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Long-term potentiation saturation in chronic cerebral hypoperfusion
Long-term potentiation saturation in chronic cerebral hypoperfusion
Lali H.S. S e k h o n 1 MB BS PhD, l a n S p e n c e 2 BSc PhD, M i c h a e l K. M o r g a n I MD FRACS, N e v i l l e C. W e b e r 3 MSc PhD 1Department of Surgery DO6, The Universityof Sydney, Sydney,NSW 2006, Australia 2Department of Pharmacology DO5, The Universityof Sydney,Sydney,NSW 2006, Australia 3School of Mathematics and Statistics FO7, The Universityof Sydney, Sydney,NSW 2006, Australia
Chronic reductions in cerebral b l o o d flow (CBF) o f between 25 and 50%, in the absence o f cerebral infarction, lead to i m p a i r m e n t s in h i p p o c a m p a l in vitro long-term potentiation (LTP). This study set out to explore s o m e o f the p r o p e r t i e s o f this i m p a i r m e n t o f LTP. LTP is an electrophysiological p r o p e r t y known to occur in the h i p p o c a m p u s , a region known to be exquisitely sensitive to hypoxic or ischemic insults. Thus, assessing LTP is a novel way o f assessing the effects o f subtle ischemic insults. Five male Sprague-Dawley rats h a d arteriovenous fistulae created surgically in the neck to induce a state o f chronic cerebral h y p o p e r f n s i o n (CCH) with the features described above. Five rats were used as age-matched controls. Twenty-six weeks after fistula formation, the animals were p r e p a r e d f o r in vitro h i p p o c a m p a l recording in a s u b m e r g e d tissue bath. Extracellular field potentials were r e c o r d e d at the Schaffer collateral-CA1 region, with a stimulus intensity that achieved a population spike amplitude o f 1 mV. After tetanic stimulation, the frequency and magnitude o f LTP was c o m p a r e d between control and fistula animals. All animals in b o t h these groups d e m o n s t r a t e d LTP in contradistinction to our previous work where LTP was impaired in fistula animals when a higher intensity o f stimulation was used. This indicates that the structures that are associated with the initiation and maintenance o f LTP (most p r o b a b l y the ischemia-sensitive CA1 pyramidal cells) are saturated as the stimulus intensity is increased. Thus, at this lower intensity o f stimulation LTP is p r e s e r v e d in the fistula animals, b u t f o u n d to be i m p a i r e d as the stimulus intensity is increased. Consequently, this study provides f u r t h e r i n f o r m a t i o n on this newly identified subtype of chronic cerebral ischemia which, in time, after f u r t h e r studies in humans, m a y help to redefine therapeutic indicators f o r the m a n a g e m e n t o f cerebral arteriovenous m a l f o r m a t i o n and severe cerebrovascular disease. Journal of Clinical Neuroscience 1998, 5 (3) : 323-328
© Harcourt Brace & Co. Ltd 1998
Keywords" arteriovenous malformation, brain slices, electrophysiology, hippocampus, ischemia, long-term potentiation
Introduction Chronic reductions in cerebral blood flow (CBF) can occur in many clinical situations, best typified by the reduction in CBF that occurs as a result of venous hypertension and cerebrovascular 'steal '1 in brain p a r e n c h y m a adjacent to cerebral arteriovenous malformations (AVMs). Numerous clinical reports have described patients who have had neurological dysfunction thought attributable to the shunting of blood from adjacent neuronal structures with cerebral AVMs, in the absence of cerebral infarction3 -7 Brown et al a n o t e d that 9 of 135 patients in their study with AVMs had a functional decline over the 4 years of follow-up, translating to a functional decline in 1.6% of patients with AVMs per year. This decline was not attributed to h e m o r r h a g e and presumably would, for the most part, be attributable to the effects of the preferential diversion of blood flow through the low resistance vessels of an AVM, creating chronic cerebral hypoperfusion (CCH) in brain regions adjacent to the AVM. CCH can also occur in severe bilateral carotid artery disease, and
may be responsible for subtle personality and intellect degradations that occur in these patients without evidence of infarction on c o m p u t e d tomography (CT) scanning. Up until the past few years it was t h o u g h t that the thresholds for n e u r o n a l dysfunction described for acute ischemic insults were applicable when hypoperfusion was sustained for a p r o l o n g e d d u r a t i o n ? -n More recently, however, we have shown that there is a new subtype of chronic cerebral ischemia which does not obey the classical thresholds for acute ischemic insults. Chronic reductions in CBF of between 25 and 50%, in the absence of cerebral infarction impair n e u r o n a l function, 12 and alter structure 13a4 with these changes present after approximately 6 m o n t h s of constant hypoperfusion. T h e changes are not present after only 10 weeks of this degree of h y p o p e r f u s i o n ] 5 which implies that whatever mechanisms are responsible for these changes take time to manifest, with a maturation process initiated prior to the induced changes b e c o m i n g overt.
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Ex,. joQ.
J
A
B
C
Fig. 1 AVF model in the rat used to create chronic hypoperfusion. (A) Schematic representation of the right carotid-jugular fistula anastomosis demonstrating some of the major connections. Angiogram of control rat (B) and AVF rat (C). The closed arrow indicates the right internal carotid artery and the open arrow indicates the transverse sinus. The large AVF is clearly visible. ICA, internal carotid artery; ECA, external carotid artery; Ext. jug. v, external jugular vein; CCA, common carotid artery; Int. jug. v, internal jugular vein.
The p h e n o m e n o n of long-term potentiation (LTP) in the rat h i p p o c a m p u s is again explored. LTP was first described in vivo in the rabbit h i p p o c a m p u s ) 6,a7 and encompasses a long-lasting e n h a n c e m e n t of synaptic efficacy following brief, repetitive stimulation of afferent pathways. ls'~9 It may play a role in short-term m e m o r y coding, but represents a specialized property of a brain region known to be exquisitely sensitive to hypoxic or ischemic insults. Using in vitro techniques, we have shown that an increase in background inhibition does not play a role in the i m p a i r m e n t of LTP after 26 weeks of CCH; 2° however, precise delineation of the mechanisms behind the impairm e n t of LTP has not been further explored. It is the p u r p o s e of this study to further explore the p h e n o m e n o n of LTP u n d e r a state of CCH. LTP u n d e r conditions of CCH was e x a m i n e d with a m u c h r e d u c e d intensity of stimulation. It is felt this would lead to the activation of a smaller n u m b e r of the excitatory afferent
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Schaffer collateral fibers, with a reduction in the subliminal fringe associated with this activation. By reducing the saturation of the total n u m b e r of possible fibers that were activated, some assessment of the relative contributions and cooperativity of the various excitatory and inhibitory inputs to the CA1 pyramidal cells could be made.
Material and methods All experimental procedures were approved by our institutional Animal Care and Ethics Committee. T h e construction of the arteriovenous fistula (AVF) has b e e n described in detail elsewhere (Fig. 1).12,21-2aFive SpragueDawley rats (250-350 g, approximately 8-10 weeks of age) were allowed to breathe spontaneously and were anesthetized with 1.6% halothane enriched with oxygen delivered at 1 l i t r e / m i n into a snout mask. Using strict aseptic technique, a midline cervical incision was m a d e
LTP in cerebral hypoperfusion u n d e r magnification. The right o m o h y o i d muscle was divided, the origin of the right external carotid artery was ligated with 7-0 m o n o f i l a m e n t polypropylene suture, and the c o m m o n carotid artery was ligated as caudally as possible. A t e m p o r a r y 3 m m aneurysm clip was placed at the carotid bifurcation and the c o m m o n carotid artery was divided above its caudal ligature. T h e external jugular vein was ligated with 7-0 Prolene as caudally as possible in the neck. A t e m p o r a r y 3 m m Mayfield aneurysm clip was placed caudally on the craniaMnost portion of the vessel. T h e external jugular vein was then divided as caudally as possible, above its ligature and s u b m e r g e d in saline. Using 10-0 m o n o f i l a m e n t nylon suture (BV 75-3 needle, Ethicon, Somerville, NJ, USA) the free ends of the external jugular vein and internal carotid artery were carefully anastomosed with i n t e r r u p t e d sutures. After this was c o m p l e t e d the t e m p o r a r y 3 m m Mayfield aneurysm clips were r e m o v e d and the cervical incision was closed in a single layer of platysma and skin with continuous 3-0 surgical braided silk r u n n i n g sutures. This f o r m e d a functional AVF between the anterior intracranial circulation and extracranial venous circulation and thus created cerebral hypoperfusion without an acute ischemic insult. Five age- and weight-matched rats were used as controls. T h e rationale for the use o f u n o p e r a t e d control animals has b e e n previously discussed. 12 T h e rats were housed after fistula f o r m a t i o n in climate-controlled facilities with 12-h d a y / n i g h t cycles. They were allowed free access to food and water and observed daily for abnormalities of activity or diet. Morgan et al 2a showed CBF was r e d u c e d f r o m a m e d i a n of 0.82-l.12 m l / g / m i n in n o n - o p e r a t e d controls to 0.46-0.68 m l / g / m i n in animals with AVFs using 14C autoradiography, with no evidence on light microscopy of infarction at 12 weeks after creation of the AVE A similar measure was attained at 26 weeks (unpublished data). T h e p r e p a r a t i o n of the h i p p o c a m p a l slices and extracellular in vitro recording technique have b e e n previously discussed) 2,15'2° Briefly, 26 weeks after fistula f o r m a t i o n the animals were anesthetized with halothane and decapitated. T h e h i p p o c a m p u s was rapidly r e m o v e d a n d 400-pm slices obtained using a McIlwain tissue chopper. T h e slices were placed into a chilled incubation c h a m b e r b u b b l e d with 95% 0 2 / 5 % CO 2 and were allowed to recover for at least 1 h prior to the transfer to a s u b m e r g e d tissue bath superfused with artificial cerebrospinal fluid (aCSF) (at 8 m l / m i n at 34 _ 0.5°C) of the following composition (in mM): NaC1, 124; KC1, 5; NaHzPO 4, 1.25; MgCI~, 1.3; CaC12, 2.5; N a H C O s, 26; Glucose, 25; p H 7.4; b u b b l e d with 95% 0 2 / 5 % CO 2. Slices were allowed a further h o u r to settle prior to recording. Extracellular field potentials were r e c o r d e d f r o m the CA1 pyramidal layer (recording electrode: glass micropipette; i m p e d a n c e 2-20 Mr2; 2M NaC1) with stimulation via the Schaffer collaterals (stimulating electrode: m o n o p o l a r stainless steel wire with paralyene coating; 125 m m 12 ° tapered tungsten tip; i m p e d a n c e 2-5 Mr2). Baseline stimulation frequency was 0.2 Hz. Unlike our previous studies, 12 control responses were taken using a stimulus intensity that achieved a response
Laboratory study of approximately 1 mY, rather than two-thirds of the maximal response (which gave approximately 2-4 mV responses). These were r e c o r d e d for 30 min. Measurements were m a d e of the amplitude and latency of the first population spike as well as the total n u m b e r of spikes. T h e population spike amplitude was measured between the peak negativity and subsequent m a x i m u m positivity whilst latency m e a s u r e d f r o m the stimulus artefact to peak negativity. Only one slice was used for recordings f r o m each e x p e r i m e n t a l animal. Once a stable response had b e e n attained, and control period recording had b e e n completed, in o r d e r to a t t e m p t to induce LTP, tetanic stimulation was delivered as 5 trains, 6 seconds apart at 50 Hz frequency (duration 400 ms) as described by Andersen et al. 24After the train, stimulation r e s u m e d at 0.2 Hz and recordings were taken for a further hour. The statistical analysis involved calculation of the m e a n _+standard deviation (s.d.) for each animal for the period of control recording and for the last 30 min of recording after tetanic stimulation. All results were then pooled. Control recordings between control and fistula animals were c o m p a r e d via two-sample t-tests to look for any differences prior to the tetanic stimulation. Twosample adjusted t-tests were used for samples with unequal variances25 For data post-train, values in the last half h o u r of recording were c o m p a r e d between control and fistula animals in the fashion previously used for the control period data. Finally, the magnitude of change as a result of tetanic stimulation was c o m p a r e d for each of the variables between control and fistula animals to assess if there was a difference in the a m o u n t of LTP which occurred between the two groups. Again, either twosample t-tests or two sample t-tests adjusting for unequal variances were utilized. For all statistical comparisons, a P value < 0.05 was r e g a r d e d as significant, with all data expressed as m e a n _+s.d. unless specified.
Results T h e results are summarized in Figure 2 and in Tables 1 and 2. T h e r e was no difference between the control and fistula animals for any of the p a r a m e t e r s studied during the control period of recording. O f the 5 control animals, 4 d e m o n s t r a t e d i m m e d i a t e post-tetanic potentiation (PTP) whilst only 1 animal had a period of post-tetanic depression (PTD). On an individual experiment-by-experiment basis, LTP was d e e m e d to have occurred in all 5 control animals and persisted for at least 1 h. All 5 slices showed at least a 10% increase in the first population spike amplitude at 1 h, with 4 of 5 showing at least a 30% increase at 1 h in that variable. C o m p a r i n g the average control period values to the average values for each variable over the last 30 min of recording after tetanic stimulation, there was a statistically signifcant difference in the amplitude of the first population spike alone (P= 0.018), which h a d risen on average by 88%. No other variable showed a significant change as a result of tetanic stimulation. Looking next at the fistula animals, all 5 fistula slices showed initial P T E and on an experiment-by-experiment basis, LTP was d e e m e d to have
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o.4,,,v L_
CONTROL
5.s Control
1 minute
10 minutes
30 minutes
60 minutes
0''V
FISTULA
5ms
Fig, 2 Representative extracellular field potentials recorded after tetanic stimulation from a control (top) and fistula hippocampal slice (bottom) during the LTP experiments which used low intensity stimulation, after 26 weeks of chronic cerebral hypoperfusion. The pretrain control response is shown to the left with the responses in minutes after the train shown to the right. Both the fistula and control slice show marked potentiation persisting for the entire hour of recording after tetanic stimulation, with a large increase in the amplitude of the first population spike and increased numbers of spikes in both the fistula and control recordings. Table 1 Mean amplitude (mY), latency (ms) and number of spikes for each group during low stimulus intensity LTP experiments 26 weeks after fistula formation (mean _+SD) Control
1st spike amplitude (mV) 1st spike latency (ms) Number of spikes
Fistula
Pre-train a (A) (n = 5)
Post-train a (B) (n = 5)
Pre-train a (D) (n = 5)
Post-train a (E) (n = 5)
A-B b
D-E b
A-D c
B-E c
1.08 _+0.35 5.97 + 0.37 ~.56 +_0.52
1.95 _+0.52 5.40 .+ 0.61 2.62 _+0.76
1.09 .+ 0.24 6.43 .+ 0.51 1.56 .+ 0.37
2.07 _+0.62 6.08 _+0.64 1.96 .+ 0.09
0.018 ~ 0.061 0.085
0.024d 0.025 d 0.086
0.998 0.141 1.00
0.732 0.124 0.126 e
C o m p a r i s o n o f g r o u p s (P v a l u e )
a = Last 30 min of recording; b = Paired t-tests; c = Two-sample t-test; d = Statistically significant, P < 0.05; e = Two-sample t-test adjusting for unequal variances.
Table 2 Comparisons of amount of change after tetanus for control (n = 5) and fistula (n = 5) groups during low stimulus intensity LTP experiments (mean (SD)
1st spike amplitude (mY) 1st spike latency (ms) 2nd spike amplitude (mV) 2nd spike latency (ms) Number of spikes
Control
Fistula
Change post-train (C = B - A ) a
Change post-train (F = E-D) a
0.86 _+0.50 -0.57 _+0.49 0.39 -+ 0.33 -0.83 _+0.62 1.06 _+ 1.04
0.99 -0.35 0.24 0.96 0.40
+_0.63 _+0.22 _+0.21 * 1.43 _+0.39
P value C-F b
0.730 0.401 0.532 0.088 0.242
a = Refer to Table 1 for key to identify columns A-F; b = Two-sample t-test; c = Statistically significant, P < 0.05.
occurred in all 5 animals. In the last 30 min of recording, all 5 fistula slices showed at least a 30% increase in the amplitude of the first population spike. When the average control period values were compared to the average values for each variable during the last 30 min after tetanic stimulation, statistically significant differences emerged in terms of the amplitude and latency of the first population spike (P-- 0.024 and 0.025, respectively). The first population spike amplitude increased on average by 98% in the last half h o u r of post-train recording, with a reduction of 6% in the first population spike latency during the same period. When the averages for
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each variable during the last 30 min of recording after tetanic stimulation were c o m p a r e d between control and fistula animals, no statistically significant differences e m e r g e d (P> 0.05). Finally, when the a m o u n t of change as a result of the tetanic stimulation in control slices was c o m p a r e d to that seen in fistula slices, no significant differences e m e r g e d for any of the variables studied. As a result, it could be concluded that not only were the control and fistula data statistically indistinguishable both before and after tetanic stimulation, but both control and fistula slices demonstrated marked and similar potentiation after tetanic stimulation.
LTP in cerebral hypoperfusion Conclusions This survey into further evaluating the properties of h i p p o c a m p a l LTP in animals u n d e r g o i n g CCH, yielded some surprising results, the possible mechanisms of which are o p e n to speculation. Synaptic transmission was intact in b o t h control and fistulas animals as shown by the similarity in responses prior to tetanic stimulation between the two groups, and in the similar incidences of PTP and PTD. LTP also o c c u r r e d with an equal frequency and to an equal degree in b o t h the control and fistula slices when a low intensity of stimulation was used, a situation quite different to that seen when a greater degree of stimulation was used, where LTP was f o u n d to be impaired in fistula animals, a2 PTD o c c u r r e d only infrequently possibly because the onset of LTP may have obscured any i m m e d i a t e PTD, a scenario that may not have o c c u r r e d in the animals that u n d e r w e n t higher intensity stimulation. Since the early 1980s the mechanisms of LTP have b e e n avidly investigated with controversy over presynaptic 26-3°versus postsynaptic al-38 control over the critical cellular processes that initiate potentiation. Most activity in the early phase of induction of LTP occurs in the vicinity of the dendritic spines. Andersen et a124 d e m o n s t r a t e d that the presynaptic afferent volley did not change after LTP induction and so inferred that LTP was not caused by a r e c r u i t m e n t of fibers during the high-frequency stimulation. It is possible, however, that in the fistula animals, by using a lower intensity of stimulation, almost all available fibers in the vicinity of the stimulating electrode are activated and already contributing to that LTE Hence, by increasing the stimulus intensity, no further fibers are available to be stimulated, a situation quite different to that envisioned by Andersen et al. 24 In fistula animals, one can conceptualize a scenario whereby when a low stimulus intensity is used, all local Schaffer collaterals and their CA1 synapses are excited. However, as the intensity of stimulation is increased, the total n u m b e r of fibres and CA1 pyramidal cells passes an u p p e r numerical threshold, and thus the n u m b e r of CA3-CA1 complexes contributing towards initiating LTP is less than in control animals. This would thus lead to a situation where in fistula animals at low intensities of stimulation, the frequency and degree of LTP generated would be similar to that seen in control animals, but with higher intensities of stimulation, the LTP apparatus would b e c o m e saturated and so the frequency and degree of LTP that was seen would be less than that seen in control animals. R e m e m b e r i n g that CA1 pyramidal cells are exquisitely sensitive to hypoxic or ischemic insults, 39-44 it is not hard to envision a situation whereby CCH leads to, for example, neuronal fall out, or reduced functional capacity in terms of the CA1 pyramidal cells, which translates into the a f o r e m e n t i o n e d electrophysiological picture. O n e other suggested hypothesis has b e e n that, as the intensity of stimulation is increased, the n u m b e r of activated n e u r o n s also increases, some of which are inhibitory fibers. This theory would be s u p p o r t e d by the relative resistance of inhibitory i n t e r n e u r o n s in the
Laboratory study h i p p o c a m p u s to ischemic or hypoxic insults. 45-~8Against this theory is our finding that an increase in b a c k g r o u n d inhibition does not occur in this state of chronic cerebral hypoperfusion. 2° This study further explores some of the mechanisms b e h i n d the i m p a i r m e n t of LTP in animals that have u n d e r g o n e chronic non-infarctional reductions in CBF of between 25 and 50%, maintained for 26 weeks. This study lends f u r t h e r credence to the assertion that this degree of CCH causes i m p a i r m e n t in the function of the CA1 pyramidal cells, with higher intensities of electrophysiological stimulation required to discern the induced changes. Further mechanistic information a b o u t the changes induced in a newly recognized subtype of chronic cerebral ischemia is presented, where a reduction in CBF of less than 50% impairs n e u r o n a l function in the absence of cerebral infarction. Chronic cerebral hypoperfusion occurs clinically in m a n y different scenarios, b u t is most clearly illustrated in patients with cerebral AVM or bilateral carotid artery disease. Whilst previously it was felt that CBF had to be r e d u c e d by greater than 50% to affect n e u r o n a l structure and function, this study and our previous reports emphasize that cerebral i m p a i r m e n t can occur with m u c h lower reductions in CBE This has potential clinical implications in terms of earlier intervention for these conditions if u n e x p l a i n e d deteriorations occur and may help to redefine therapeutic criteria for treating these disorders on the basis of the effects of CCH.
Acknowledgments This research is part of an ongoing project investigating the pathophysiology of chronic cerebral hypoperfusion and intracranial arteriovenous malformations. The work was supported in part by the following: National Health and Medical Research Council; Clive and Vera Ramaciotti Research Foundation; Royal Australasian College of Surgeons and the Australian Brain Foundation. Received26 June 1996; Accepted 3 March 1997
Correspondence and offprint requests:Associate Professor M. K. Morgan, Department of Neurosurgery, Level 7, RoyalNorth Shore Hospital, St Leonards, NSW 2065, Australia, Tel: 61 2 9926 8756, Fax: 61 2 9437 5172 References 1. MurphyJP. Cerebrovascular Disease. Chicago: Yearbook, 1954: 242-262. 2. Norlen G. Arteriovenous aneurysms of the brain. Report of 10 cases of total removal of the lesion. J Neurosurg 1949; 6: 475-494. 3. PatersonJH, McKissQck W. A clinical survey of intracranial aneurysms with special reference to their mode of progression and surgical treatment: a report of 110 cases. Brain 1956; 79: 233-266. 4. KusskeJA, Kelly WA. Embolisation and reduction of the 'steal' syndrome in cerebral arteriovenous malformations. J Neurosurg 1974; 40: 313-321. 5. Luessenhop AJ, PresperJH. Surgical embolization of cerebral arteriovenous malformations through internal carotid and vertebral arteries. Long-term results. J Neurosurg 1975; 42: 443-451.
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LTP in cerebral hypoperfusion 27. Bliss TV. Maintenance is presynaptic. Nature 1990; 346: 698-699. 28. Bliss TV, Errington ML, Lynch MA, WilliamsJH. Presynaptic mechanisms in hippocampal long-term potentiation. Cold Spring Harb Symp Quant Biol 1990; 55: 119-129. 29. Malinow R, Tsien RW. Presynaptic enhancement shown by whole-cell recordings of long-term potentiation in hippocampal slices. Nature 1990; 346: 177-180. 30. Larkman A, Hannay T, Stratford K, JackJ. Presynaptic release probability influences the locus of long-term potentiation. Nature 1992; 360: 70-73. 31. Douglas RM, Goddard GV, Riives M. Inhibitory modulation of long-term potentiation: evidence for a postsynaptic locus of control. Brain Res 1982; 240: 259-272. 32. Wigstr6m H, Gustafsson B. Postsynaptic control of hippocampal long-term potentiation. J Physiol (Paris) 1986; 81: 228-236. 33. KauerJA, Malenka RC, Nicoll RA. A persistent postsynaptic modification mediates long-term potentiation in the hippocampus. Neuron 1988; 1: 911-917. 34. Muller D, Joly M, Lynch G. Contributions of quisqualate and NMDA receptors to the induction and expression of LTP. Science 1988; 242: 1694-1697. 35. Malenka RC. Postsynaptic factors control the duration of synaptic enhancement in area CA1 of the hippocampus. Neuron 1991; 6: 53-60. 36. Pockett S, Slack JR. Presynaptic activity in the period immediately after conditioning is not necessary for longterm potentiation. Neurosci Lett 1991; 121: 81-82. 37. Hanse E, Gustafsson B. Postsynaptic, but not presynaptic, activity controls the early time course of long-term potentiation in the dentate gyrus.J Neurosci 1992; 12: 3226-3240. 38. Manabe T, Renner P, Nicoll RA. Postsynaptic contribution to long-term potentiation revealed by the analysis of miniature synaptic currents. Nature 1992; 355: 50-55. 39. Sommer W. Erkrankung des Ammonshorns als aetiologisches Moment der Epilepsie. Arch Psychiat Nervenkr 1880; 10: 631-675. 40. Bratz E, Grossman W. Uber Ammonshornsklerose. Z ges Neurol Psychiat 1923; 81: 45-61. 41. Spielmeyer W. Die Pathogenese des epiletischen Krampfes. Histopathologischer Teil. Z ges Neurol Psychiat 1927; 109: 501-520. 42. Bodechtel G. Die Topik der Ammonshornsch/idigung. Z ges Neurol Psychiat 1930; 123: 485-535. 43. Vogt C, Vogt O. Sitz und Wesen der Krankheiten im Lichte der topistischen Hirnforschung und des Variierens der Tiete.J Psychol Neurol 1937; 47: 237-457. 44. Friede RL. The histochemical architecture of the Ammon's horn as related to its selective vulnerability. Acta Neuropathol (Berl) 1966; 6: 1-13. 45. Johansen FE Jorgensen MK, Diemer NH. Cerebral ischemia and interneuron. In: Battistini N, Fiorani P, Coubier R, Plum F, Fieschi C (eds). Acute Brain Ischemia Medical and Surgical Therapy. New York: Raven Press, 1986: 53-56. 46. Nitsch C, Goping G, Klatzo I. Preservation of GABAergic perikarya and boutons after transient ischemia in the gerbil hippocampal CA1 field. Brain Res 1989; 495: 243-252. 47. Johansen FF, Christensen T, Jensen MS et al. Inhibition in postischemic rat hippocampus: GABAreceptors, GABA release and inhibitory postsynaptic potentials. Exp Brain Res 1991; 84: 529-537. 48. Fukuda T, Nakano S, Yoshiya I, Hashimoto PH. Persistent degenerative state of non-pyramidal neurons in the CA1 region of the gerbil hippocampus following transient forebrain ischemia. Neuroscience 1993; 53: 23-38.
Lali H.S. S e k h o n 1 MB BS PhD, l a n S p e n c e 2 BSc PhD, M i c h a e l K. M o r g a n I MD FRACS, N e v i l l e C. W e b e r 3 MSc PhD 1Department of Surgery DO6, The Universityof Sydney, Sydney,NSW 2006, Australia 2Department of Pharmacology DO5, The Universityof Sydney,Sydney,NSW 2006, Australia 3School of Mathematics and Statistics FO7, The Universityof Sydney, Sydney,NSW 2006, Australia
Chronic reductions in cerebral b l o o d flow (CBF) o f between 25 and 50%, in the absence o f cerebral infarction, lead to i m p a i r m e n t s in h i p p o c a m p a l in vitro long-term potentiation (LTP). This study set out to explore s o m e o f the p r o p e r t i e s o f this i m p a i r m e n t o f LTP. LTP is an electrophysiological p r o p e r t y known to occur in the h i p p o c a m p u s , a region known to be exquisitely sensitive to hypoxic or ischemic insults. Thus, assessing LTP is a novel way o f assessing the effects o f subtle ischemic insults. Five male Sprague-Dawley rats h a d arteriovenous fistulae created surgically in the neck to induce a state o f chronic cerebral h y p o p e r f n s i o n (CCH) with the features described above. Five rats were used as age-matched controls. Twenty-six weeks after fistula formation, the animals were p r e p a r e d f o r in vitro h i p p o c a m p a l recording in a s u b m e r g e d tissue bath. Extracellular field potentials were r e c o r d e d at the Schaffer collateral-CA1 region, with a stimulus intensity that achieved a population spike amplitude o f 1 mV. After tetanic stimulation, the frequency and magnitude o f LTP was c o m p a r e d between control and fistula animals. All animals in b o t h these groups d e m o n s t r a t e d LTP in contradistinction to our previous work where LTP was impaired in fistula animals when a higher intensity o f stimulation was used. This indicates that the structures that are associated with the initiation and maintenance o f LTP (most p r o b a b l y the ischemia-sensitive CA1 pyramidal cells) are saturated as the stimulus intensity is increased. Thus, at this lower intensity o f stimulation LTP is p r e s e r v e d in the fistula animals, b u t f o u n d to be i m p a i r e d as the stimulus intensity is increased. Consequently, this study provides f u r t h e r i n f o r m a t i o n on this newly identified subtype of chronic cerebral ischemia which, in time, after f u r t h e r studies in humans, m a y help to redefine therapeutic indicators f o r the m a n a g e m e n t o f cerebral arteriovenous m a l f o r m a t i o n and severe cerebrovascular disease. Journal of Clinical Neuroscience 1998, 5 (3) : 323-328
© Harcourt Brace & Co. Ltd 1998
Keywords" arteriovenous malformation, brain slices, electrophysiology, hippocampus, ischemia, long-term potentiation
Introduction Chronic reductions in cerebral blood flow (CBF) can occur in many clinical situations, best typified by the reduction in CBF that occurs as a result of venous hypertension and cerebrovascular 'steal '1 in brain p a r e n c h y m a adjacent to cerebral arteriovenous malformations (AVMs). Numerous clinical reports have described patients who have had neurological dysfunction thought attributable to the shunting of blood from adjacent neuronal structures with cerebral AVMs, in the absence of cerebral infarction3 -7 Brown et al a n o t e d that 9 of 135 patients in their study with AVMs had a functional decline over the 4 years of follow-up, translating to a functional decline in 1.6% of patients with AVMs per year. This decline was not attributed to h e m o r r h a g e and presumably would, for the most part, be attributable to the effects of the preferential diversion of blood flow through the low resistance vessels of an AVM, creating chronic cerebral hypoperfusion (CCH) in brain regions adjacent to the AVM. CCH can also occur in severe bilateral carotid artery disease, and
may be responsible for subtle personality and intellect degradations that occur in these patients without evidence of infarction on c o m p u t e d tomography (CT) scanning. Up until the past few years it was t h o u g h t that the thresholds for n e u r o n a l dysfunction described for acute ischemic insults were applicable when hypoperfusion was sustained for a p r o l o n g e d d u r a t i o n ? -n More recently, however, we have shown that there is a new subtype of chronic cerebral ischemia which does not obey the classical thresholds for acute ischemic insults. Chronic reductions in CBF of between 25 and 50%, in the absence of cerebral infarction impair n e u r o n a l function, 12 and alter structure 13a4 with these changes present after approximately 6 m o n t h s of constant hypoperfusion. T h e changes are not present after only 10 weeks of this degree of h y p o p e r f u s i o n ] 5 which implies that whatever mechanisms are responsible for these changes take time to manifest, with a maturation process initiated prior to the induced changes b e c o m i n g overt.
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Ex,. joQ.
J
A
B
C
Fig. 1 AVF model in the rat used to create chronic hypoperfusion. (A) Schematic representation of the right carotid-jugular fistula anastomosis demonstrating some of the major connections. Angiogram of control rat (B) and AVF rat (C). The closed arrow indicates the right internal carotid artery and the open arrow indicates the transverse sinus. The large AVF is clearly visible. ICA, internal carotid artery; ECA, external carotid artery; Ext. jug. v, external jugular vein; CCA, common carotid artery; Int. jug. v, internal jugular vein.
The p h e n o m e n o n of long-term potentiation (LTP) in the rat h i p p o c a m p u s is again explored. LTP was first described in vivo in the rabbit h i p p o c a m p u s ) 6,a7 and encompasses a long-lasting e n h a n c e m e n t of synaptic efficacy following brief, repetitive stimulation of afferent pathways. ls'~9 It may play a role in short-term m e m o r y coding, but represents a specialized property of a brain region known to be exquisitely sensitive to hypoxic or ischemic insults. Using in vitro techniques, we have shown that an increase in background inhibition does not play a role in the i m p a i r m e n t of LTP after 26 weeks of CCH; 2° however, precise delineation of the mechanisms behind the impairm e n t of LTP has not been further explored. It is the p u r p o s e of this study to further explore the p h e n o m e n o n of LTP u n d e r a state of CCH. LTP u n d e r conditions of CCH was e x a m i n e d with a m u c h r e d u c e d intensity of stimulation. It is felt this would lead to the activation of a smaller n u m b e r of the excitatory afferent
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Schaffer collateral fibers, with a reduction in the subliminal fringe associated with this activation. By reducing the saturation of the total n u m b e r of possible fibers that were activated, some assessment of the relative contributions and cooperativity of the various excitatory and inhibitory inputs to the CA1 pyramidal cells could be made.
Material and methods All experimental procedures were approved by our institutional Animal Care and Ethics Committee. T h e construction of the arteriovenous fistula (AVF) has b e e n described in detail elsewhere (Fig. 1).12,21-2aFive SpragueDawley rats (250-350 g, approximately 8-10 weeks of age) were allowed to breathe spontaneously and were anesthetized with 1.6% halothane enriched with oxygen delivered at 1 l i t r e / m i n into a snout mask. Using strict aseptic technique, a midline cervical incision was m a d e
LTP in cerebral hypoperfusion u n d e r magnification. The right o m o h y o i d muscle was divided, the origin of the right external carotid artery was ligated with 7-0 m o n o f i l a m e n t polypropylene suture, and the c o m m o n carotid artery was ligated as caudally as possible. A t e m p o r a r y 3 m m aneurysm clip was placed at the carotid bifurcation and the c o m m o n carotid artery was divided above its caudal ligature. T h e external jugular vein was ligated with 7-0 Prolene as caudally as possible in the neck. A t e m p o r a r y 3 m m Mayfield aneurysm clip was placed caudally on the craniaMnost portion of the vessel. T h e external jugular vein was then divided as caudally as possible, above its ligature and s u b m e r g e d in saline. Using 10-0 m o n o f i l a m e n t nylon suture (BV 75-3 needle, Ethicon, Somerville, NJ, USA) the free ends of the external jugular vein and internal carotid artery were carefully anastomosed with i n t e r r u p t e d sutures. After this was c o m p l e t e d the t e m p o r a r y 3 m m Mayfield aneurysm clips were r e m o v e d and the cervical incision was closed in a single layer of platysma and skin with continuous 3-0 surgical braided silk r u n n i n g sutures. This f o r m e d a functional AVF between the anterior intracranial circulation and extracranial venous circulation and thus created cerebral hypoperfusion without an acute ischemic insult. Five age- and weight-matched rats were used as controls. T h e rationale for the use o f u n o p e r a t e d control animals has b e e n previously discussed. 12 T h e rats were housed after fistula f o r m a t i o n in climate-controlled facilities with 12-h d a y / n i g h t cycles. They were allowed free access to food and water and observed daily for abnormalities of activity or diet. Morgan et al 2a showed CBF was r e d u c e d f r o m a m e d i a n of 0.82-l.12 m l / g / m i n in n o n - o p e r a t e d controls to 0.46-0.68 m l / g / m i n in animals with AVFs using 14C autoradiography, with no evidence on light microscopy of infarction at 12 weeks after creation of the AVE A similar measure was attained at 26 weeks (unpublished data). T h e p r e p a r a t i o n of the h i p p o c a m p a l slices and extracellular in vitro recording technique have b e e n previously discussed) 2,15'2° Briefly, 26 weeks after fistula f o r m a t i o n the animals were anesthetized with halothane and decapitated. T h e h i p p o c a m p u s was rapidly r e m o v e d a n d 400-pm slices obtained using a McIlwain tissue chopper. T h e slices were placed into a chilled incubation c h a m b e r b u b b l e d with 95% 0 2 / 5 % CO 2 and were allowed to recover for at least 1 h prior to the transfer to a s u b m e r g e d tissue bath superfused with artificial cerebrospinal fluid (aCSF) (at 8 m l / m i n at 34 _ 0.5°C) of the following composition (in mM): NaC1, 124; KC1, 5; NaHzPO 4, 1.25; MgCI~, 1.3; CaC12, 2.5; N a H C O s, 26; Glucose, 25; p H 7.4; b u b b l e d with 95% 0 2 / 5 % CO 2. Slices were allowed a further h o u r to settle prior to recording. Extracellular field potentials were r e c o r d e d f r o m the CA1 pyramidal layer (recording electrode: glass micropipette; i m p e d a n c e 2-20 Mr2; 2M NaC1) with stimulation via the Schaffer collaterals (stimulating electrode: m o n o p o l a r stainless steel wire with paralyene coating; 125 m m 12 ° tapered tungsten tip; i m p e d a n c e 2-5 Mr2). Baseline stimulation frequency was 0.2 Hz. Unlike our previous studies, 12 control responses were taken using a stimulus intensity that achieved a response
Laboratory study of approximately 1 mY, rather than two-thirds of the maximal response (which gave approximately 2-4 mV responses). These were r e c o r d e d for 30 min. Measurements were m a d e of the amplitude and latency of the first population spike as well as the total n u m b e r of spikes. T h e population spike amplitude was measured between the peak negativity and subsequent m a x i m u m positivity whilst latency m e a s u r e d f r o m the stimulus artefact to peak negativity. Only one slice was used for recordings f r o m each e x p e r i m e n t a l animal. Once a stable response had b e e n attained, and control period recording had b e e n completed, in o r d e r to a t t e m p t to induce LTP, tetanic stimulation was delivered as 5 trains, 6 seconds apart at 50 Hz frequency (duration 400 ms) as described by Andersen et al. 24After the train, stimulation r e s u m e d at 0.2 Hz and recordings were taken for a further hour. The statistical analysis involved calculation of the m e a n _+standard deviation (s.d.) for each animal for the period of control recording and for the last 30 min of recording after tetanic stimulation. All results were then pooled. Control recordings between control and fistula animals were c o m p a r e d via two-sample t-tests to look for any differences prior to the tetanic stimulation. Twosample adjusted t-tests were used for samples with unequal variances25 For data post-train, values in the last half h o u r of recording were c o m p a r e d between control and fistula animals in the fashion previously used for the control period data. Finally, the magnitude of change as a result of tetanic stimulation was c o m p a r e d for each of the variables between control and fistula animals to assess if there was a difference in the a m o u n t of LTP which occurred between the two groups. Again, either twosample t-tests or two sample t-tests adjusting for unequal variances were utilized. For all statistical comparisons, a P value < 0.05 was r e g a r d e d as significant, with all data expressed as m e a n _+s.d. unless specified.
Results T h e results are summarized in Figure 2 and in Tables 1 and 2. T h e r e was no difference between the control and fistula animals for any of the p a r a m e t e r s studied during the control period of recording. O f the 5 control animals, 4 d e m o n s t r a t e d i m m e d i a t e post-tetanic potentiation (PTP) whilst only 1 animal had a period of post-tetanic depression (PTD). On an individual experiment-by-experiment basis, LTP was d e e m e d to have occurred in all 5 control animals and persisted for at least 1 h. All 5 slices showed at least a 10% increase in the first population spike amplitude at 1 h, with 4 of 5 showing at least a 30% increase at 1 h in that variable. C o m p a r i n g the average control period values to the average values for each variable over the last 30 min of recording after tetanic stimulation, there was a statistically signifcant difference in the amplitude of the first population spike alone (P= 0.018), which h a d risen on average by 88%. No other variable showed a significant change as a result of tetanic stimulation. Looking next at the fistula animals, all 5 fistula slices showed initial P T E and on an experiment-by-experiment basis, LTP was d e e m e d to have
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o.4,,,v L_
CONTROL
5.s Control
1 minute
10 minutes
30 minutes
60 minutes
0''V
FISTULA
5ms
Fig, 2 Representative extracellular field potentials recorded after tetanic stimulation from a control (top) and fistula hippocampal slice (bottom) during the LTP experiments which used low intensity stimulation, after 26 weeks of chronic cerebral hypoperfusion. The pretrain control response is shown to the left with the responses in minutes after the train shown to the right. Both the fistula and control slice show marked potentiation persisting for the entire hour of recording after tetanic stimulation, with a large increase in the amplitude of the first population spike and increased numbers of spikes in both the fistula and control recordings. Table 1 Mean amplitude (mY), latency (ms) and number of spikes for each group during low stimulus intensity LTP experiments 26 weeks after fistula formation (mean _+SD) Control
1st spike amplitude (mV) 1st spike latency (ms) Number of spikes
Fistula
Pre-train a (A) (n = 5)
Post-train a (B) (n = 5)
Pre-train a (D) (n = 5)
Post-train a (E) (n = 5)
A-B b
D-E b
A-D c
B-E c
1.08 _+0.35 5.97 + 0.37 ~.56 +_0.52
1.95 _+0.52 5.40 .+ 0.61 2.62 _+0.76
1.09 .+ 0.24 6.43 .+ 0.51 1.56 .+ 0.37
2.07 _+0.62 6.08 _+0.64 1.96 .+ 0.09
0.018 ~ 0.061 0.085
0.024d 0.025 d 0.086
0.998 0.141 1.00
0.732 0.124 0.126 e
C o m p a r i s o n o f g r o u p s (P v a l u e )
a = Last 30 min of recording; b = Paired t-tests; c = Two-sample t-test; d = Statistically significant, P < 0.05; e = Two-sample t-test adjusting for unequal variances.
Table 2 Comparisons of amount of change after tetanus for control (n = 5) and fistula (n = 5) groups during low stimulus intensity LTP experiments (mean (SD)
1st spike amplitude (mY) 1st spike latency (ms) 2nd spike amplitude (mV) 2nd spike latency (ms) Number of spikes
Control
Fistula
Change post-train (C = B - A ) a
Change post-train (F = E-D) a
0.86 _+0.50 -0.57 _+0.49 0.39 -+ 0.33 -0.83 _+0.62 1.06 _+ 1.04
0.99 -0.35 0.24 0.96 0.40
+_0.63 _+0.22 _+0.21 * 1.43 _+0.39
P value C-F b
0.730 0.401 0.532 0.088 0.242
a = Refer to Table 1 for key to identify columns A-F; b = Two-sample t-test; c = Statistically significant, P < 0.05.
occurred in all 5 animals. In the last 30 min of recording, all 5 fistula slices showed at least a 30% increase in the amplitude of the first population spike. When the average control period values were compared to the average values for each variable during the last 30 min after tetanic stimulation, statistically significant differences emerged in terms of the amplitude and latency of the first population spike (P-- 0.024 and 0.025, respectively). The first population spike amplitude increased on average by 98% in the last half h o u r of post-train recording, with a reduction of 6% in the first population spike latency during the same period. When the averages for
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each variable during the last 30 min of recording after tetanic stimulation were c o m p a r e d between control and fistula animals, no statistically significant differences e m e r g e d (P> 0.05). Finally, when the a m o u n t of change as a result of the tetanic stimulation in control slices was c o m p a r e d to that seen in fistula slices, no significant differences e m e r g e d for any of the variables studied. As a result, it could be concluded that not only were the control and fistula data statistically indistinguishable both before and after tetanic stimulation, but both control and fistula slices demonstrated marked and similar potentiation after tetanic stimulation.
LTP in cerebral hypoperfusion Conclusions This survey into further evaluating the properties of h i p p o c a m p a l LTP in animals u n d e r g o i n g CCH, yielded some surprising results, the possible mechanisms of which are o p e n to speculation. Synaptic transmission was intact in b o t h control and fistulas animals as shown by the similarity in responses prior to tetanic stimulation between the two groups, and in the similar incidences of PTP and PTD. LTP also o c c u r r e d with an equal frequency and to an equal degree in b o t h the control and fistula slices when a low intensity of stimulation was used, a situation quite different to that seen when a greater degree of stimulation was used, where LTP was f o u n d to be impaired in fistula animals, a2 PTD o c c u r r e d only infrequently possibly because the onset of LTP may have obscured any i m m e d i a t e PTD, a scenario that may not have o c c u r r e d in the animals that u n d e r w e n t higher intensity stimulation. Since the early 1980s the mechanisms of LTP have b e e n avidly investigated with controversy over presynaptic 26-3°versus postsynaptic al-38 control over the critical cellular processes that initiate potentiation. Most activity in the early phase of induction of LTP occurs in the vicinity of the dendritic spines. Andersen et a124 d e m o n s t r a t e d that the presynaptic afferent volley did not change after LTP induction and so inferred that LTP was not caused by a r e c r u i t m e n t of fibers during the high-frequency stimulation. It is possible, however, that in the fistula animals, by using a lower intensity of stimulation, almost all available fibers in the vicinity of the stimulating electrode are activated and already contributing to that LTE Hence, by increasing the stimulus intensity, no further fibers are available to be stimulated, a situation quite different to that envisioned by Andersen et al. 24 In fistula animals, one can conceptualize a scenario whereby when a low stimulus intensity is used, all local Schaffer collaterals and their CA1 synapses are excited. However, as the intensity of stimulation is increased, the total n u m b e r of fibres and CA1 pyramidal cells passes an u p p e r numerical threshold, and thus the n u m b e r of CA3-CA1 complexes contributing towards initiating LTP is less than in control animals. This would thus lead to a situation where in fistula animals at low intensities of stimulation, the frequency and degree of LTP generated would be similar to that seen in control animals, but with higher intensities of stimulation, the LTP apparatus would b e c o m e saturated and so the frequency and degree of LTP that was seen would be less than that seen in control animals. R e m e m b e r i n g that CA1 pyramidal cells are exquisitely sensitive to hypoxic or ischemic insults, 39-44 it is not hard to envision a situation whereby CCH leads to, for example, neuronal fall out, or reduced functional capacity in terms of the CA1 pyramidal cells, which translates into the a f o r e m e n t i o n e d electrophysiological picture. O n e other suggested hypothesis has b e e n that, as the intensity of stimulation is increased, the n u m b e r of activated n e u r o n s also increases, some of which are inhibitory fibers. This theory would be s u p p o r t e d by the relative resistance of inhibitory i n t e r n e u r o n s in the
Laboratory study h i p p o c a m p u s to ischemic or hypoxic insults. 45-~8Against this theory is our finding that an increase in b a c k g r o u n d inhibition does not occur in this state of chronic cerebral hypoperfusion. 2° This study further explores some of the mechanisms b e h i n d the i m p a i r m e n t of LTP in animals that have u n d e r g o n e chronic non-infarctional reductions in CBF of between 25 and 50%, maintained for 26 weeks. This study lends f u r t h e r credence to the assertion that this degree of CCH causes i m p a i r m e n t in the function of the CA1 pyramidal cells, with higher intensities of electrophysiological stimulation required to discern the induced changes. Further mechanistic information a b o u t the changes induced in a newly recognized subtype of chronic cerebral ischemia is presented, where a reduction in CBF of less than 50% impairs n e u r o n a l function in the absence of cerebral infarction. Chronic cerebral hypoperfusion occurs clinically in m a n y different scenarios, b u t is most clearly illustrated in patients with cerebral AVM or bilateral carotid artery disease. Whilst previously it was felt that CBF had to be r e d u c e d by greater than 50% to affect n e u r o n a l structure and function, this study and our previous reports emphasize that cerebral i m p a i r m e n t can occur with m u c h lower reductions in CBE This has potential clinical implications in terms of earlier intervention for these conditions if u n e x p l a i n e d deteriorations occur and may help to redefine therapeutic criteria for treating these disorders on the basis of the effects of CCH.
Acknowledgments This research is part of an ongoing project investigating the pathophysiology of chronic cerebral hypoperfusion and intracranial arteriovenous malformations. The work was supported in part by the following: National Health and Medical Research Council; Clive and Vera Ramaciotti Research Foundation; Royal Australasian College of Surgeons and the Australian Brain Foundation. Received26 June 1996; Accepted 3 March 1997
Correspondence and offprint requests:Associate Professor M. K. Morgan, Department of Neurosurgery, Level 7, RoyalNorth Shore Hospital, St Leonards, NSW 2065, Australia, Tel: 61 2 9926 8756, Fax: 61 2 9437 5172 References 1. MurphyJP. Cerebrovascular Disease. Chicago: Yearbook, 1954: 242-262. 2. Norlen G. Arteriovenous aneurysms of the brain. Report of 10 cases of total removal of the lesion. J Neurosurg 1949; 6: 475-494. 3. PatersonJH, McKissQck W. A clinical survey of intracranial aneurysms with special reference to their mode of progression and surgical treatment: a report of 110 cases. Brain 1956; 79: 233-266. 4. KusskeJA, Kelly WA. Embolisation and reduction of the 'steal' syndrome in cerebral arteriovenous malformations. J Neurosurg 1974; 40: 313-321. 5. Luessenhop AJ, PresperJH. Surgical embolization of cerebral arteriovenous malformations through internal carotid and vertebral arteries. Long-term results. J Neurosurg 1975; 42: 443-451.
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