsequently harvested onto glass fiber filters, and incorporation of [3H]thymidine was determined in a liquid scintillation counter. For statistical analysis, Dunnett's multiple test was used and results were compared with those of the vehicletreated group. While the brain water contents, measured by wet and dry weight method, in the MCA, the DCE and VCP area of normal rats was 79.0+0.1, 76.6+0.3, and 76.6+0.2%, respectively (mean + S.E.M.), 24 h after reperfusion, when brain edema in each area was significantly higher than that of normal rats, it was 85.5+0.3, 86.0_+0.3, and

MCA

DCP

VCP

l~ig. 2. Water contents on the occluded side 24 h after 60 mm ol lschemia. ZnPP as IL- 1 blocker at doses of 1, 10/ag/2/al was injected into the lateral cerebroventricle immediately after reperfusion. Results are the mean _+ S.E.M. (n=5 for each group). Note the dose-dependent amelioration of brain edema by injection of ZnPR ** P<0.01 compared to each vehicle-treated group (Dunnett's multiple analysis).

83.9+0.4%, respectively. On the other hand, injection of rhIL-lfl caused significant enhancement of brain edema in the DCP and the VCR but not in the MCA 24 h after reperfusion as compared to vehicle- treatment. The water content on the occluded side was 86.2+0.7, 88.4_+0.7, and 86.6_+0.4%, respectively (Fig. 1). Since rats treated with rhIL-lfl tended to show higher body temperature, we kept the body temperature of vehicle-treated rats the same as that of the rats treated with rhlL-lfl using a heating pad. Thus, augmentation of brain edema on the ischemic side could not be caused by hyperthermia. We confirmed the anti-IL-I activity of ZnPP in the murine thymocytes bioassay system. In this system, ZnPP suppressed IL-1 activated [3H]thymidine incorporation dose-dependently, as shown in Table 1. The maxiTABLE 1 [3H]THYMIDINE INCORPORATION MOCYTES (2 x 106 CELLS/WELL)

IN

MURINE

THY-

rhIL-l~ at a dose of 10 U/ml stimulated the incorporation. Results are the mean +_ S.E.M. from three separate experiments. Note the dosedependent suppressive efl~ct of ZnPP on [3H]thymidine incorporation. [3H]Thymidine incorporation (cpm/2xl06 cells) Vehicle ZnPP (,aM)

Inhibition (%)

14310.1 _+ 1272.8 0.03 0.3 3

6638.9 _+ 911.2"* 3733.2 _+ 402.3** 2869.7 +_ 178.6"*

53.6 73.9 79.9

**P<0.01 compared to vehicle-treated group (Dunnett's multiple analysis).

47

mum suppressive effect of ZnPP (80%) was obtained at a dose of 3/~M. In addition, ZnPP showed weak anti-inflammatory activities (data not shown). ZnPP dose-dependently (1 or 10 yg) ameliorated brain edema in the DCP and VCP 24 h after ischemia. In rats treated with ZnPP at a dose of 10 yg/brain, the water content in the DCP and VCP was 84.5_+0.4%, and 83.6_+0.3%, respectively, which were significant lower values than those in saline-treated rats. However, the water content in the MCA area was not reduced (Fig. 2). 1L-I is involved in the responses after the CNS injury. For instance, IL-I level increases around stab wounds and at sites of penetrating brain injury [4]. Furthermore, high doses of exogenous IL-1 have been shown to induce brain edema and astrogliosis in the normal brain [6, 7]. These findings indicate that IL-I plays an important role during edema formation and helps to mediate astrogliosis after brain damage. We found that exogenous rhlk1 augmented post-ischemic brain edema formation, and that the IL-I blocker could reduce the brain edema. ZnPP was first found to show anti-lL-1 activity in vitro, and has been shown to be effective on collagen-induced arthritis in rats [14]. Our present findings indicate that ZnPP has 1k-I blocking activity both in vitro and in ViVO. The contribution of [L-1 in post-ischemic pathology is not well understood. However, an abnormal increase of IL-1 immunoreactivity in microglia has been recently reported in Alzheimer's disease [8]. Astrocytes and microglia, as well as blood-borne macrophages and leukocytes, have been reported to produce IL-1. We could detect microglia and reactive astrocytes even 24 h after reperfusion in this model (data not shown). The local leukocyte accumulation suggests that local synthesis of IL-1 is an important factor in the brain damage after a stroke [9]. We also reported that neutrophils play a critical role in the formation of brain edema using the same model [16]. The resultant astrogliosis may prevent the recovery of neuronal function by inhibiting the regeneration of neuronal processes. While the contribution of IL-1 is obvious at the early stages of brain edema, further studies are required to confirm whether I L-I-mediated processes determine the final outcome of ischemic brain damage,

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