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Naloxone or TRH fails to improve neurologic deficits in gerbil models of “stroke”
Life Sciences, Vol. 31, pp. 385-392 Printed in the U.S.A.
Pergamon Press
NALOXONE OR T R H F A I L S TO IMPROVE NEUROLOGIC DEFICITS IN GERBIL MODELS OF "STROKE" John W. Holaday and R o b e r t J . D'Amato Neuropharmacology Branch Department o f M e d l c a l N e u r o s c t e n c e s D l v l s i o n of N e u r o p s y c h l a t r y W a l t e r Reed Army I n s t i t u t e o f R e s e a r c h Washington D.C. 20012 (Received in final form May 13, 1982) S~.-.-ry The e f f e c t s of n a l o x o n e or t h y r o t r o p l n r e l e a s i n g hormone (TRH) upon n e u r o l o g l c outcome were e v a l u a t e d I n g e r b i l models o f c e r e b r a l Ischemla. Following temporary bilateral carotid occluslon, hypotenslon was transiently reversed by these endorphln antagonists. However, neither drug altered time to awaken, tlme to death, or the severity of neurologlc signs (ptosls, movement, retracted paws, circling, righting reflexes, seizures, or o p l s t h o t o n u s ) when e v a l u a t e d by a b l l n d e d r a t e r . 6or p l a t e e s c a p e and r o t o - r o d p e r f o r m a n c e were a l s o u n a l t e r e d by n a l o x o n e o r TRH; TRH, but not n a l o x o n e , i n c r e a s e d r e s p i r a t o r y rates. Thus, t h e transient improvement o f c a r d l o r e s p l r a t o r y f u n c t i o n produced by t h e s e drugs I s u n r e l a t e d t o t h e m o r b i d i t y and m o r t a l i t y a s s o c i a t e d w l t h t em p o r ar y c e r e b r a l I s c h e ~ t a i n t h e g e r b l l . Additional studies e v a l u a t i n g t h e e f f e c t s o f n a l o x o n e o r TRH upon n e u r o l o g l c outcome f o l l o w i n g permanent u n i l a t e r a l carotid occluslon also failed to show any t h e r a p e u t i c e f f e c t s o f t h e s e d r u g s . Both morphine and TRH e x a c e r b a t e d th e e f f e c t s of I s c h e m l a . Of g e r b l l s which d e v e l o p e d n e u r o l o g l c I m p a i r m e n t , t h e d e f i c i t was u s u a l l y i p s l l a t e r a l to the occluded carotid. Collectively, these results indicate that neither n a l o x o n e nor TRH p r e v e n t s tschemtc deflcits In t h e gerbll. Further studles wlth different cerebral Ischemla models In other species are requlred to clarify the possible therapeutic e f f e c t s o f t h e s e drugs In e x p e r l m e n t a l s t r o k e . We have shown in a variety of species that the opiate antagonist naloxone as well as the trlpeptlde thyrotropln releaslng hormone (TRR) reverses the cardlovascular depreselon of circulatory shock and significantly improves survival (1,2,3). Addltlonally, the antl-endorphln effects of these substances were associated wlth a decrease in paralysis followlng experlmental spinal c o r d trauma (4,5,6). Because e p l n a l cord i n j u r y and c e r t a i n c e r e b r o v a s c u l a r d i s o r d e r s s h a r e I n c o ~ o n an i s c h e m l c e t i o l o g y , t h e p r e s e n t s t u d i e s were co n d u c t e d t o l n v e s t l g a t e t h e h y p o t h e s i s t h a t n a l o x o n e o r TRH would improve n e u r o l o g l c outcome I n t h e g e r b i l model o f c e r e b r a l I s c h e m l a and stroke. C a r o t i d o c c l u s l o n I n t h e Mongolian g e r b i l has s e r v e d as a model o f c e r e b r a l l e c h e m l a s i n c e t h l s s p e c l e e , u n l i k e many ma~als, l a c k s p o s t e r i o r co~-,nlcatlng arteries. Thus, I n t h e a b s e n c e o f a c o m p l e t e c l r c l e o f W l l l l s , 0024-3205/82/040385-08503.00/0
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Naloxone and TRH in Gerbil Stroke Models
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forebrain perfusion is entirely dependent upon carotid blood flow (7,8). Ischemic injury to the brain, induced by temporary bilateral occlusion of both common carotid arteries, results in neurologlc deficits and mortality (8). By contrast, permanent carotid occlusion of a single carotid artery produces neurologlc signs and death in only 30-40% of gerbils; non-responders maintain adequate Ipsilateral blood flow via shunting through a functional anterior communicating artery. Since our primary interest was in the pharmacologlcal management of the acute Ischem/c period, we chose to evaluate temporary bilateral carotid occlusion instead of permanent unllateral occlusion for three reasons: I. Theoretically, it appeared that permanent carotid occlusion would prevent vascular access of injected drugs or improved blood flow to affect the ischem/c areas of the brain. 2. The permanent unilateral carotid occlusion model produces neurologic signs in only 30-40% of gerbils. 3. Permanent carotid occluslon is more relevant to problems of infarction and cerebral edema (8,9). Nonetheless, in studies using both experimental ischem/c models, naloxone was shown to have no therapeutic effects~ whereas both TRH and morphine further exacerbated neurologlc signs and mortality. Materials and Methods Temporary bilateral occlusion: Adult male Mongolian gerbils (50-60g; Tumble Brook Farms, HD), previously maintained on rat chow and ad-libltum water, were anesthetized with 30 mg/kg intraperitoneal (ip) pentobarbltal. Both common carotid arteries were exposed through a cervical incision and carefully dissected away from adjacent nerve sheaths. Temporary bilateral carotid occlusion was accomplished with Mayfield (aneurism) cllps for 15, 20, or 30 ,tin according to the method of Harrison et al
(10).
In a few animals, the ventral tail artery and right external Jugular vein were catheterized with PE 10 tubing to allow for cardiovascular monitoring and intravenous (iv) drug delivery according to methods previously used in this laboratory (I). In these studies, 15 ,tin of bilateral carotid occlusion was followed by an additional 15 m/n wait until blood pressure stabilized at hypotense levels. At this time, cardiovascular effects of iv naloxone (5 mg/kg; Endo Labs.), TRH (2 mg/kg; Beckman Labs.), or sallne were compared. The neurologlc effects resulting from 20 or 30 min of temporary bilateral carotid occlusion were evaluated in additional animals. In randomly selected gerbils, drugs were injected immediately following cllp removal. Treatment groups w e r e concurrently studied following ip saline plus subcutaneous (sc) placebo pellet, naloxone (10 mg/kg) plus I0 mg naloxone pellet, or TRH (8 mg/kg) plus 2 mg TRH in 0.1 ml gelatin. Intraperitoneal drugs were injected in a volume of 0.10-0.12 ml, depending upon gerbil size. Pellets were placed sc over the hip; TR}Pgel was injected in the same region. Neurologic signs were evaluated by a rater who was blinded as to treatment groups. Measures included time to awaken (return of righting and ambulation) and time to death. Five hours following clip release and treatment, each gerbil was rated for presence or absence of ptosis, movement, retracted paws, circling, righting reflexes, seizures, and opisthotonus. Each neurologic sign was given a value of "I" if present, and slngle scores were added for each Indlvldual gerbil. Respiratory rates were observatlonally determined. In addition, animals were tested for the ability to escape from a 55°C hot plate onto the 5" high rim of a plexiglas cylinder as well as the ability to walk on a rotating rod (3" diameter, 30 RPM).
Vol. 31, No. 4, 1982
Naloxone and TRH in Gerbil Stroke Models
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Permanent unilateral occlusion: Using surgical p r o c e d u r e s d e s c r i b e d a b o v e , t h e r i g h t common c a r o t i d was doubly ltgated w i t h 4 - 0 s u t u r e a n d t h e wound was c l o s e d . Treatment groups were as described above, except an additional series of gerbils received ip morphine (10 mg/kg; Mallinckrodt) a n d one 75 mg m o r p h i n e p e l l e t immediately following carotid occlusion. Neurologtc signs as well as pharmacological testing were performed 5 hrs after occlusion and treatment as above except that lateralizatton of neurologic deficits (ptosis, circling, and retract paw) was n o t e d . Individual gerbils were once again compared across treatment groups in a blinded fashion. Results Temporary bllateral occluslon: In pilot studies, 15 min of bilateral occlusion resulted in the usual baroreceptor-medlated sustained hypertension associated wlth decreased carotid blood flow. As others have reported (I0), release of aneurism cllps resulted in a significant fall of mean arterial pressure (MAP) and pulse pressure (PP) below the baseline values. MAP dropped 30-40 mm HE below basellne over the next 15 min; PP decreased to 25% of control values. Both naloxone and TRH lmproved MAP by 15 mm HE, however this pressor effect was not sustained (<1520 m/n; data not shown). Table I: Effects of treatment on w a k e - u p t i m e a n d following 30 min of temporary bilateral carotid occlusion.
survival
Time to wake up (mln)
Tlme t o death* (mln)
Saline (n-10)
120 ± 18
310 ± 30 (n=6)
2/10
Naloxone (n=10)
134 f
12
387 f 23 (n=8)
2/10
TRH (n=10)
112 ± 7
3 2 8 f 27 (n=5)
2/10
Treatment
groups
One week survlval (alive/dead)
*represents only animals which dled during the first 8 hours. As seen in Table I, although TRH-treated gerbils were the first to awaken, no significant differences were observed across treatment groups. After awakening, many gerbils relapsed into a comatose state and died before neurologlcal evaluations could be performed at 5 hrs post-treatment. Of the gerbils which died during the first day, no significant differences in time to death were observed. Consistent with the results of Harrison et al (I0), 30 min of bilateral carotid occluslon was 80% lethal. Neurologlc impairment 5 hrs post occlusion and treatment was the same in all three groups of gerbils (Figure 1). W i t h o n e o r two e x c e p t i o n s , none of the gerbils with compromised neurologic function were able to escape from the h o t p l a t e o r w a l k on t h e r o t o - r o d . Consistent with the tachypulc effects of TRH i n c o n t r o l a n i m a l s , r e s p i r a t o r y rates were significantly elevated only In TRH-treated gerbils at 10, 80, a n d 300 m i n ( F i g u r e 2, ANOVA, 1 ) < . 0 1 ) . Otherwise, salineand naloxone-treated animals were breathing a t t h e same rate. Respiratory rates in all three groups of animals were greatest at 300 min ( F i g u r e 2).
388
Naloxone and TRH in Gerbil Stroke Models
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IIIEUROL061CSlGIHS 6HIIS POST TREATMENT
4
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0 ~UlNE
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F IGURE 1 Heurologic scores 5 h r s . a f t e r temporary b i l a t e r a l c a r o t i d o c c l u s i o n and t r e a t m e n t . S e v e r a l g e r b i l s d i e d b e f o r e n e u r o l o g i c e v a l u a t i o n . Seven d i f f e r e n t n e u r o l o g i c s i g n s were s e p a r a t e l y e v a l u a t e d ( s e e m e t h o d s ) ; d a t a p o i n t s r e p r e s e n t s i m p l e sum o f i n d i v i d u a l p o s i t i v e s i g n s . Histograms i n d i c a t e a v e r a g e n e u r o l o g i c s i g n s f o r each group o f a n i m a l s . No d i f f e r e n c e s among t r e a t m e n t groups were s e e n .
RESPIRATORYRATES FOLLOWING CUP RELEASEAND TREATMENT
i'°°t 75
50,
SAL NAL TRH @OF 6ERDLS-- 10 10 10 IOta
SAL NAL T'RH 10 10 10 8011i
S~ NAL 11~H 8 4 7 300 MIN
FIGURE 2 H i s t o g r a m r e p r e s e n t a v e r a g e r e s p i r a t o r y r a t e s ~SEH as measured a t various intervals f o l l o w i n g c l i p r e l e a s e and t r e a t a e n t . ANOVA i n d i c a t e s t h a t o n l y T R H i n c r e a s e s r e s p i r a t o r y r a t e s (p<0.01)
Vol. 31, No. 4, 1982
Naloxone and TRH in Gerbil Stroke Models
389
An a d d i t o n a l s t u d y was p e r f o r m e d t o e v a l u a t e t h e e f f e c t s o f n a l o x o n e f o l l o w i n g l e s s - s e v e r e i s c h e s i a (20 s i n as opposed t o 30 ndn of o c c l u s l o n ) . Twenty min o f b i l a t e r a l o c c l u s i o n r e s u l t e d i n a l e s s e r i n c i d e n c e o f n e u r o l o g l c s i g n s , and o n l y 20g m o r t a l i t y . However, none of t h e s e measures were a l t e r e d by n e l o x o n e t r e a t m e n t ( d a t a n o t shown) Permanent u n i l a t e r a l
occlusion:
As o t h e r s have r e p o r t e d ( 7 , 8 ) permanent u n i l a t e r a l o c c l u s i o n o f one common carotid artery produced neurologic signs in about 35-40Z o f g e r b i l s (Table 2). With few e x c e p t i o n s , none o f t h e g e r b i l s which d e m o n s t r a t e d n e u r o l o g l c s l g n s were a b l e t o e s c a p e from t h e h o t p l a t e o r walk on t h e r o t o rod. All gerbils are included in Figure 3 (top) to indicate total incidence of s i g n s ; a b s o l u t e l y no d i f f e r e n c e s between s a l i n e and n a l o x o n e a n i m a l s were s e e n on any n e u r o l o g l c measure ( T a b l e 2; F i g u r e 3, top) or on s u r v i v a l ( T a b l e 2; F i g u r e 3, b o t t o m ) . Most o f th e g e r b i l s which d e m o n s t r a t e d n e u r o l o g t c s i g n s u l t i m e t e d d i e d , w i t h t h e a v e r a g e t i m e t o d e a t h b e i n g 2 days ( F l g u r e 3, b o t t o m ) . Table 2: Summary o f s e l e c t e d n e u r o l o g i c f i n d i n g s 5 h r s a f t e r t r e a t m e n t o f g e r b i l s f o l l o w i n g permanent u n i l a t e r a l c a r o t i d o c c l u s i o n . treatment group
number of gerbils
resp. rate neurologlc ptosts (breaths/min) signs* (g) (Z)
h o t p l a t e r o t o - r o d one week escape 30 RPM s u r v i v a l w / I n 60 s e c (%) (%)
(z)
Saline
27
1 3 5 :1 :6
37
30
41
67
78
Naloxone
28
140 ± 4
36
29
33
64
75
TRH
I0
128 ± 7
70
50
20
30
30
Horphine
10
70
60
0
0
30
88 • 5 t • excluding ptosls
t p<.01, ANOVA
By c o n t r a s t w l t h s a l i n e and n a l o x o n e g r o u p s , b o t h TRH and morphine t r e a t e d g e r b i l s were s i g n i f i c a n t l y more i m p a i r e d and had a much lower s u r v i v a l r a t e ( T a b l e 2; F i g u r e 3 ) . In b o t h o f . t h e s e groups o f a n i m a l s , 70g o f t h e g e r b i l s had n e u r o l o g t c s i g n s and s u b s e q u e n t l y d i e d ( F i g u r e 3, b o t t o m ) . Although TRHt r e a t e d g e r b i l s d i d n o t have e l e v a t e d r e s p i r a t o r y r a t e s i n t h e s e s t u d i e s , morphine i n j e c t i o n s d i d produce s i g n i f i c a n t r e s p i r a t o r y d e p r e s s i o n ( T a b l e 2 ) . F o l l o w i n g u n i l a t e r a l c a r o t i d l i g a t l o n , n e u r o l o g t c impairment was u s u a l l y lpsilateral to the side of o c c l u s i o n . Of 29 g e r b i l s d e m o n s t r a t i n g a g r e a t e r p t o s i s i n one e y e , 22 e x p e r i e n c e d t h e d e f i c i t on t h e o c c l u d e d s i d e . Circling was i p s i l a t e r a l i n 13 of 19 g e r b i l s which d e m o n s t r a t e d t h i s b e h a v i o r . In a l l 13 g e r b i l s w i t h r e t r a c t e d f o r e p a w s , t h e impairment was i p s i l a t e r a l to the occluded carotid artery. None o f t h e drug t r e a t m e n t s a l t e r e d t h i s i p s i l a t e r a l pattern of neurologic deficits. In a f i n a l s e r i e s o f g e r b l l s , t r e a t m e n t w i t h e i t h e r s a l l n e o r u a l o x o n e was w i t h h e l d u n t i l S-6 h r s a f t e r permanent r l g h t - c a r o t i d o c c l u s i o n . A cco r d i n g t o t h e method o f Bosobuchl e t e l ( 1 1 ) , 1.0 o r 5.0 Lg/kg n a l o x o n e i n j e c t i o n s were c o z p a r e d t o e q u l v o l u m e Ip s a l i n e t r e a t m e n t , however n e u r o l o g l c a s n s e s s m e n t was by • b l i n d e d r a t e r . R e g a r d l e s s o f t r e a t m e n t , h a n d l i n g a l o n e produced had an i n c r e a s e i n a c t i v i t y and a p p a r e n t d e c r e a s e i n n e u r o l o g i c d e f i c i t s which l a s t e d 20-30 s i n . Again, n a l o x o n e was w i t h o u t b e n e f i t [ e . g . a t lmg/kg, n e u r o l o g t c s c o r e s ( p o p u l a t i o n ) were 7 ( 1 ) , 6 ( 1 ) , 2 ( 2 ) , 1 ( 3 ) and 0 ( 3 ) ; compare w l t h F i g u r e 3 ] .
390
Naloxone and TRB In Gerbil Stroke Models
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NEUROLOGICSIGNS 5HRS POST TREATMENT =w,
6 5
o .,J
000
00
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on "
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NALOXONE 25%
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F IGURE 3
N e u r o l o g l c s i g n s and tlme t o d e a t h 5 h r s f o l l o w l n g permanent r l g h t c a r o t i d o c c l u s l o n . As b e f o r e , each p o l n t r e p r e s e n t s t h e sum o f I n d l v l d u a l n e u r o l o g l c s i g n s o b s e r v e d out o f s e v e n p o s s l b l e t o t a l points. Histograms a r e a v e r a g e v a l u e s . A l l g e r b l l s s t u d i e d a r e r e p r e s e n t e d ; g e r b l l s w l t h p o i n t s a t "0" appeared normal. Naloxone t r e a t m e n t was w i t h o u t e f f e c t , whereas b o t h TRH and morphine r e s u l t e d I n an r e l a t l v e i n c r e a s e I n t h e number o f i m p a i r e d g e r b i l s ( t o p ; Hann-Whltney "U", p>0.30) and a g r e a t e r t o t a l Z I o r t a l l t y (bottom; Mann-Whltney "U", p > 0 . 3 0 ) .
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Naloxone and TRH in Gerbil Stroke Models
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Discussion The r e s u l t s of this study fail to demonstrate any therapeutic effects of naloxone upon neurologlc signs, re8piratory rates, or survival following temporary bilateral occlusion or permanent unilateral occlusion of the carotid arteries in Mongolian gerbils. T h u s , n a l o x o n e was w i t h o u t b e n e f i t i n b l o c k i n g or reversing the neurologic deficits i n t h e s e m o d e l s o f s t r o k e ( F i g u r e s 1&3 ; T a b l e s 1&2). Although naloxone transiently improved cardiovascular function when injected following temporary carotid occlusion and TRH improved respiratory rates as well as blood pressure, these effects are clearly not associated with the morbidity and mortality which accompany this model of cerebral lschemia. In f a c t , TRH e x a c e r b a t e d neurologic s i g n s and m o r t a l i t y when a d m i n i s t e r e d f o l l o w i n g p e r m a n e n t u n i l a t e r a l carotid ligation. Our prior work had indicated that neuronal ischemia following blunt injury to the cervical spinal cord was alleviated by subsequent treatment with n a l o x o n e a s w e l l a s TRH ( 4 - 6 ) . In t h o s e s t u d i e s , i t was shown t h a t b o t h naloxone a n d TRH i m p r o v e d t h e post-injury hypotension and significantly prevented the development of quadrlparesis associated with cervical spinal trauma. Circulating endorphin levels were elevated following injury, and naloxone treated animals demonstrated a significant improvement of spinal cord blood flow. These collective results prompted the hypothesis that central n e r v o u s s y s t e m (CNS) i s c h e m i a r e s u l t i n g from other causes would also involve an endorphin etiology and thus respond to receptor level (naloxone) or physiological (TRH) o p i a t e a n t a g o n i s t s . The obvious conclusion from the various experiments described above and previously (4-6) is that different forms of CNS ischemla have variable etiologies and hence variable pharmacological responses. Certainly, cardiorespiratory improvement alone probably cannot alleviate the ischemic-lnduced neurologic impairment in gerbil models of stroke. Perfuslon of the brain and spinal cord is normally independent of peripheral arterial pressure due to predominant autoregulatory influences on CNS vascular beds (12). Perhaps differences in autoregulatory responses following traumatic injury versus ischemia induced by other causes may be an important factor which distinguishes these forms of ischemic insult as well as their responses to drugs. As d e m o n s t r a t e d above, our initial results with the gerbil model of temporary bilateral c a r o t i d o c c l u s i o n f a i l e d t o show a n y t h e r a p e u t i c effects of n a l o x o n e o r TRH. However, r e c e n t l y H o s o b u c h i a n d a s s o c i a t e s (11) have reported that naloxone temporarily improved neurologic signs and survival in gerbils subjected to permanent unilateral carotid occlusion. We a t t e m p t e d a d i r e c t replication o f t h a t work a n d o b t a i n e d n e g a t i v e r e s u l t s when n e u r o l o g i c signs were evaluated across treatment groups in a single-blind fashion (vide supra). Our f u r t h e r e x p e r i m e n t s u s i n g d i f f e r e n t doses and treatment schedules described above also failed to uncover therapeutic effects of naloxone in gerbil models of cerebral lschemia. At p r e s e n t , we a r e u n c e r t a i n as to the reason for these discrepancies. C o n s o n a n t w i t h t h e f i n d i n g s o f H o s o b u c h i e t a l ( 1 1 ) , we a l s o o b s e r v e d t h a t morphine exacerbated the signs of "stroke". Indeed, the pharmacological effects of opiates closely resemble neurologic deficits seen in gerbils following carotid occlusion (e.g. decreased locomotion, decreased responsiveness, loss of righting reflexes, and lower respiratory rates). However, pharmacological responses to injected o p i a t e s do n o t s e r v e t o v e r i f y an involvement of endogenous opiates and associated receptors in surgicallyinduced cerebral ischemia. Since permanent carotid occlusion is a model of cerebral edema, the effects o f n a l o x o n e a n d m o r p h i n e on b r a i n edema i n t h i s gerbil model should be directly investigated.
392
Naloxone and TRH in Gerbil Stroke Models
Vol. 31, No. 4, 1982
Baskin and Hosobuchi (13) have r e p o r t e d t h a t n a l o x o n e t e m p o r a r i l y r e v e r s e d neurologic deficits i n two c a s e s o f human c e r e b r a l l s c h e m i a where permanent c e r e b r a l i n f a r c t i o n was n o t p r e s e n t . In one a d d i t i o n a l c a s e , where a permanent i n f a r c t i o n was r a d i o l o g i c a l l y v e r i f i e d , n a l o x o n e was w i t h o u t e f f e c t ( 1 3 ) . This c l i n i c a l f i n d i n g q u e s t i o n s t h e p r e d i c t i v e v a l u e o f s t u d i e s u t i l i z i n g permanent c a r o t i d o c c l u s i o n i n th e g e r b i l s i n c e t h i s model would presumably s i m u l a t e a permanent i n f a r c t s i t u a t i o n . S i n c e c e r e b r a l l s c h e m / a can r e s u l t from many c a u s e s o f both c e n t r a l and p e r i p h e r a l o r i g i n , t h e l a c k o f e f f e c t of n a l o x o n e i n t h e g e r b i l models we used may not be g e n e r a l i z e d t o o t h e r f o r l s of c e r e b r a l i s c h e e t t a i n d i f f e r e n t s p e c i e s , i n c l u d i n g man. For example, p o s t - i s c h e m / c poor p e r f u e l o n (no r e f l o v ) may be a p r i m a r y f a c t o r i n t h e c o ~ p l i c a t i o n s o f human c e r e b r a l i s c h e a d a ( 1 4 ) . By c o n t r a s t , "no r e f l o w " i s an i n f r e q u e n t o c c u r r e n c e i n g e r b i l models o f cerebral £schemla, a f i n d i n g which l i m i t s the u s e f u l n e s s of g e r b i l s in e x t r a p o l a t i n g to the c l i n i c a l environment (10,15). R e c e n t l y , Paden e t e l , have demonstrated that n a l o x o n e improves b o t h l o c a l cerebral b l o o d f l o w and s o m a t o s e n s o r y r e s p o n s e s i n e x p e r i m e n t a l s t r o k e i n dogs u s i n g an a i r embolism model o f c e r e b r a l £echemia ( 1 6 ) . Thus, as v a r i o u s forms of c e r e b r a l i s c h e ~ a a r e e x p e r i m e n t a l l y a p p r o x i m a t e d i n animal s p e c i e s , t h e p o s s i b l e u t i l i t y of n a l o x o n e i n t h e t r e a t m e n t o f CNS £schemic d i s o r d e r s w i l l be c l a r i f i e d . Acknovle~emants We thank C h a r l e s G l a t t , J u l i e Kenner, Barry Ruvio, Lydia R o b l e s , and C l i f f Johns o n f o r t e c h n i c a l h e l p and Pat Conners f o r p r e p a r a t i o n o f t h e m a n u s c r i p t . References I. 2. 3.
4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16.
J.W. HOIADAY and A.I. FADEN, Nature 275 450-451 (1978). J.W. ROLADAY, R.J. D'AMATO, and A.l. FADEN, Science 213 216-218 (1981). J.W. HOLADAY and A . I . FADEN, Advances i n P h a r u a c o l o g y and T h e r a p e u t i c s I I , H. Yoshida, Y. H a g i h a r a , and S. E b a s h i ( E d s . ) , pp. 45-55, Pergamon P r e s s , Oxford (1982). A . I . FADEN, T.P. JACOBS, and J.W. HOLADAY, S c i e n c e 211 493-494 ( 1 9 8 1 ) . A . I . FADEN, T.P. JACOBS, E.H. MOUGRY, and J.W. HOIADAY, Annals o f N e u r o l . 10 326-332 ( 1 9 8 1 ) . A . I . FADEN, T.P. JACOBS, and J.W. HOL%DAY, New Eng. J . Med. 305 1063-1067 (1981). S. LEVINE and D. SOHN, Arch. P a t h . 87 315-317 ( 1 9 6 9 ) . K. KAHN, N e u r o l . 22 510-515 (1972). M.J.G. HARRISON, J . ARNOLD, L. SRDAL, and R. W. ROSS RUSSELL, J . N eu r o l . N e u r o s u r g . Psych. 38 1194-1196 (1975). M.J.G. HARRISON, L. SEDAL, J . ARNOLD, and R.W. ROSS RUSSELL, J . N e u r o l . N e u r o s u r g . Psych. 38 1190-1193 (1975). Y. NOSOBUCHI, D.S. BASK]N, and S.K. WOO, S c i e n c e 215 69-71 ( 1 9 8 2 ) . J . L . OSTERHOIM, J . Neurosurg. 40 5-33 (1974). D.S. BASK]N and Y. HOSOBUCHI, l a n c e t i 272-275 ( 1 9 8 1 ) . T.W. IANGFITT, J . Neurosurg. 40 561-575 ( 1 9 7 4 ) . D.E. LEVY, J . B . BRIERLEY, and F. PLUM, J . N e u r o l . Neurosurg. Psych. 38 1197-1205 (1975). A . L FADRN, J . RALI.ENBE(X, and C. BROWN, N e u r o l . ( i n p r e s s ) .
Pergamon Press
NALOXONE OR T R H F A I L S TO IMPROVE NEUROLOGIC DEFICITS IN GERBIL MODELS OF "STROKE" John W. Holaday and R o b e r t J . D'Amato Neuropharmacology Branch Department o f M e d l c a l N e u r o s c t e n c e s D l v l s i o n of N e u r o p s y c h l a t r y W a l t e r Reed Army I n s t i t u t e o f R e s e a r c h Washington D.C. 20012 (Received in final form May 13, 1982) S~.-.-ry The e f f e c t s of n a l o x o n e or t h y r o t r o p l n r e l e a s i n g hormone (TRH) upon n e u r o l o g l c outcome were e v a l u a t e d I n g e r b i l models o f c e r e b r a l Ischemla. Following temporary bilateral carotid occluslon, hypotenslon was transiently reversed by these endorphln antagonists. However, neither drug altered time to awaken, tlme to death, or the severity of neurologlc signs (ptosls, movement, retracted paws, circling, righting reflexes, seizures, or o p l s t h o t o n u s ) when e v a l u a t e d by a b l l n d e d r a t e r . 6or p l a t e e s c a p e and r o t o - r o d p e r f o r m a n c e were a l s o u n a l t e r e d by n a l o x o n e o r TRH; TRH, but not n a l o x o n e , i n c r e a s e d r e s p i r a t o r y rates. Thus, t h e transient improvement o f c a r d l o r e s p l r a t o r y f u n c t i o n produced by t h e s e drugs I s u n r e l a t e d t o t h e m o r b i d i t y and m o r t a l i t y a s s o c i a t e d w l t h t em p o r ar y c e r e b r a l I s c h e ~ t a i n t h e g e r b l l . Additional studies e v a l u a t i n g t h e e f f e c t s o f n a l o x o n e o r TRH upon n e u r o l o g l c outcome f o l l o w i n g permanent u n i l a t e r a l carotid occluslon also failed to show any t h e r a p e u t i c e f f e c t s o f t h e s e d r u g s . Both morphine and TRH e x a c e r b a t e d th e e f f e c t s of I s c h e m l a . Of g e r b l l s which d e v e l o p e d n e u r o l o g l c I m p a i r m e n t , t h e d e f i c i t was u s u a l l y i p s l l a t e r a l to the occluded carotid. Collectively, these results indicate that neither n a l o x o n e nor TRH p r e v e n t s tschemtc deflcits In t h e gerbll. Further studles wlth different cerebral Ischemla models In other species are requlred to clarify the possible therapeutic e f f e c t s o f t h e s e drugs In e x p e r l m e n t a l s t r o k e . We have shown in a variety of species that the opiate antagonist naloxone as well as the trlpeptlde thyrotropln releaslng hormone (TRR) reverses the cardlovascular depreselon of circulatory shock and significantly improves survival (1,2,3). Addltlonally, the antl-endorphln effects of these substances were associated wlth a decrease in paralysis followlng experlmental spinal c o r d trauma (4,5,6). Because e p l n a l cord i n j u r y and c e r t a i n c e r e b r o v a s c u l a r d i s o r d e r s s h a r e I n c o ~ o n an i s c h e m l c e t i o l o g y , t h e p r e s e n t s t u d i e s were co n d u c t e d t o l n v e s t l g a t e t h e h y p o t h e s i s t h a t n a l o x o n e o r TRH would improve n e u r o l o g l c outcome I n t h e g e r b i l model o f c e r e b r a l I s c h e m l a and stroke. C a r o t i d o c c l u s l o n I n t h e Mongolian g e r b i l has s e r v e d as a model o f c e r e b r a l l e c h e m l a s i n c e t h l s s p e c l e e , u n l i k e many ma~als, l a c k s p o s t e r i o r co~-,nlcatlng arteries. Thus, I n t h e a b s e n c e o f a c o m p l e t e c l r c l e o f W l l l l s , 0024-3205/82/040385-08503.00/0
386
Naloxone and TRH in Gerbil Stroke Models
Vol. 31, No. 4, 1982
forebrain perfusion is entirely dependent upon carotid blood flow (7,8). Ischemic injury to the brain, induced by temporary bilateral occlusion of both common carotid arteries, results in neurologlc deficits and mortality (8). By contrast, permanent carotid occlusion of a single carotid artery produces neurologlc signs and death in only 30-40% of gerbils; non-responders maintain adequate Ipsilateral blood flow via shunting through a functional anterior communicating artery. Since our primary interest was in the pharmacologlcal management of the acute Ischem/c period, we chose to evaluate temporary bilateral carotid occlusion instead of permanent unllateral occlusion for three reasons: I. Theoretically, it appeared that permanent carotid occlusion would prevent vascular access of injected drugs or improved blood flow to affect the ischem/c areas of the brain. 2. The permanent unilateral carotid occlusion model produces neurologic signs in only 30-40% of gerbils. 3. Permanent carotid occluslon is more relevant to problems of infarction and cerebral edema (8,9). Nonetheless, in studies using both experimental ischem/c models, naloxone was shown to have no therapeutic effects~ whereas both TRH and morphine further exacerbated neurologlc signs and mortality. Materials and Methods Temporary bilateral occlusion: Adult male Mongolian gerbils (50-60g; Tumble Brook Farms, HD), previously maintained on rat chow and ad-libltum water, were anesthetized with 30 mg/kg intraperitoneal (ip) pentobarbltal. Both common carotid arteries were exposed through a cervical incision and carefully dissected away from adjacent nerve sheaths. Temporary bilateral carotid occlusion was accomplished with Mayfield (aneurism) cllps for 15, 20, or 30 ,tin according to the method of Harrison et al
(10).
In a few animals, the ventral tail artery and right external Jugular vein were catheterized with PE 10 tubing to allow for cardiovascular monitoring and intravenous (iv) drug delivery according to methods previously used in this laboratory (I). In these studies, 15 ,tin of bilateral carotid occlusion was followed by an additional 15 m/n wait until blood pressure stabilized at hypotense levels. At this time, cardiovascular effects of iv naloxone (5 mg/kg; Endo Labs.), TRH (2 mg/kg; Beckman Labs.), or sallne were compared. The neurologlc effects resulting from 20 or 30 min of temporary bilateral carotid occlusion were evaluated in additional animals. In randomly selected gerbils, drugs were injected immediately following cllp removal. Treatment groups w e r e concurrently studied following ip saline plus subcutaneous (sc) placebo pellet, naloxone (10 mg/kg) plus I0 mg naloxone pellet, or TRH (8 mg/kg) plus 2 mg TRH in 0.1 ml gelatin. Intraperitoneal drugs were injected in a volume of 0.10-0.12 ml, depending upon gerbil size. Pellets were placed sc over the hip; TR}Pgel was injected in the same region. Neurologic signs were evaluated by a rater who was blinded as to treatment groups. Measures included time to awaken (return of righting and ambulation) and time to death. Five hours following clip release and treatment, each gerbil was rated for presence or absence of ptosis, movement, retracted paws, circling, righting reflexes, seizures, and opisthotonus. Each neurologic sign was given a value of "I" if present, and slngle scores were added for each Indlvldual gerbil. Respiratory rates were observatlonally determined. In addition, animals were tested for the ability to escape from a 55°C hot plate onto the 5" high rim of a plexiglas cylinder as well as the ability to walk on a rotating rod (3" diameter, 30 RPM).
Vol. 31, No. 4, 1982
Naloxone and TRH in Gerbil Stroke Models
387
Permanent unilateral occlusion: Using surgical p r o c e d u r e s d e s c r i b e d a b o v e , t h e r i g h t common c a r o t i d was doubly ltgated w i t h 4 - 0 s u t u r e a n d t h e wound was c l o s e d . Treatment groups were as described above, except an additional series of gerbils received ip morphine (10 mg/kg; Mallinckrodt) a n d one 75 mg m o r p h i n e p e l l e t immediately following carotid occlusion. Neurologtc signs as well as pharmacological testing were performed 5 hrs after occlusion and treatment as above except that lateralizatton of neurologic deficits (ptosis, circling, and retract paw) was n o t e d . Individual gerbils were once again compared across treatment groups in a blinded fashion. Results Temporary bllateral occluslon: In pilot studies, 15 min of bilateral occlusion resulted in the usual baroreceptor-medlated sustained hypertension associated wlth decreased carotid blood flow. As others have reported (I0), release of aneurism cllps resulted in a significant fall of mean arterial pressure (MAP) and pulse pressure (PP) below the baseline values. MAP dropped 30-40 mm HE below basellne over the next 15 min; PP decreased to 25% of control values. Both naloxone and TRH lmproved MAP by 15 mm HE, however this pressor effect was not sustained (<1520 m/n; data not shown). Table I: Effects of treatment on w a k e - u p t i m e a n d following 30 min of temporary bilateral carotid occlusion.
survival
Time to wake up (mln)
Tlme t o death* (mln)
Saline (n-10)
120 ± 18
310 ± 30 (n=6)
2/10
Naloxone (n=10)
134 f
12
387 f 23 (n=8)
2/10
TRH (n=10)
112 ± 7
3 2 8 f 27 (n=5)
2/10
Treatment
groups
One week survlval (alive/dead)
*represents only animals which dled during the first 8 hours. As seen in Table I, although TRH-treated gerbils were the first to awaken, no significant differences were observed across treatment groups. After awakening, many gerbils relapsed into a comatose state and died before neurologlcal evaluations could be performed at 5 hrs post-treatment. Of the gerbils which died during the first day, no significant differences in time to death were observed. Consistent with the results of Harrison et al (I0), 30 min of bilateral carotid occluslon was 80% lethal. Neurologlc impairment 5 hrs post occlusion and treatment was the same in all three groups of gerbils (Figure 1). W i t h o n e o r two e x c e p t i o n s , none of the gerbils with compromised neurologic function were able to escape from the h o t p l a t e o r w a l k on t h e r o t o - r o d . Consistent with the tachypulc effects of TRH i n c o n t r o l a n i m a l s , r e s p i r a t o r y rates were significantly elevated only In TRH-treated gerbils at 10, 80, a n d 300 m i n ( F i g u r e 2, ANOVA, 1 ) < . 0 1 ) . Otherwise, salineand naloxone-treated animals were breathing a t t h e same rate. Respiratory rates in all three groups of animals were greatest at 300 min ( F i g u r e 2).
388
Naloxone and TRH in Gerbil Stroke Models
Vol. 31, No. 4, 1982
IIIEUROL061CSlGIHS 6HIIS POST TREATMENT
4
oo •
2
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0 ~UlNE
• ~.OXO~
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F IGURE 1 Heurologic scores 5 h r s . a f t e r temporary b i l a t e r a l c a r o t i d o c c l u s i o n and t r e a t m e n t . S e v e r a l g e r b i l s d i e d b e f o r e n e u r o l o g i c e v a l u a t i o n . Seven d i f f e r e n t n e u r o l o g i c s i g n s were s e p a r a t e l y e v a l u a t e d ( s e e m e t h o d s ) ; d a t a p o i n t s r e p r e s e n t s i m p l e sum o f i n d i v i d u a l p o s i t i v e s i g n s . Histograms i n d i c a t e a v e r a g e n e u r o l o g i c s i g n s f o r each group o f a n i m a l s . No d i f f e r e n c e s among t r e a t m e n t groups were s e e n .
RESPIRATORYRATES FOLLOWING CUP RELEASEAND TREATMENT
i'°°t 75
50,
SAL NAL TRH @OF 6ERDLS-- 10 10 10 IOta
SAL NAL T'RH 10 10 10 8011i
S~ NAL 11~H 8 4 7 300 MIN
FIGURE 2 H i s t o g r a m r e p r e s e n t a v e r a g e r e s p i r a t o r y r a t e s ~SEH as measured a t various intervals f o l l o w i n g c l i p r e l e a s e and t r e a t a e n t . ANOVA i n d i c a t e s t h a t o n l y T R H i n c r e a s e s r e s p i r a t o r y r a t e s (p<0.01)
Vol. 31, No. 4, 1982
Naloxone and TRH in Gerbil Stroke Models
389
An a d d i t o n a l s t u d y was p e r f o r m e d t o e v a l u a t e t h e e f f e c t s o f n a l o x o n e f o l l o w i n g l e s s - s e v e r e i s c h e s i a (20 s i n as opposed t o 30 ndn of o c c l u s l o n ) . Twenty min o f b i l a t e r a l o c c l u s i o n r e s u l t e d i n a l e s s e r i n c i d e n c e o f n e u r o l o g l c s i g n s , and o n l y 20g m o r t a l i t y . However, none of t h e s e measures were a l t e r e d by n e l o x o n e t r e a t m e n t ( d a t a n o t shown) Permanent u n i l a t e r a l
occlusion:
As o t h e r s have r e p o r t e d ( 7 , 8 ) permanent u n i l a t e r a l o c c l u s i o n o f one common carotid artery produced neurologic signs in about 35-40Z o f g e r b i l s (Table 2). With few e x c e p t i o n s , none o f t h e g e r b i l s which d e m o n s t r a t e d n e u r o l o g l c s l g n s were a b l e t o e s c a p e from t h e h o t p l a t e o r walk on t h e r o t o rod. All gerbils are included in Figure 3 (top) to indicate total incidence of s i g n s ; a b s o l u t e l y no d i f f e r e n c e s between s a l i n e and n a l o x o n e a n i m a l s were s e e n on any n e u r o l o g l c measure ( T a b l e 2; F i g u r e 3, top) or on s u r v i v a l ( T a b l e 2; F i g u r e 3, b o t t o m ) . Most o f th e g e r b i l s which d e m o n s t r a t e d n e u r o l o g t c s i g n s u l t i m e t e d d i e d , w i t h t h e a v e r a g e t i m e t o d e a t h b e i n g 2 days ( F l g u r e 3, b o t t o m ) . Table 2: Summary o f s e l e c t e d n e u r o l o g i c f i n d i n g s 5 h r s a f t e r t r e a t m e n t o f g e r b i l s f o l l o w i n g permanent u n i l a t e r a l c a r o t i d o c c l u s i o n . treatment group
number of gerbils
resp. rate neurologlc ptosts (breaths/min) signs* (g) (Z)
h o t p l a t e r o t o - r o d one week escape 30 RPM s u r v i v a l w / I n 60 s e c (%) (%)
(z)
Saline
27
1 3 5 :1 :6
37
30
41
67
78
Naloxone
28
140 ± 4
36
29
33
64
75
TRH
I0
128 ± 7
70
50
20
30
30
Horphine
10
70
60
0
0
30
88 • 5 t • excluding ptosls
t p<.01, ANOVA
By c o n t r a s t w l t h s a l i n e and n a l o x o n e g r o u p s , b o t h TRH and morphine t r e a t e d g e r b i l s were s i g n i f i c a n t l y more i m p a i r e d and had a much lower s u r v i v a l r a t e ( T a b l e 2; F i g u r e 3 ) . In b o t h o f . t h e s e groups o f a n i m a l s , 70g o f t h e g e r b i l s had n e u r o l o g t c s i g n s and s u b s e q u e n t l y d i e d ( F i g u r e 3, b o t t o m ) . Although TRHt r e a t e d g e r b i l s d i d n o t have e l e v a t e d r e s p i r a t o r y r a t e s i n t h e s e s t u d i e s , morphine i n j e c t i o n s d i d produce s i g n i f i c a n t r e s p i r a t o r y d e p r e s s i o n ( T a b l e 2 ) . F o l l o w i n g u n i l a t e r a l c a r o t i d l i g a t l o n , n e u r o l o g t c impairment was u s u a l l y lpsilateral to the side of o c c l u s i o n . Of 29 g e r b i l s d e m o n s t r a t i n g a g r e a t e r p t o s i s i n one e y e , 22 e x p e r i e n c e d t h e d e f i c i t on t h e o c c l u d e d s i d e . Circling was i p s i l a t e r a l i n 13 of 19 g e r b i l s which d e m o n s t r a t e d t h i s b e h a v i o r . In a l l 13 g e r b i l s w i t h r e t r a c t e d f o r e p a w s , t h e impairment was i p s i l a t e r a l to the occluded carotid artery. None o f t h e drug t r e a t m e n t s a l t e r e d t h i s i p s i l a t e r a l pattern of neurologic deficits. In a f i n a l s e r i e s o f g e r b l l s , t r e a t m e n t w i t h e i t h e r s a l l n e o r u a l o x o n e was w i t h h e l d u n t i l S-6 h r s a f t e r permanent r l g h t - c a r o t i d o c c l u s i o n . A cco r d i n g t o t h e method o f Bosobuchl e t e l ( 1 1 ) , 1.0 o r 5.0 Lg/kg n a l o x o n e i n j e c t i o n s were c o z p a r e d t o e q u l v o l u m e Ip s a l i n e t r e a t m e n t , however n e u r o l o g l c a s n s e s s m e n t was by • b l i n d e d r a t e r . R e g a r d l e s s o f t r e a t m e n t , h a n d l i n g a l o n e produced had an i n c r e a s e i n a c t i v i t y and a p p a r e n t d e c r e a s e i n n e u r o l o g i c d e f i c i t s which l a s t e d 20-30 s i n . Again, n a l o x o n e was w i t h o u t b e n e f i t [ e . g . a t lmg/kg, n e u r o l o g t c s c o r e s ( p o p u l a t i o n ) were 7 ( 1 ) , 6 ( 1 ) , 2 ( 2 ) , 1 ( 3 ) and 0 ( 3 ) ; compare w l t h F i g u r e 3 ] .
390
Naloxone and TRB In Gerbil Stroke Models
Vol. 31, No. 4, 1982
NEUROLOGICSIGNS 5HRS POST TREATMENT =w,
6 5
o .,J
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NALOXONE 25%
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F IGURE 3
N e u r o l o g l c s i g n s and tlme t o d e a t h 5 h r s f o l l o w l n g permanent r l g h t c a r o t i d o c c l u s l o n . As b e f o r e , each p o l n t r e p r e s e n t s t h e sum o f I n d l v l d u a l n e u r o l o g l c s i g n s o b s e r v e d out o f s e v e n p o s s l b l e t o t a l points. Histograms a r e a v e r a g e v a l u e s . A l l g e r b l l s s t u d i e d a r e r e p r e s e n t e d ; g e r b l l s w l t h p o i n t s a t "0" appeared normal. Naloxone t r e a t m e n t was w i t h o u t e f f e c t , whereas b o t h TRH and morphine r e s u l t e d I n an r e l a t l v e i n c r e a s e I n t h e number o f i m p a i r e d g e r b i l s ( t o p ; Hann-Whltney "U", p>0.30) and a g r e a t e r t o t a l Z I o r t a l l t y (bottom; Mann-Whltney "U", p > 0 . 3 0 ) .
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Discussion The r e s u l t s of this study fail to demonstrate any therapeutic effects of naloxone upon neurologlc signs, re8piratory rates, or survival following temporary bilateral occlusion or permanent unilateral occlusion of the carotid arteries in Mongolian gerbils. T h u s , n a l o x o n e was w i t h o u t b e n e f i t i n b l o c k i n g or reversing the neurologic deficits i n t h e s e m o d e l s o f s t r o k e ( F i g u r e s 1&3 ; T a b l e s 1&2). Although naloxone transiently improved cardiovascular function when injected following temporary carotid occlusion and TRH improved respiratory rates as well as blood pressure, these effects are clearly not associated with the morbidity and mortality which accompany this model of cerebral lschemia. In f a c t , TRH e x a c e r b a t e d neurologic s i g n s and m o r t a l i t y when a d m i n i s t e r e d f o l l o w i n g p e r m a n e n t u n i l a t e r a l carotid ligation. Our prior work had indicated that neuronal ischemia following blunt injury to the cervical spinal cord was alleviated by subsequent treatment with n a l o x o n e a s w e l l a s TRH ( 4 - 6 ) . In t h o s e s t u d i e s , i t was shown t h a t b o t h naloxone a n d TRH i m p r o v e d t h e post-injury hypotension and significantly prevented the development of quadrlparesis associated with cervical spinal trauma. Circulating endorphin levels were elevated following injury, and naloxone treated animals demonstrated a significant improvement of spinal cord blood flow. These collective results prompted the hypothesis that central n e r v o u s s y s t e m (CNS) i s c h e m i a r e s u l t i n g from other causes would also involve an endorphin etiology and thus respond to receptor level (naloxone) or physiological (TRH) o p i a t e a n t a g o n i s t s . The obvious conclusion from the various experiments described above and previously (4-6) is that different forms of CNS ischemla have variable etiologies and hence variable pharmacological responses. Certainly, cardiorespiratory improvement alone probably cannot alleviate the ischemic-lnduced neurologic impairment in gerbil models of stroke. Perfuslon of the brain and spinal cord is normally independent of peripheral arterial pressure due to predominant autoregulatory influences on CNS vascular beds (12). Perhaps differences in autoregulatory responses following traumatic injury versus ischemia induced by other causes may be an important factor which distinguishes these forms of ischemic insult as well as their responses to drugs. As d e m o n s t r a t e d above, our initial results with the gerbil model of temporary bilateral c a r o t i d o c c l u s i o n f a i l e d t o show a n y t h e r a p e u t i c effects of n a l o x o n e o r TRH. However, r e c e n t l y H o s o b u c h i a n d a s s o c i a t e s (11) have reported that naloxone temporarily improved neurologic signs and survival in gerbils subjected to permanent unilateral carotid occlusion. We a t t e m p t e d a d i r e c t replication o f t h a t work a n d o b t a i n e d n e g a t i v e r e s u l t s when n e u r o l o g i c signs were evaluated across treatment groups in a single-blind fashion (vide supra). Our f u r t h e r e x p e r i m e n t s u s i n g d i f f e r e n t doses and treatment schedules described above also failed to uncover therapeutic effects of naloxone in gerbil models of cerebral lschemia. At p r e s e n t , we a r e u n c e r t a i n as to the reason for these discrepancies. C o n s o n a n t w i t h t h e f i n d i n g s o f H o s o b u c h i e t a l ( 1 1 ) , we a l s o o b s e r v e d t h a t morphine exacerbated the signs of "stroke". Indeed, the pharmacological effects of opiates closely resemble neurologic deficits seen in gerbils following carotid occlusion (e.g. decreased locomotion, decreased responsiveness, loss of righting reflexes, and lower respiratory rates). However, pharmacological responses to injected o p i a t e s do n o t s e r v e t o v e r i f y an involvement of endogenous opiates and associated receptors in surgicallyinduced cerebral ischemia. Since permanent carotid occlusion is a model of cerebral edema, the effects o f n a l o x o n e a n d m o r p h i n e on b r a i n edema i n t h i s gerbil model should be directly investigated.
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Baskin and Hosobuchi (13) have r e p o r t e d t h a t n a l o x o n e t e m p o r a r i l y r e v e r s e d neurologic deficits i n two c a s e s o f human c e r e b r a l l s c h e m i a where permanent c e r e b r a l i n f a r c t i o n was n o t p r e s e n t . In one a d d i t i o n a l c a s e , where a permanent i n f a r c t i o n was r a d i o l o g i c a l l y v e r i f i e d , n a l o x o n e was w i t h o u t e f f e c t ( 1 3 ) . This c l i n i c a l f i n d i n g q u e s t i o n s t h e p r e d i c t i v e v a l u e o f s t u d i e s u t i l i z i n g permanent c a r o t i d o c c l u s i o n i n th e g e r b i l s i n c e t h i s model would presumably s i m u l a t e a permanent i n f a r c t s i t u a t i o n . S i n c e c e r e b r a l l s c h e m / a can r e s u l t from many c a u s e s o f both c e n t r a l and p e r i p h e r a l o r i g i n , t h e l a c k o f e f f e c t of n a l o x o n e i n t h e g e r b i l models we used may not be g e n e r a l i z e d t o o t h e r f o r l s of c e r e b r a l i s c h e e t t a i n d i f f e r e n t s p e c i e s , i n c l u d i n g man. For example, p o s t - i s c h e m / c poor p e r f u e l o n (no r e f l o v ) may be a p r i m a r y f a c t o r i n t h e c o ~ p l i c a t i o n s o f human c e r e b r a l i s c h e a d a ( 1 4 ) . By c o n t r a s t , "no r e f l o w " i s an i n f r e q u e n t o c c u r r e n c e i n g e r b i l models o f cerebral £schemla, a f i n d i n g which l i m i t s the u s e f u l n e s s of g e r b i l s in e x t r a p o l a t i n g to the c l i n i c a l environment (10,15). R e c e n t l y , Paden e t e l , have demonstrated that n a l o x o n e improves b o t h l o c a l cerebral b l o o d f l o w and s o m a t o s e n s o r y r e s p o n s e s i n e x p e r i m e n t a l s t r o k e i n dogs u s i n g an a i r embolism model o f c e r e b r a l £echemia ( 1 6 ) . Thus, as v a r i o u s forms of c e r e b r a l i s c h e ~ a a r e e x p e r i m e n t a l l y a p p r o x i m a t e d i n animal s p e c i e s , t h e p o s s i b l e u t i l i t y of n a l o x o n e i n t h e t r e a t m e n t o f CNS £schemic d i s o r d e r s w i l l be c l a r i f i e d . Acknovle~emants We thank C h a r l e s G l a t t , J u l i e Kenner, Barry Ruvio, Lydia R o b l e s , and C l i f f Johns o n f o r t e c h n i c a l h e l p and Pat Conners f o r p r e p a r a t i o n o f t h e m a n u s c r i p t . References I. 2. 3.
4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16.
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