Pre-ischemic depletion of brain norepinephrine decreases infarct size in normothermic rats exposed to transient focal cerebral ischemia

Neuroscience Letters 275 (1999) 167±170 www.elsevier.com/locate/neulet

Pre-ischemic depletion of brain norepinephrine decreases infarct size in normothermic rats exposed to transient focal cerebral ischemia Bengt NellgaÊrd a, G. Burkhard Mackensen a, Shiva Sarraf-Yazdi b, Yoshihide Miura a, Robert Pearlstein c, David S. Warner a,* a

Department of Anesthesiology, P.O. Box 3094, Duke University Medical Center, Durham, NC 27710, USA b Duke University School of Medicine, Durham, NC 27710, USA c Department of Surgery, P.O. Box 3094, Duke University Medical Center, Durham, NC 27710, USA Received 28 June 1999; received in revised form 6 September 1999; accepted 7 September 1999

Abstract This study examined the importance of brain norepinephrine concentration on outcome from a focal ischemic insult. Fasted temperature-controlled male Wistar rats pretreated with DSP-4, (N-(chloroethyl)-N-ethyl-2-bromobenzylamine), to selectively deplete brain norepinephrine, were subjected to reversible ®lament occlusion of the middle cerebral artery for 75 min in the awake state. After 3 days recovery, total infarct volume in DSP-4 treated rats (185 ^ 107 mm 3) was reduced vs. untreated control animals (242 ^ 71 mm 3, P ˆ 0:04). Subcortical infarct volume was also smaller in the DSP-4 group (93 ^ 44 vs. 121 ^ 28 mm 3, P ˆ 0:02). Cortical infarct volume was not statistically different between groups. Neurologic function correlated with infarct-size. These ®ndings suggest that brain norepinephrine affects stroke development either by direct neuronal toxicity and/or through in¯uences on the penumbral circulation. Dampening of the central stress response induced by the onset of stroke may thus be advantageous. q 1999 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Brain; Ischemia; Focal; Rats; Norepinephrine; Blockade; (N-(Chloroethyl)-N-ethyl-2-bromobenzylamine)

Cerebral ischemia increases extracellular catecholamine concentrations as demonstrated by microdialysis in the rat brain [5]. The effects of central and/or plasma epinephrine and norepinephrine release on neuronal outcome following global cerebral ischemia have been investigated, albeit with contradictory results [3,15]. No work has investigated the signi®cance of central norepinephrine concentration on outcome from focal ischemia. Accordingly, this study examined the histologic and behavioral effects of selective brain noradrenergic depletion by pretreatment with DSP-4 (N-(chloroethyl)-N-ethyl-2-bromobenzylamine) in temperature-controlled awake rats exposed to 75 min of reversible ®lament-induced occlusion of the right middle cerebral artery (MCAO). This study was approved by the Duke University Animal Care and Use Committee. Male Wistar rats (age 8±10 weeks), were randomly assigned to receive either DSP-4, * Corresponding author. Tel.: 11-919-684-6633; fax: 11-919684-6692. E-mail address: [email protected] (D.S. Warner)

50 mg/kg in 1 ml of saline i.p. over 1 min (n ˆ 23), or no treatment (control animals, n ˆ 22). Four days later, the same animals were anesthetized with 50 mg/kg i.p. pentobarbital and positioned in a stereotactic head frame. The scalp was incised. A burr hole was drilled 2 mm lateral to midline and 3 mm anterior to bregma. A radiotelemetry thermistor (Brain Probe, model XM-FH-BP, Minimitter CO., Inc. Sunriver, OR), accuracy ^0:18C, was placed on the skull with the tip positioned on the dura. The probe was ®xed in place. The wound was closed and the animals were allowed to recover. The thermistor had been previously calibrated (range: 35.0±40.08C) allowing extrapolation of temperatures in accordance with the radiofrequency emitted by the probe. Probe radiofrequency signals were received, digitized and processed through a computer with software allowing monitoring and automated control of brain temperature by surface heating or cooling. Three days later, the same rats were fasted overnight but allowed free access to water. Rats were then anesthetized

0304-3940/99/$ - see front matter q 1999 Elsevier Science Ireland Ltd. All rights reserved. PII: S03 04 - 394 0( 9 9) 00 74 3- 0

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with halothane (3±5%) in 100% O2. Following tracheal intubation, inspired halothane concentration was reduced to 1.0±1.5% in 30% O2/balance N2 and the lungs were mechanically ventilated. The tail artery was cannulated. The animals were then prepared for MCAO [17]. A midline cervical incision was made and the right common carotid artery was identi®ed. The external and internal carotid arteries were isolated. A 20-min interval was then allowed for physiological stabilization. Mean arterial blood pressure (MABP) was continuously recorded. Arterial blood gases, hematocrit, plasma glucose, epinephrine and norepinephrine were measured 10 min before and 45 min after MCAO onset. Samples (200 ml) for catecholamine analysis were collected in tubes containing sodium metabisulfate and EDTA. Plasma norepinephrine and epinephrine were extracted on aluminum and samples were injected into a reverse phase high pressure liquid chromatography equipped with a C-18 column and an electrochemical detector. The values were corrected for extraction ef®ciency. To achieve MCAO, a 0.25-mm diameter nylon ®lament coated with poly-l-lysine was inserted into the stump of the external carotid artery and passed 23 mm distally through the internal carotid artery until a slight resistance was felt. The ®lament was secured and the wound was closed. Halothane was abruptly discontinued and the animals were awakened. After recovery of spontaneous ventilation the trachea was extubated and the animals were placed in a clear acrylic box containing 100% O2. The total duration of MCAO was 75 min. Five minutes prior to onset of recirculation, rats were reanesthetized with 0.7±1.0% halothane. The occlusive ®lament and the tail artery catheter were removed and the wounds closed. Halothane was discontinued and rats were awakened. Epidural temperature was controlled at 37:5 ^ 0:18C prior to, during and for 22 h after MCAO. Three days later, rats underwent a neurologic examination [4]. With the observer blinded to group assignment, spontaneous activity, movement symmetry, forepaw outstretching, climbing, body proprioception and response to vibrissae touch were scored. The total score was the sum of all six scores, three being the minimum (worst) and 18 being the maximum (best). Animals were then weighed, anesthetized with 3% halothane and decapitated. The brains were removed, frozen and stored at 2708C. Serial 20-mm thick coronal sections were taken using a cryotome at 660 mm intervals over the rostral-caudal extent of the infarct. The sections were dried and stained with hematoxylin and eosin. Infarct volume was measured by digitally sampling stained sections with a video camera controlled by an image analyzer. The digitized image was then displayed on a video monitor. With the observer blinded to experimental conditions, infarct borders in both cortex and subcortex were individually outlined (corpus callosum excluded). The area of infarct (mm 2) was determined within the

outlined regions of interest. Infarct volumes (mm 3) were computed as running sums of infarct area multiplied by the known interval (e.g. 660 mm) between sections over the extent of the infarct calculated as an orthogonal projection. To assure that brain size was not affected by DSP-4 treatment, a section from each animal, taken at bregma 1 0.48 mm was also imaged. Cross-sectional area of the hemisphere contralateral to the ischemic area was measured and compared with groups. Physiologic values, infarct sizes, contralateral hemispheric cross-sectional area and catecholamine concentrations were compared with groups by the Student's t-test and are presented as mean ^ SD. Neurologic scores were compared by the Mann±Whitney U-statistic and are presented as median ^ interquartile range. Physiologic values are summarized in Table 1. No significant differences were present between groups. At all time points throughout ischemia and 22 h of reperfusion, epidural temperature remained within 37:5 ^ 0:18C. Plasma concentrations of epinephrine and norepinephrine are presented in Table 1. No signi®cant differences were present between groups. All animals were awake during MCAO and had left foreTable 1 Physiologic values and cerebral infarct volumes in rats exposed to 75 min of normothermic transient focal cerebral ischemia a

10 min pre-ischemia MABP (mm Hg) Arterial pH PaCO2 (mm Hg) PaO2 (mm Hg) Glucose (mg/dl) Hematocrit (%) Plasma norepinephrine (ng/ml) Plasma epinephrine (ng/ml) 45 min intra-ischemia MABP (mm Hg) Arterial pH PaCO2 (mm Hg) PaO2 (mm Hg) Glucose (mg/dl) Hematocrit (%) Plasma norepinephrine (ng/ml) Plasma epinephrine (ng/ml) Total infarct volume (mm 3) Subcortical infarct volume (mm 3) Cortical infarct volume (mm 3) a

Control (n ˆ 22)

DSP-4 (n ˆ 23)

93 ^ 6 7.40 ^ .04 38 ^ 3 153 ^ 34 122 ^ 20 39 ^ 2 0.9 ^ 0.3

94 ^ 10 7.40 ^ 0.03 38 ^ 3 144 ^ 29 121 ^ 21 40 ^ 1 0.9 ^ 0.4

0.4 ^ 0.3

0.5 ^ 0.5

125 ^ 2 7.41 ^ 0.04 38 ^ 5 228 ^ 83 117 ^ 11 39 ^ 2 1.5 ^ 1.5

130 ^ 10 7.43 ^ 0.04 36 ^ 4 187 ^ 75 121 ^ 21 40 ^ 2 1.0 ^ 0.4

0.6 ^ 0.3

0.6 ^ 0.5

242 ^ 71 121 ^ 28

185 ^ 107* 93 ^ 44*

121 ^ 48

92 ^ 67

MABP ˆ mean arterial blood pressure, PaCO2 ˆ arterial blood carbon dioxide partial pressure, PaO2 ˆ arterial blood oxygen partial pressure. Values ˆ mean ^ standard deviation. *P , 0:05.

B. NellgaÊrd et al. / Neuroscience Letters 275 (1999) 167±170

limb paresis. Neurologic scores 3 days later were similar for the two groups (DSP-4 ˆ 11 ^ 3; control ˆ 11 ^ 2, P ˆ 0:46). A correlation between neurological de®cit and stroke-size was present (P ˆ 0:0005; Spearman rank correlation coef®cient). Total infarct volumes of DSP-4 treated animals were 24% smaller than those in control animals (185 ^ 107 vs. 242 ^ 71 mm 3, P ˆ 0:04). DSP-4 reduced subcortical infarct size by 23% (93 ^ 44 vs. 121 ^ 28 mm 3, P ˆ 0:02). Cortical infarct volumes of DSP-4 and control animals were not statistically different (92 ^ 67 vs. 121 ^ 48 mm 3, respectively; P ˆ 0:10). Cross-sectional area at bregma 1 0.48 mm was not different between groups (control ˆ 46:1 ^ 4:1 mm 2; DSP-4 ˆ 46:2 ^ 4:5 mm 2, P ˆ 0:94). We have demonstrated that DSP-4 pretreatment in normothermic rats exposed to conscious transient MCAO modestly reduces total and subcortical infarct-size without affecting plasma catecholamine concentrations. DSP-4 was chosen because it selectively reduces brain norepinephrine concentrations by degrading norepinephrine terminals originating from the locus coeruleus (LC) [7]. The DSP-4 treatment used in the current study has been shown to reduce immediate pre-ischemic rat brain norepinephrine concentrations by 90±95% in both cortex (control: 297 ^ 59; DSP-4: 16 ^ 9 ng/g wet weight) and hippocampus (control: 416 ^ 167; DSP-4: 30 ^ 24 ng/g wet weight) without altering plasma norepinephrine concentrations [12]. Furthermore, recent work in a model of severe forebrain ischemia found no increases in brain norepinephrine in rats identically treated with DSP-4 in contrast to untreated controls in which massive increases in norepinephrine were observed [12]. This is supported by ®ndings in the current experiment that DSP-4 did not alter plasma catecholamines before or during the focal ischemic insult. Brain catecholamines were not measured in the current study because the tissue was saved for histologic evaluation. The effect of ischemia duration was addressed in pilot studies where rats exposed to 90 min of reversible MCAO in the awake state had a high mortality rate, while 60 min of MCAO gave a variable subcortical infarct. Seventy-®ve minutes of MCAO gave consistent necrosis in both neocortex and striatum and was therefore selected for study. Anesthetics are known to affect the adrenergic response to brain ischemia [10] and outcome from focal ischemic insults [14]. In the present study the effect of anesthetics on outcome was minimized as all animals were awakened immediately after ®lament occlusion. This procedure also allowed intra-ischemic assessment of neurological de®cits to con®rm appropriate positioning of the occlusive ®lament. Previously described intra-ischemic increases in brain temperature in awake rats subjected to MCAO [9] were absent in the current study. Given these conditions, we found a modest (24%) reduction in brain injury resulting from MCAO in rats depleted of central norepinephrine. The mechanism by which brain norepinephrine might cause this protection is unknown.

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Norepinephrine plays an important role in normal brain physiology acting as a neurotransmitter, vasoregulator and anticonvulsant. Brain noradrenergic innervation originates from brain-stem nuclei, most prominently the LC. Additionally, noradrenergic projections from the superior cervical ganglia innervate cerebral arteries. However, the LC is also involved in the regulation of cerebral blood ¯ow (CBF). Electrical/chemical LC stimulation vasoconstricts cerebral vessels [6]. Pathophysiologic responses to ischemia include downregulation of brain adrenoceptors [11]. In contrast, DSP-4 treatment causes an approximate 50% upregulation of a 1-, a 2- and b -adrenoceptors within 7 days of treatment [16]. In models of global brain ischemia, preischemic depletion of central norepinephrine has given contradictory results. Bilateral lesions of the ascending ®bers from LC performed two weeks prior to cardiac arrest in the rat, increased hippocampal CA1 and neocortical damage [3]. In gerbils subjected to forebrain ischemia, DSP-4 pre-treatment augmented neuronal damage in hippocampal CA3 and CA4, but not in CA1 [13]. Both investigations lacked adequate control of physiologic parameters. In a recent study, we could not detect any difference in neuronal damage in hippocampus, striatum, or neocortex between DSP-4 treated and control rats subjected to global cerebral ischemia [12]. In that study, pericranial temperature and other physiological parameters were tightly controlled. It has been speculated that the increased number of spontaneous depolarizations seen in the ischemic penumbra during focal ischemia increases neuronal necrosis [1]. As norepinephrine can serve as an anticonvulsant [8], cerebral noradrenergic depletion theoretically could be detrimental. To the contrary, DSP-4 treatment reduced infarct size. Therefore, it seems unlikely that loss of inhibitory tone from DSP-4 treatment was important in de®ning ischemic outcome on the basis of changes in frequency of spontaneous depolarizations. It is possible that DSP-4 induced depletion of brain norepinephrine resulted in an increase of CBF in our MCAO model, which might bene®t collateral circulation in the ischemic penumbra. Consistent with this, chemical lesions in the LC have been associated with increased CBF in a variety of structures including neocortex and caudoputamen [2]. This might explain the modest neuroprotection observed by norepinephrine depletion in focal ischemia which is held in contrast to the lack of neuroprotection by DSP-4 in severe forebrain ischemia [12]. In forebrain ischemia, blood ¯ow is reduced to near zero by a combination of bilateral carotid occlusion and systemic hypotension. Perfusion changes caused by perturbation of vasomotor tone would be expected to have little effect on the magnitude of that ischemic insult. Further investigation using intraischemic autoradiographic CBF analysis in the focal and global models in the presence/absence of DSP-4 would be useful in examining this issue. In conclusion, we have examined the importance of

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central norepinephrine on stroke development. Depletion of cerebral norepinephrine by DSP-4 pretreatment modestly decreased focal ischemic brain injury. The results indicate that dampening of the central adrenergic response to stroke may be advantageous. The Laerdal Foundation for Acute Medicine, Swedish Stroke Society and Swedish Research Counsel supported B.N. G.B.M. was supported by a post-doctoral stipend through the German Academic Exchange Service (DAAD). S.S.-Y. was supported by an American Heart Association Student Scholarship in Cerebrovascular Disease. This study was supported by NIH Grant RO1GM39771±13. The authors are grateful to Ann D. Brinkhous for expert technical assistance. [1] Back, T., Kohno, K. and Hossman, K.-A., Cortical negative DC de¯ections following middle cerebral artery occlusion and KCl-induced spreading depression: effect on blood ¯ow, tissue oxygenation and electroencephalogram. J. Cereb. Blood Flow Metab., 14 (1994) 12±19. [2] Blomqvist, P., Lindvall, O. and Wieloch, T., Delayed postischemic hypoperfusion: evidence against involvement of the noradrenergic locus ceruleus system. J. Cereb. Blood Flow Metab., 4 (1984) 425±429. [3] Blomqvist, P., Lindvall, O. and Wieloch, T., Lesions of the locus coeruleus system aggravate ischemic damage in the rat brain. Neurosci. Lett., 38 (1985) 353±358. [4] Garcia, J.H., Wagner, S., Liu, K.F. and Hu, X.J., Neurological de®cit and extent of neuronal necrosis attributable to middle cerebral artery occlusion in rats: statistical validation. Stroke, 26 (1995) 627±635. [5] Globus, M.Y., Busto, R., Dietrich, W.D., Martinez, E., Valdes, I. and Ginsberg, M.D., Direct evidence for acute and massive norepinephrine release in the hippocampus during transient ischemia. J. Cereb. Blood Flow Metab., 9 (1989) 892±896. [6] Goadsby, P.J., Lambert, G.A. and Lance, J.W., The mechanism of cerebrovascular vasoconstriction in response to locus coeruleus stimulation. Brain Res., 326 (1985) 213±217. [7] Jonsson, G., Hallman, H., Ponzo, F. and Ross, S., DSP4

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