- Email: [email protected]
Effect of intracerebral norepinephrine depletion on outcome from severe forebrain ischemia in the rat
Brain Research 847 Ž1999. 262–269 www.elsevier.comrlocaterbres
Research report
Effect of intracerebral norepinephrine depletion on outcome from severe forebrain ischemia in the rat Bengt M.G. Nellgard ˚ a , Yoshihide Miura a , G. Burkhard Mackensen a , Robert D. Pearlstein b , David S. Warner a,b,) a
Department of Anesthesiology, Duke UniÕersity Medical Center, Durham, NC 27710, USA b Department of Surgery, Duke UniÕersity Medical Center, Durham, NC 27710, USA Accepted 31 August 1999
Abstract Manipulations of plasma catecholamine concentrations influence outcome from ischemic brain insults. It has been suggested that these effects are mediated by influences on brain catecholamine concentrations. This study examined whether major changes in brain norepinephrine concentrations can alter outcome from severe forebrain ischemia. Sprague–Dawley rats were administered 50 mgrkg i.p. N-Žchloroethyl.-N-ethyl-2-bromobenzylamine ŽDSP-4. or were left untreated Žcontrol.. One week later, these rats were subjected to either 7 or 8 min of normothermic forebrain ischemia Žbilateral carotid occlusion and MABPs 30 mmHg. and allowed to recover for 4 days. Histologic damage was then evaluated. In other control and DSP-4-treated animals, hippocampal microdialysate norepinephrine concentrations were measured before, during and after 8 min of forebrain ischemia. Norepinephrine concentrations were also determined in brain homogenates from non-ischemic DSP-treated and control rats. A 95% depletion of norepinephrine was observed in brain homogenates from non-ischemic DSP-4-treated rats compared with control. During ischemia, microdialysate norepinephrine concentrations increased in control but not in DSP-4-treated rats Ž P s 0.002.. For plasma, intra-ischemic epinephrine concentrations increased 8–10-fold and returned to baseline values post-ischemia with no differences between groups. Plasma norepinephrine values remained unchanged in both groups. Histologic damage resulting from either 7 or 8 min of ischemia in hippocampal structures, caudoputamen, and neocortex was similar between DSP-4-treated and control groups. This study could not identify any effect of major changes in brain norepinephrine concentrations on ischemic brain damage. These data indicate that peripheral catecholamine effects on near-complete forebrain ischemic outcome are unlikely to be mediated by effects on central catecholamine concentrations. q 1999 Elsevier Science B.V. All rights reserved. Keywords: Rat; Brain; Ischemia; Norepinephrine; Microdialysis; Histology
1. Introduction Cerebral ischemia is known to elicit responses from the sympathetic nervous system. Microdialysis studies of rat brain subjected to global ischemia have shown that extracellular norepinephrine becomes markedly increased during the insult w6,24x. Similarly, concentrations of plasma catecholamines increase during ischemia w14,29,31x. The effect of norepinephrine on ischemic outcome remains controversial. In rats subjected to forebrain ischemia, Koide et al. w14x found that pre-ischemic administration of trimethaphan, a sympathetic ganglionic antago) Corresponding author. Department of Anesthesiology, Box 3094, Duke University Medical Center, Durham, NC 27710, USA. Fax: q1919-684-6692; e-mail: [email protected]
nist, worsened histologic damage. Intravenous infusion of epinephrine and norepinephrine reversed this adverse effect. Conversely, Werner et al. w31x, using a model of unilateral hemispheric ischemia, found that hexamethonium, also a sympathetic ganglionic antagonist, improved ischemic outcome. Intravenous infusions of epinephrine and norepinephrine reversed the beneficial effect of hexamethonium. A similar dichotomy exists for compounds having direct effects on brain norepinephrine activity. Agonism of the central a 2-adrenoceptor by the anesthetic dexmedetomidine has been shown to decrease damage resulting from both unilateral hemispheric ischemia in the rat w10x and middle cerebral artery occlusion in the rabbit w17x. However, antagonism of the same receptor with idazoxan also improved ischemic outcome in the rat w7,8x.
0006-8993r99r$ - see front matter q 1999 Elsevier Science B.V. All rights reserved. PII: S 0 0 0 6 - 8 9 9 3 Ž 9 9 . 0 2 0 5 9 - 4
B.M.G. Nellgard ˚ et al.r Brain Research 847 (1999) 262–269
Although most of the studies cited above were performed in the absence of brain temperature regulation, cumulatively, these studies suggest important effects of norepinephrine on ischemic brain damage. It has been postulated that these effects are predominantly attributable to adrenergic modulation of central activity w6,14,31x. If so, substantial changes in brain norepinephrine concentrations should influence outcome from an ischemic insult. To test this hypothesis, we examined outcome from severe forebrain ischemia in normothermic rats pre-treated with DSP-4 Ž N-Žchloroethyl.-N-ethyl-2-bromobenzylamine., a compound which is known to cause selective depletion of brain norepinephrine. 2. Materials and methods The following studies were approved by the Duke University Animal Care and Use Committee. Male Sprague–Dawley rats Ž8–10 weeks, Harlan Sprague–Dawley, Indianapolis, IN., weighing between 270–340 g Ž8–11 weeks of age. were investigated. 2.1. Experiment 1 To investigate the effects of DSP-4 on brain catecholamine concentrations, animals were randomly assigned to two groups Ž n s 4 per group.: 1. DSP-4-treated: 50 mgrkg dissolved in 1 ml 0.9% NaCl i.p. ŽDSP-4 was given within 1 min after reconstitution in solution.; 2. Control: These rats received no pretreatment. One week after group-assignmentrtreatment, the rats were anesthetized with 4%–5% halothane and decapitated. This corresponds to the time point in later experiments at which rats were subjected to forebrain ischemia. Samples Ž2–30 mg. dissected from the neocortex and hippocampus were homogenized by a microsonicator Ž50 W. in 200 ml of 0.2 M perchloric acid ŽPCA. for 5 s. The homogenate was then centrifuged at 10,000 rpm for 1 min. Catecholamines were extracted on aluminum and analyzed by a reverse phase HPLC equipped with a C-18 column and an electrochemical detector. 2.2. Experiment 2 Sprague–Dawley rats were randomized into DSP-4treated and untreated control groups as described in Experiment 1 Ž n s 4 per group.. One week after group-assignmentrtreatment, these rats were fasted Ž12–16 h. but allowed free access to water. The animals were then anesthetized with 4%–5% halothane in oxygen. After orotracheal intubation, the lungs were mechanically ventilated Ž30%–40% O 2rbalance N2 .. The inspired concentration of halothane Ž1%–2%. was then adjusted to maintain mean arterial blood pressure ŽMABP. within 80–120 mmHg. Surgery was performed using an aseptic technique and all wounds were infiltrated with 1% lidocaine. The tail artery
263
was cannulated and used for blood pressure measurement and for arterial blood sampling. Via a neck incision the right jugular vein was cannulated. The common carotid arteries were then exposed and encircled with sutures, leaving the vagus nerves and cervical sympathetic plexus intact. The animals were positioned in a stereotaxic head frame, a midline scalp incision was made, and the skull was exposed. Using a 2-mm trephine drill with continuous saline irrigation, a burr hole was drilled over the left hemisphere above the dorsal hippocampus Žbregma y4.2 mm, lateral 2.0 mm.. The dura was opened and a guide cannula ŽCMAr12 guide cannula; CMArMicrodialysis, Acton, MA. was inserted through the cranial opening and stereotactically positioned with the tip at the brain surface Ž108 from perpendicular.. The guide was fixed in place with a cranial screw, and the burr hole was sealed with orthodontic cement. The wound was closed with sutures and the animal was unmounted from the head frame. Cortical EEG was continuously monitored during the experiment from active subdermal electrodes positioned over the parietal cortex bilaterally and a ground lead positioned in the tail. A 22-gauge needle thermistor ŽModel 524; YSI, Yellow Springs, OH. was subcutaneously placed adjacent to the skull beneath the temporalis muscle and the temperature was servoregulated ŽModel 73ATA Indicating Controller; YSI, Yellow Springs. at 37.5 " 0.18C with surface heating or cooling. A 2.0 mm membrane microdialysis probe ŽCMAr12 microdialysis probe; CMArMicrodialysis. was prepared according to manufacturer’s recommendations. Immediately before each experiment, the probe was connected to a microinfusion pump ŽCMAr102 microdialysis pump; CMArMicrodialysis. and perfused at a flow rate of 2 mlrmin with artificial cerebrospinal fluid ŽACSF; 122 mM NaCl, 3 mM KCl, 1.2 mM MgSO4 , 0.4 mM KH 2 PO4 , 25 mM NaHCO 3 and 1.2 mM CaCl 2 , pH 7.4, filtered through a 0.2-mm filter and degassed before use.. Membrane efficiency was determined prior to each use by dialyzing against a 1-ngrmg norepinephrine standard solution in 0.2 N PCA. Microdialysate norepinephrine and epinephrine concentrations were corrected for membrane efficiency. The probe was then inserted through the guide cannula into the dorsal hippocampus with the tip of the probe positioned at a depth of 4 mm from the brain surface. ACSF was perfused through the probe for 2 h. Two microdialysate samples were then collected prior to ischemia over 15 min intervals. One 10-min sample was collected during ischemia, and five 15-min samples were collected after restoring circulation. Microdialysate samples were collected in tubes containing 0.2 N PCA to prevent catecholamine auto-oxidation. Microdialysate samples were maintained on ice and analyzed within 3 h of collection. Norepinephrine and epinephrine concentrations were measured by HPLC. Microdialysate samples were taken before, during and after ischemia and directly injected Žno extraction. into the chromatographic column.
264
B.M.G. Nellgard ˚ et al.r Brain Research 847 (1999) 262–269
Following surgical preparation, the halothane concentration was decreased to 0.8%. Ventilation was adjusted to produce normocapnia and mild hyperoxemia. Muscle paralysis was provided by a 1-mg i.v. bolus of succinylcholine, repeated when necessary, to allow control of ventilation during ischemia. Heparin Ž50 IU. was given intravenously. Near complete global cerebral ischemia was then induced by exsanguination through the jugular venous catheter to a MABP of 30 mmHg w5x. The carotid arteries were then occluded using temporary aneurysm clips w29x. The onset of ischemia was defined as the time point when the MABP had been stable at 30 mmHg for 30 s. After 8 min, ischemia was terminated by removal of the carotid clips and reinfusion of shed blood. NaHCO 3 Ž0.3 mEq i.v.. was given to minimize systemic acidosis. Animals remained anesthetized an additional 2 h Ž0.8% halothane. with pericranial temperature servoregulated at 37.5 " 0.18C. The animals were then euthanized with a high dose of halothane. Brains were removed and probe locations were verified at necropsy. 2.3. Experiment 3 To investigate the effect of DSP-4 on ischemic histologic outcome, Sprague–Dawley rats were randomized into DSP-4-treated and untreated control groups as described in Experiment 1. Rats were fasted but allowed free access to water for 12–16 h and then anesthetized with halothane and surgically prepared for forebrain ischemia as described in Experiment 2, but microdialysis probes were not implanted. Rats from each group were then subjected to either 7 or 8 min of forebrain ischemia as described in Experiment 2. Blood gases and plasma norepinephrine, epinephrine and glucose were measured 10 min pre-ischemia, 4 min after ischemia onset and 10 min post-ischemia. Blood samples Ž200 ml volume. were collected in pre-cooled tubes containing sodium metabisulfite and EDTA to prevent oxidation of norepinephrine and epinephrine. Plasma catecholamines were then extracted on aluminum and samples were injected into a reverse phase HPLC equipped with a C-18 column and an electrochemical detector. Values for plasma norepinephrine and epinephrine were corrected for extraction efficiency. Ischemia was terminated by removal of the carotid clips and reinfusion of shed blood. NaHCO 3 Ž0.3 mEq i.v.. was given to minimize systemic acidosis. Animals remained anesthetized an additional 2 h Ž0.8% halothane. with pericranial temperature servoregulated at 37.5 " 0.18C. Upon recovery of spontaneous ventilation, the trachea was extubated. After recovery of the righting reflex, the animals were returned to their cages. Four days post-ischemia, the animals were again anesthetized with halothane, intubated and mechanically ventilated followed by intracardiac injection of saline followed by buffered 10% formalin. After 24 h, the brains were
removed and stored in 10% formalin. Paraffin-embedded brain sections were serially cut Ž5 mm thick. and stained with acid fuchsinrcelestine blue. With the investigator blinded to group assignment, injury to the hippocampus, striatum and cortex was measured using light microscopy. All viable and nonviable neurons in the CA1 sector of hippocampus were manually counted and the percentage of dead neurons was calculated at a coronal level approximately 3.8 mm caudal to bregma. Damage to neurons in the hilus of dentate gyrus, or CA4 were examined at the same coronal level and graded on a 1–4 scale, Ž1 s 0%– 25% neurons damaged; 2 s 25%–50% neurons damaged; 3 s 50%–75% neurons damaged and 4 s 75%–100% neurons damaged.. Striatum was examined in a central circular area of 400-mm diameter, at a level 0.3 mm caudal to bregma. Damage was calculated as percent dead neurons per field. Neocortical damage was defined as the number of dead neurons in a circular area with a 400-mm diameter dorsal to the entorhinal fissure at 3.8 mm caudal to bregma. Values from the hemisphere with the worst damage in each animal were used for statistical analyses. Parametric values including histologic injury in the hippocampal CA1, neocortex and caudoputamen and norepinephrinerepinephrine concentrations were analyzed by analysis of variance. When indicated by a significant F ratio, between group differences were defined using post hoc protected least squares difference analysis. Values are reported as mean " S.D. Non-parametric values Že.g., histologic scores. were compared by the Kruskal–Wallis H statistic and are reported as median" interquartile deviation. Significance was assumed when p - 0.05. 3. Results 3.1. Experiment 1 Treatment with DSP-4 reduced tissue norepinephrine concentration in non-ischemic brain by approximately 95% Table 1 Physiologic values for rats exposed to an 8-min forebrain ischemic insult while undergoing hippocampal microdialysis for norepinephrine ŽExperiment 2.. Values are mean"S.D. Control Ž ns 4.
DSP-4 Ž ns 4.
10 min pre-ischemia MABP ŽmmHg. pH a PaCO 2 ŽmmHg. PaO 2 ŽmmHg. Glucose Žmgrdl. Hematocrit Ž%.
100"9 7.38"0.02 41"2 141"7 179"9 43"1
98"16 7.38"0.02 40"1 123"7 230"20 46"3
10 min post-ischemia MABP ŽmmHg. pH a PaCO 2 ŽmmHg. PaO 2 ŽmmHg.
125"19 7.31"0.11 43"7 149"24
123"17 7.29"0.10 45"12 154"31
B.M.G. Nellgard ˚ et al.r Brain Research 847 (1999) 262–269
265
observed in the DSP-4 group Ž P s 0.07.. The difference in intra-ischemic microdialysate norepinephrine concentration between groups was significant Ž P s 0.0005.. No difference between Control and DSP-4 groups could be detected at other measurement intervals. Microdialysate epinephrine was under the limit of detection. 3.3. Experiment 3
Fig. 1. Microdialysate from hippocampal CA1 in animals exposed to 8 min of forebrain ischemia. Extracellular brain norepinephrine concentrations increased during ischemia in control animals Žwhite triangles., but not in DSP-4-treated animals Žfilled circles.. At all other time points, a difference between control and DSP-4 animals could not be detected. Microdialysate epinephrine was under the limit of detection. Valuess mean"S.D. w Difference between groups for intra-ischemic microdialysate norepinephrine concentration Ž P s 0.0005..
in both cortex ŽControl: 297 " 59; DSP-4: 16 " 9 ngrg wet weight, P s- 0.0001. and hippocampus ŽControl: 416 " 167; DSP-4: 30 " 24 ngrg wet weight, P s 0.004.. Epinephrine was under the limit of detection in both groups. 3.2. Experiment 2 Physiologic values are reported in Table 1 for rats undergoing hippocampal microdialysis. Pericranial temperature was held between 37.5 " 0.18C throughout the experimental procedure. Baseline microdialysate norepinephrine concentrations were similar between control and DSP-4treated animals ŽFig. 1.. During ischemia, microdialysate norepinephrine increased from baseline in control animals Ž P s 0.001., while no statistically significant change was
Physiologic values were similar between groups in the histologic outcome studies ŽTable 2.. Pericranial temperature was held between 37.5 " 0.18C throughout the experimental procedure. In animals subjected to 8 min of forebrain ischemia, damage in hippocampal CA1 ŽControls 78 " 22; DSP-4s 73 " 28% dead neurons, P s 0.48.; caudoputamen ŽControl s 63 " 20; DSP-4 s 64 " 21% dead neurons, P s 0.91. and neocortex ŽControls 15 " 4; DSP-4s 14 " 6 necrotic neuronsrhigh power field, P s 0.63. was similar between groups ŽFig. 2.. No necrotic neurons were present in the hippocampal CA3 sector or dentate gyrus in either group. Median" interquartile range histologic scores in hippocampal CA4 were not different between groups ŽControls 3 " 1; DSP-4s 3 " 1, P s 0.66.. No differences were observed between groups for plasma epinephrine or norepinephrine prior to, during, or after ischemia ŽFig. 3.. The change in plasma norepinephrine between pre-ischemic and intra-ischemic measurement intervals was not significant in either the control or DSP-4 groups. In contrast, ischemia caused an increase in plasma epinephrine in both groups Ž P s 0.002.. Similar findings were present for animals exposed to 7 min of ischemia. Although overall damage was numerically decreased relative to the 8-min insult, there was no difference between groups for percent dead neurons in hippocampal CA1% ŽControls 57 " 27%; DSP-4s 57 " 34%, P s 0.99. or caudoputamen ŽControls 38 " 25%; DSP-4s 47 " 23%, P s 0.45.. The number of necrotic cortical neuronsrhigh power field in the Control Ž14 " 5.
Table 2 Physiologic values for rats undergoing 7 or 8 min of forebrain ischemia and analysis of histologic outcome ŽExperiment 3.. All values are mean" S.D. Controlr7 min Ž n s 10.
DSP-4r7 min Ž n s 12.
Controlr8 min Ž n s 22.
DSP-4r8 min Ž n s 19.
10 min pre-ischemia MABP ŽmmHg. pH a PaCO 2 ŽmmHg. PaO 2 ŽmmHg. Glucose Žmgrdl. Hematocrit Ž%.
97 " 14 7.32 " 0.05 38 " 2 153 " 22 134 " 36 38 " 2
97 " 12 7.32 " 0.05 37 " 4 155 " 23 137 " 26 39 " 2
88 " 11 7.36 " 0.03 38 " 2 165 " 53 117 " 18 39 " 2
100 " 15 7.36 " 0.04 38 " 3 178 " 45 140 " 23 39 " 2
10 min post-ischemia MABP ŽmmHg. pH a PaCO 2 ŽmmHg. PaO 2 ŽmmHg. Glucose Žmgrdl. Hematocrit Ž%.
105 " 15 7.33 " 0.05 39 " 3 149 " 24 146 " 43 37 " 2
110 " 17 7.35 " 0.05 38 " 2 154 " 31 137 " 40 39 " 2
114 " 11 7.35 " 0.04 40 " 2 163 " 42 110 " 24 39 " 2
124 " 11 7.35 " 0.05 39 " 2 177 " 26 139 " 44 40 " 2
266
B.M.G. Nellgard ˚ et al.r Brain Research 847 (1999) 262–269
Fig. 3. Plasma norepinephrine and epinephrine concentrations in control animals Žopen triangles. and DSP-4-treated animals Žfilled circles. exposed to 8 min of forebrain ischemia. No differences were present between groups at any interval for either catecholamine. Ischemia did not cause a significant change in plasma norepinephrine in either group, but epinephrine was increased in both groups during ischemia. w Difference between pre-ischemic and intra-ischemic values for both the DSP-4-treated and control groups Ž P s 0.002.. Valuess mean"S.D.
and DSP-4 Ž16 " 5, P s 0.36. groups was similar. No necrotic neurons were present in the hippocampal CA3 sector or dentate gyrus in either group. Median " interquartile range histologic scores in hippocampal CA4 were not different between groups ŽControls 3 " 1; DSP4 s 3 " 1, P s 0.29.. Plasma norepinephrine and epinephrine concentrations were similar to those observed in the 8-min group Ždata not shown..
4. Discussion We have demonstrated in the rat that DSP-4 pretreatment: Ž1. markedly reduces brain norepinephrine concentration; Ž2. inhibits intra-ischemic increases in brain extracellular norepinephrine but does not effect plasma catecholamine concentrations; and Ž3. fails to modulate delayed neuronal necrosis resulting from different durations of normothermic forebrain ischemia in the hippocampus, striatum, and neocortex. DSP-4 was chosen for this study because it selectively reduces brain norepinephrine concentration but exerts negligible influence on dopaminergic or serotonergic neurons w13,16,32x. DSP-4 readily penetrates the blood–brain barrier and selectively degrades noradrenergic terminals originating in the locus coeruleus but leaves cell bodies intact w13x. In contrast to reserpine, the effects of DSP-4 on brain are long lasting, while the extracerebral noradrenergic
Fig. 2. Histologic damage was similar in hippocampal CA1 Ž P s 0.48., caudoputamen Ž P s 0.91., and neocortex Ž P s 0.63. in control Ž ns 22. and DSP-4-treated Ž ns19. animals exposed to 8 min of forebrain ischemia. Open circles depict values for individual animals and bars indicate the group mean. HPF s high power field.
B.M.G. Nellgard ˚ et al.r Brain Research 847 (1999) 262–269
effects are transient and normalized after 1 week w11,13x. The regimen of DSP-4 administration utilized in this study was similar to that used by others w1x and resulted in reduction of neocortical and hippocampal norepinephrine concentrations to less than 10% of normal at the time of ischemia induction. The trial of DSP-4-treated vs. control animals exposed to 8-min ischemia showed severe damage in hippocampal CA1 in both groups. However, given the severe CA1 damage observed after 8-min ischemia, there was little apparent opportunity for DSP-4 to exhibit a worsened outcome should that have been a potential effect of norepinephrine depletion. Accordingly, the study was repeated with 7 min of forebrain ischemia. Although the two studies were not performed concurrently, CA1 damage in the control group was reduced Ž7 min s 57 " 34% dead neurons; 8 min s 78 " 22% dead neurons, P s 0.041.. Despite this, no difference between DSP-4-treated and control animals was seen in any structure examined. The failure of DSP-4 to modulate delayed neuronal necrosis in our study is somewhat surprising. There is reason to believe that brain parenchymal norepinephrine plays a role in the eventual histologicrbehavioral outcome from ischemia. In the gerbil, a reduced number of brain a 1-, a 2-, and b-adrenoceptors has been demonstrated as early as 24 h after unilateral carotid artery occlusion w18x. The magnitude of this effect is both anatomically and receptor subtype specific. Consistent with this specificity, a 1-adrenergic receptor binding is reduced by approximately 50% in forebrain homogenates of post-ischemic gerbils while b 1 and b 2 binding undergoes little or no change w21x. In contrast, DSP-4 treatment causes an approximate 50% upregulation of a 1-, a 2-, and b-adrenoceptors within 7 days of treatment w32x. Therefore, if changes in receptor function are important in ischemic brain, it would be expected that an effect of DSP-4 treatment on histologic outcome would have been evident. Others have examined effects of depletion of central norepinephrine on histologic outcome from cerebral ischemia. Blomqvist et al. w2x lesioned the locus coeruleus ascending fibers by bilateral injections of 6-hydroxydopamine 2–3 weeks prior to a cardiac arrest insult in the rat. Although brain norepinephrine was not measured during the ischemic insult, frontal cortex was sampled from recovery animals at 7 days post-ischemia and a near-total depletion of norepinephrine was documented. In lesioned animals, enhanced neuronal necrosis was observed in the dorsal hippocampal CA1 and in neocortex. Furthermore, Nishino et al. w22x utilized DSP-4-treated Mongolian gerbils in a paradigm of global cerebral ischemia and reported that DSP-4 treatment augmented CA3 and CA4, but not CA1 injury. In contrast, our investigation found no effect of norepinephrine depletion on outcome from global ischemia in any of these structures. The studies by both Blomqvist et al. w2x and Nishino et al. w22x were performed prior to widespread appreciation of
267
the effects of brain temperature and plasma glucose on ischemic outcome w3,26x. Both studies monitored rectal temperature only and both groups examined non-fasted animals. Further, the gerbils studied for histologic outcome by Nishino et al. w22x did not undergo physiologic monitoring and were allowed to breathe spontaneously while anesthetized halothane. The effects of locus coeruleus lesioning and DSP-4-treatment on these well-described physiologic determinants of ischemic outcome are unknown. Presumably, the current study, which incorporated strict physiologic regulation during and after ischemia, was devoid of these potential confounding influences. The absence of effect of central norepinephrine depletion on histologic outcome from forebrain ischemia observed in the current experiment has implications for the interpretation of other studies, which have associated either circulating catecholamine concentrations or centrally-acting noradrenergic compounds with ischemic outcome. It has been suggested that peripheral catecholamines might alter outcome by transiting the blood–brain barrier during the ischemic insult w6,14,31x. However, little change in plasma norepinephrine was observed. In contrast, despite a six- to eight-fold intra-ischemic increase in plasma epinephrine, brain epinephrine remained below the limit of detection, which is consistent with prior work, which demonstrated little or no blood–brain barrier leakage resulting from global ischemia w25x. It has also been suggested that drugs such as idazoxan or dexmedetomidine exert effects on ischemic outcome by either antagonizing or agonizing central a 2-adrenoceptors w6,14,31x. This appears unlikely, because the results of the current experiment indicate that near-total depletion of brain norepinephrine has no effect on ischemic outcome. Alternatively, circulating catecholamines, or drugs such as idazoxan and dexmedetomidine, may indirectly influence ischemic outcome by effects on extracranial neurohumoral events, which then influence neurons and glia. Both idazoxan and dexmedetomidine have potent effects on peripheral adrenergic receptors w19,27x. For example, increases in catecholamines induce release of corticosteroids, which in turn can cross the blood–brain barrier and bind to intracellular receptors w12x. Inhibition of corticosterone synthesis has been shown in various models to improve outcome from ischemicrhypoxic insults w15,30x. Furthermore, adrenergic responses to stress can interact with plasma concentrations of interleukin-6 w23x, interleukin-1b w9x, and tumor necrosis factor w4x — factors having potential to influence outcome from brain ischemic insults w20,28,33x. In conclusion, there is a substantial body of evidence that norepinephrine and drugs, which affect the adrenergic nervous system can alter outcome from cerebral ischemic insults. This study examined whether major changes in brain norepinephrine concentration were sufficient to alter histologic damage resulting from severe forebrain ischemia. Pre-ischemic administration of DSP-4 served to
268
B.M.G. Nellgard ˚ et al.r Brain Research 847 (1999) 262–269
deplete brain norepinephrine by 95% and inhibited increases in brain norepinephrine during ischemia, but had no effect on plasma concentrations of norepinephrine or epinephrine. Ischemic intervals of 7 and 8 min caused substantive delayed neuronal necrosis but damage was similar in DSP-4-treated and control rats. These results suggest that central noradrenergic events are unlikely to explain effects of either exogenous catecholamines or noradrenergic compounds on ischemic outcome. Acknowledgements This study was supported by NIH Grant RO1 GM39771-12. Bengt Nellgard ˚ was supported by The Laerdal Foundation for Acute Medicine and Swedish Society of Physicians. G. Burkhard Mackensen was supported by a post-doctoral stipend through the German Academic Exchange Service ŽDAAD.. The authors are grateful to Ann D. Brinkhous for expert technical assistance. References w1x S.S.A. Al-Zahrani, A.S.A. Al-Ruwaitea, M.Y. Ho, C.M. Bradshaw, E. Szabadi, Destruction of central noradrenergic neurones with DSP4 impairs the acquisition of temporal discrimination but does not affect memory for duration in a delayed conditional discrimination task, Psychopharmacology 130 Ž1997. 166–173. w2x P. Blomqvist, O. Lindvall, T. Wieloch, Lesions of the locus coeruleus system aggravate ischemic damage in the rat brain, Neurosci. Lett. 38 Ž1985. 353–358. w3x R. Busto, W.D. Dietrich, M.Y.T. Globus, I. Valdes, ´ P. Scheinberg, M.D. Ginsberg, Small differences in intra-ischemic brain temperature critically determine the extent of neuronal injury, J. Cereb. Blood Flow Metab. 7 Ž1987. 729–738. w4x G. Fantuzzi, E. Di Santo, S. Sacco, F. Benigni, P. Ghezzi, Role of the hypothalamic–pituitary–adrenal axis in the regulation of TNF production in mice, J. Immunol. 155 Ž1995. 3552–3555. w5x T.X. Gionet, D.S. Warner, M. Verhaegen, J.D. Thomas, M.M. Todd, Effects of intra-ischemic blood pressure on outcome from 2-vessel occlusion forebrain ischemia in the rat, Brain Res. 586 Ž1992. 188–194. w6x M.Y. Globus, R. Busto, W.D. Dietrich, E. Martinez, I. Valdes, M.D. Ginsberg, Direct evidence for acute and massive norepinephrine release in the hippocampus during transient ischemia, J. Cereb. Blood Flow Metab. 9 Ž1989. 892–896. w7x I. Gustafson, Y. Miyauchi, T.W. Wieloch, Post-ischemic administration of idazoxan, an a 2 -adrenergic receptor antagonist, decreases neuronal damage in the rat brain, J. Cereb. Blood Flow Metab. 9 Ž1989. 171–174. w8x I. Gustafson, E. Westerberg, T. Wieloch, Protection against ischemia-induced neuronal damage by the a 2-adrenoceptor antagonist idazoxan: influence of time of administration and possible mechanisms of action, J. Cereb. Blood Flow Metab. 10 Ž1990. 885–894. w9x A.R. Gwosdow, Mechanisms of interleukin-1-induced hormone secretion from the rat adrenal gland, Endocr. Res. 21 Ž1995. 25–37. w10x W.E. Hoffman, E. Kochs, C. Werner, R.F. Albrecht, Dexmedetomidine improves neurologic outcome from incomplete cerebral ischemia in the rat, Anesthesiology 75 Ž1991. 328–332. w11x G. Jaim-Etcheverry, L.M. Zieher, DSP-4: a novel compound with neurotoxic effects on noradrenergic neurons of adult and developing rats, Brain Res. 188 Ž1980. 513–523.
w12x M. Joels, ¨ E.R. de Kloet, Control of neuronal excitability by corticosteroid hormones, Trend Neurosci. 15 Ž1992. 25–30. w13x G. Jonsson, H. Hallman, F. Ponzo, S. Ross, DSP4 Ž N-Ž2-chloroethyl.-N-ethyl-2-bromobenzylamine. — a useful denervation tool for central and peripheral noradrenaline neurons, Eur. J. Pharmacol. 72 Ž1981. 173–188. w14x T. Koide, T. Wieloch, B.K. Siesjo, ¨ Circulating catecholamines modulate ischemic brain damage, J. Cereb. Blood Flow Metab. 6 Ž1986. 559–565. w15x H.J. Krugers, R.H.A. Kemper, J. Korf, G.J. Ter Horst, S. Knooema, Metyrapone reduces rat brain damage and seizures after hypoxia– ischemia: an effect independent of modulation of plasma corticosterone levels?, J. Cereb. Blood Flow Metab. 18 Ž1998. 386–390. w16x K.J. Lookingland, D.S. Chapin, D.W. McKay, K.E. Moore, Comparative effects of the neurotoxins N-chloroethyl-N-ethyl-2-bromobenzylamine hydrochloride ŽDSP4. and 6-hydroxydopamine on hypothalamic noradrenergic, dopaminergic and 5-hydroxytryptaminergic neurons in the male rat, Brain Res. 365 Ž1986. 228–234. w17x C. Maier, G.K. Steinberg, G.H. Sun, G.T. Zhi, M. Maze, Neuroprotection by the a 2-adrenoreceptor agonist dexmedetomidine in a focal model of cerebral ischemia, Anesthesiology 79 Ž1993. 306– 312. w18x T. Mizuki, H. Kobasyashi, S. Ueno, Y. Nakashima, A. Kuroiwa, F. Izumi, Differential changes in a- and b-adrenoceptors in the cerebral cortex and hippocampus of the Mongolian gerbil after unilateral brain ischemia, Stroke 26 Ž1995. 2333–2336. w19x G.J. Molderings, J. Likungu, J. Jakschik, M. Gothert, Presynaptic imidazole receptors and non-adrenoceptor w3 Hx-idazoxan binding sites in human cardiovascular tissues, Br. J. Pharmacol. 1997 Ž1997. 43–50. w20x H. Nawashiro, K. Tasaki, C.A. Ruetzler, J.M. Hallenbeck, TNF-alpha pretreatment induces protective effects against focal cerebral ischemia in mice, J. Cereb. Blood Flow Metab. 17 Ž1997. 483–490. w21x K. Nishino, J.N. Davis, Rapid reduction in w3 Hxprazosin binding to gerbil forebrain membranes during bilateral common carotid artery occlusion, J. Cereb. Blood Flow Metab. 9 Ž1989. 358–363. w22x K. Nishino, C.S. Lin, J.K. Morse, J.N. Davis, DSP4 treatment worsens hippocampal pyramidal cell damage after transient ischemia, Neuroscience 43 Ž1991. 361–367. w23x S. Noisakran, W.P. Halford, L. Veress, D.J.J. Carr, Role of the hypothalamic pituitary adrenal axis and IL-6 in stress-induced reactivation of latent herpes simplex virus type I, J. Immunol. 160 Ž1998. 5441–5447. w24x C. Perego, S. Gatti, G.C. Vetrugno, F. Marzatico, S. Algeri, Correlation between electroencephalogram isoelectric time and hippocampal norepinephrine levels, measured by microdialysis, during ischemia in rats, J. Neurochem. 59 Ž1992. 1257–1262. w25x C.K. Petito, W.A. Pulsinelli, G. Jacobson, F. Plum, Edema and vascular permeability in cerebral ischemia: comparison between ischemic neuronal damage and infarction, J. Neuropathol. Exp. Neurol. 41 Ž1982. 423–436. w26x W. Pulsinelli, S. Waldman, D. Rawlinson, F. Plum, Moderate hyperglycemia augments ischemic brain damage: a neuropathologic study in the rat, Neurology 32 Ž1982. 1239–1246. w27x H. Scheinin, R. Aantaa, M. Anttila, P. Hakola, A. Helminen, S. Karhuvaara, Reversal of the sedative and sympatholytic effects of dexmedetomidine with a specific alpha 2-adrenoceptor antagonist atipamezole: a pharmacodynamic and kinetic study in healthy volunteers, Anesthesiology 89 Ž1998. 574–584. w28x G.P. Schielke, G.Y. Yang, B.D. Shivers, A.L. Betz, Reduced ischemic brain injury in interleukin-1b converting enzyme-deficient mice, J. Cereb. Blood Flow Metab. 18 Ž1998. 180–185. w29x M.-L. Smith, G. Bendek, N. Dahlgren, I. Rosen, T. Wieloch, B.K. Siesjo, ¨ Models for studying long-term recovery following forebrain ischemia in the rat: 2. A 2-vessel occlusion model, Acta Neurol. Scand. 69 Ž1984. 385–401. w30x V.L. Smith-Swintosky, L.C. Pettigrew, R.M. Sapolsky, C. Phares,
B.M.G. Nellgard ˚ et al.r Brain Research 847 (1999) 262–269 S.D. Craddock, S.M. Brooke, M.P. Mattson, Metyrapone, an inhibitor of glucocorticoid production, reduces brain injury induced by focal and global ischemia and seizures, J. Cereb. Blood Flow Metab. 16 Ž1996. 585–598. w31x C. Werner, W.E. Hoffman, C. Thomas, D.J. Miletich, R.F. Albrecht, Ganglionic blockade improves neurologic outcome from incomplete ischemia in rats-partial reversal by exogenous catecholamines, Anesthesiology 73 Ž1990. 923–929.
269
w32x C. Wolfman, V. Abo, D. Calvo, J. Medina, F. Dajas, R. Silveria, Recovery of central noradrenergic neurons one year after the administration of the neurotoxin DSP4, Neurochem. Int. 25 Ž1994. 395– 400. w33x M. Yamada, H. Hatanaka, Interleukin-6 protects cultured rat hippocampal neurons against glutamate-induced cell death, Brain Res. 643 Ž1994. 173–180.
Research report
Effect of intracerebral norepinephrine depletion on outcome from severe forebrain ischemia in the rat Bengt M.G. Nellgard ˚ a , Yoshihide Miura a , G. Burkhard Mackensen a , Robert D. Pearlstein b , David S. Warner a,b,) a
Department of Anesthesiology, Duke UniÕersity Medical Center, Durham, NC 27710, USA b Department of Surgery, Duke UniÕersity Medical Center, Durham, NC 27710, USA Accepted 31 August 1999
Abstract Manipulations of plasma catecholamine concentrations influence outcome from ischemic brain insults. It has been suggested that these effects are mediated by influences on brain catecholamine concentrations. This study examined whether major changes in brain norepinephrine concentrations can alter outcome from severe forebrain ischemia. Sprague–Dawley rats were administered 50 mgrkg i.p. N-Žchloroethyl.-N-ethyl-2-bromobenzylamine ŽDSP-4. or were left untreated Žcontrol.. One week later, these rats were subjected to either 7 or 8 min of normothermic forebrain ischemia Žbilateral carotid occlusion and MABPs 30 mmHg. and allowed to recover for 4 days. Histologic damage was then evaluated. In other control and DSP-4-treated animals, hippocampal microdialysate norepinephrine concentrations were measured before, during and after 8 min of forebrain ischemia. Norepinephrine concentrations were also determined in brain homogenates from non-ischemic DSP-treated and control rats. A 95% depletion of norepinephrine was observed in brain homogenates from non-ischemic DSP-4-treated rats compared with control. During ischemia, microdialysate norepinephrine concentrations increased in control but not in DSP-4-treated rats Ž P s 0.002.. For plasma, intra-ischemic epinephrine concentrations increased 8–10-fold and returned to baseline values post-ischemia with no differences between groups. Plasma norepinephrine values remained unchanged in both groups. Histologic damage resulting from either 7 or 8 min of ischemia in hippocampal structures, caudoputamen, and neocortex was similar between DSP-4-treated and control groups. This study could not identify any effect of major changes in brain norepinephrine concentrations on ischemic brain damage. These data indicate that peripheral catecholamine effects on near-complete forebrain ischemic outcome are unlikely to be mediated by effects on central catecholamine concentrations. q 1999 Elsevier Science B.V. All rights reserved. Keywords: Rat; Brain; Ischemia; Norepinephrine; Microdialysis; Histology
1. Introduction Cerebral ischemia is known to elicit responses from the sympathetic nervous system. Microdialysis studies of rat brain subjected to global ischemia have shown that extracellular norepinephrine becomes markedly increased during the insult w6,24x. Similarly, concentrations of plasma catecholamines increase during ischemia w14,29,31x. The effect of norepinephrine on ischemic outcome remains controversial. In rats subjected to forebrain ischemia, Koide et al. w14x found that pre-ischemic administration of trimethaphan, a sympathetic ganglionic antago) Corresponding author. Department of Anesthesiology, Box 3094, Duke University Medical Center, Durham, NC 27710, USA. Fax: q1919-684-6692; e-mail: [email protected]
nist, worsened histologic damage. Intravenous infusion of epinephrine and norepinephrine reversed this adverse effect. Conversely, Werner et al. w31x, using a model of unilateral hemispheric ischemia, found that hexamethonium, also a sympathetic ganglionic antagonist, improved ischemic outcome. Intravenous infusions of epinephrine and norepinephrine reversed the beneficial effect of hexamethonium. A similar dichotomy exists for compounds having direct effects on brain norepinephrine activity. Agonism of the central a 2-adrenoceptor by the anesthetic dexmedetomidine has been shown to decrease damage resulting from both unilateral hemispheric ischemia in the rat w10x and middle cerebral artery occlusion in the rabbit w17x. However, antagonism of the same receptor with idazoxan also improved ischemic outcome in the rat w7,8x.
0006-8993r99r$ - see front matter q 1999 Elsevier Science B.V. All rights reserved. PII: S 0 0 0 6 - 8 9 9 3 Ž 9 9 . 0 2 0 5 9 - 4
B.M.G. Nellgard ˚ et al.r Brain Research 847 (1999) 262–269
Although most of the studies cited above were performed in the absence of brain temperature regulation, cumulatively, these studies suggest important effects of norepinephrine on ischemic brain damage. It has been postulated that these effects are predominantly attributable to adrenergic modulation of central activity w6,14,31x. If so, substantial changes in brain norepinephrine concentrations should influence outcome from an ischemic insult. To test this hypothesis, we examined outcome from severe forebrain ischemia in normothermic rats pre-treated with DSP-4 Ž N-Žchloroethyl.-N-ethyl-2-bromobenzylamine., a compound which is known to cause selective depletion of brain norepinephrine. 2. Materials and methods The following studies were approved by the Duke University Animal Care and Use Committee. Male Sprague–Dawley rats Ž8–10 weeks, Harlan Sprague–Dawley, Indianapolis, IN., weighing between 270–340 g Ž8–11 weeks of age. were investigated. 2.1. Experiment 1 To investigate the effects of DSP-4 on brain catecholamine concentrations, animals were randomly assigned to two groups Ž n s 4 per group.: 1. DSP-4-treated: 50 mgrkg dissolved in 1 ml 0.9% NaCl i.p. ŽDSP-4 was given within 1 min after reconstitution in solution.; 2. Control: These rats received no pretreatment. One week after group-assignmentrtreatment, the rats were anesthetized with 4%–5% halothane and decapitated. This corresponds to the time point in later experiments at which rats were subjected to forebrain ischemia. Samples Ž2–30 mg. dissected from the neocortex and hippocampus were homogenized by a microsonicator Ž50 W. in 200 ml of 0.2 M perchloric acid ŽPCA. for 5 s. The homogenate was then centrifuged at 10,000 rpm for 1 min. Catecholamines were extracted on aluminum and analyzed by a reverse phase HPLC equipped with a C-18 column and an electrochemical detector. 2.2. Experiment 2 Sprague–Dawley rats were randomized into DSP-4treated and untreated control groups as described in Experiment 1 Ž n s 4 per group.. One week after group-assignmentrtreatment, these rats were fasted Ž12–16 h. but allowed free access to water. The animals were then anesthetized with 4%–5% halothane in oxygen. After orotracheal intubation, the lungs were mechanically ventilated Ž30%–40% O 2rbalance N2 .. The inspired concentration of halothane Ž1%–2%. was then adjusted to maintain mean arterial blood pressure ŽMABP. within 80–120 mmHg. Surgery was performed using an aseptic technique and all wounds were infiltrated with 1% lidocaine. The tail artery
263
was cannulated and used for blood pressure measurement and for arterial blood sampling. Via a neck incision the right jugular vein was cannulated. The common carotid arteries were then exposed and encircled with sutures, leaving the vagus nerves and cervical sympathetic plexus intact. The animals were positioned in a stereotaxic head frame, a midline scalp incision was made, and the skull was exposed. Using a 2-mm trephine drill with continuous saline irrigation, a burr hole was drilled over the left hemisphere above the dorsal hippocampus Žbregma y4.2 mm, lateral 2.0 mm.. The dura was opened and a guide cannula ŽCMAr12 guide cannula; CMArMicrodialysis, Acton, MA. was inserted through the cranial opening and stereotactically positioned with the tip at the brain surface Ž108 from perpendicular.. The guide was fixed in place with a cranial screw, and the burr hole was sealed with orthodontic cement. The wound was closed with sutures and the animal was unmounted from the head frame. Cortical EEG was continuously monitored during the experiment from active subdermal electrodes positioned over the parietal cortex bilaterally and a ground lead positioned in the tail. A 22-gauge needle thermistor ŽModel 524; YSI, Yellow Springs, OH. was subcutaneously placed adjacent to the skull beneath the temporalis muscle and the temperature was servoregulated ŽModel 73ATA Indicating Controller; YSI, Yellow Springs. at 37.5 " 0.18C with surface heating or cooling. A 2.0 mm membrane microdialysis probe ŽCMAr12 microdialysis probe; CMArMicrodialysis. was prepared according to manufacturer’s recommendations. Immediately before each experiment, the probe was connected to a microinfusion pump ŽCMAr102 microdialysis pump; CMArMicrodialysis. and perfused at a flow rate of 2 mlrmin with artificial cerebrospinal fluid ŽACSF; 122 mM NaCl, 3 mM KCl, 1.2 mM MgSO4 , 0.4 mM KH 2 PO4 , 25 mM NaHCO 3 and 1.2 mM CaCl 2 , pH 7.4, filtered through a 0.2-mm filter and degassed before use.. Membrane efficiency was determined prior to each use by dialyzing against a 1-ngrmg norepinephrine standard solution in 0.2 N PCA. Microdialysate norepinephrine and epinephrine concentrations were corrected for membrane efficiency. The probe was then inserted through the guide cannula into the dorsal hippocampus with the tip of the probe positioned at a depth of 4 mm from the brain surface. ACSF was perfused through the probe for 2 h. Two microdialysate samples were then collected prior to ischemia over 15 min intervals. One 10-min sample was collected during ischemia, and five 15-min samples were collected after restoring circulation. Microdialysate samples were collected in tubes containing 0.2 N PCA to prevent catecholamine auto-oxidation. Microdialysate samples were maintained on ice and analyzed within 3 h of collection. Norepinephrine and epinephrine concentrations were measured by HPLC. Microdialysate samples were taken before, during and after ischemia and directly injected Žno extraction. into the chromatographic column.
264
B.M.G. Nellgard ˚ et al.r Brain Research 847 (1999) 262–269
Following surgical preparation, the halothane concentration was decreased to 0.8%. Ventilation was adjusted to produce normocapnia and mild hyperoxemia. Muscle paralysis was provided by a 1-mg i.v. bolus of succinylcholine, repeated when necessary, to allow control of ventilation during ischemia. Heparin Ž50 IU. was given intravenously. Near complete global cerebral ischemia was then induced by exsanguination through the jugular venous catheter to a MABP of 30 mmHg w5x. The carotid arteries were then occluded using temporary aneurysm clips w29x. The onset of ischemia was defined as the time point when the MABP had been stable at 30 mmHg for 30 s. After 8 min, ischemia was terminated by removal of the carotid clips and reinfusion of shed blood. NaHCO 3 Ž0.3 mEq i.v.. was given to minimize systemic acidosis. Animals remained anesthetized an additional 2 h Ž0.8% halothane. with pericranial temperature servoregulated at 37.5 " 0.18C. The animals were then euthanized with a high dose of halothane. Brains were removed and probe locations were verified at necropsy. 2.3. Experiment 3 To investigate the effect of DSP-4 on ischemic histologic outcome, Sprague–Dawley rats were randomized into DSP-4-treated and untreated control groups as described in Experiment 1. Rats were fasted but allowed free access to water for 12–16 h and then anesthetized with halothane and surgically prepared for forebrain ischemia as described in Experiment 2, but microdialysis probes were not implanted. Rats from each group were then subjected to either 7 or 8 min of forebrain ischemia as described in Experiment 2. Blood gases and plasma norepinephrine, epinephrine and glucose were measured 10 min pre-ischemia, 4 min after ischemia onset and 10 min post-ischemia. Blood samples Ž200 ml volume. were collected in pre-cooled tubes containing sodium metabisulfite and EDTA to prevent oxidation of norepinephrine and epinephrine. Plasma catecholamines were then extracted on aluminum and samples were injected into a reverse phase HPLC equipped with a C-18 column and an electrochemical detector. Values for plasma norepinephrine and epinephrine were corrected for extraction efficiency. Ischemia was terminated by removal of the carotid clips and reinfusion of shed blood. NaHCO 3 Ž0.3 mEq i.v.. was given to minimize systemic acidosis. Animals remained anesthetized an additional 2 h Ž0.8% halothane. with pericranial temperature servoregulated at 37.5 " 0.18C. Upon recovery of spontaneous ventilation, the trachea was extubated. After recovery of the righting reflex, the animals were returned to their cages. Four days post-ischemia, the animals were again anesthetized with halothane, intubated and mechanically ventilated followed by intracardiac injection of saline followed by buffered 10% formalin. After 24 h, the brains were
removed and stored in 10% formalin. Paraffin-embedded brain sections were serially cut Ž5 mm thick. and stained with acid fuchsinrcelestine blue. With the investigator blinded to group assignment, injury to the hippocampus, striatum and cortex was measured using light microscopy. All viable and nonviable neurons in the CA1 sector of hippocampus were manually counted and the percentage of dead neurons was calculated at a coronal level approximately 3.8 mm caudal to bregma. Damage to neurons in the hilus of dentate gyrus, or CA4 were examined at the same coronal level and graded on a 1–4 scale, Ž1 s 0%– 25% neurons damaged; 2 s 25%–50% neurons damaged; 3 s 50%–75% neurons damaged and 4 s 75%–100% neurons damaged.. Striatum was examined in a central circular area of 400-mm diameter, at a level 0.3 mm caudal to bregma. Damage was calculated as percent dead neurons per field. Neocortical damage was defined as the number of dead neurons in a circular area with a 400-mm diameter dorsal to the entorhinal fissure at 3.8 mm caudal to bregma. Values from the hemisphere with the worst damage in each animal were used for statistical analyses. Parametric values including histologic injury in the hippocampal CA1, neocortex and caudoputamen and norepinephrinerepinephrine concentrations were analyzed by analysis of variance. When indicated by a significant F ratio, between group differences were defined using post hoc protected least squares difference analysis. Values are reported as mean " S.D. Non-parametric values Že.g., histologic scores. were compared by the Kruskal–Wallis H statistic and are reported as median" interquartile deviation. Significance was assumed when p - 0.05. 3. Results 3.1. Experiment 1 Treatment with DSP-4 reduced tissue norepinephrine concentration in non-ischemic brain by approximately 95% Table 1 Physiologic values for rats exposed to an 8-min forebrain ischemic insult while undergoing hippocampal microdialysis for norepinephrine ŽExperiment 2.. Values are mean"S.D. Control Ž ns 4.
DSP-4 Ž ns 4.
10 min pre-ischemia MABP ŽmmHg. pH a PaCO 2 ŽmmHg. PaO 2 ŽmmHg. Glucose Žmgrdl. Hematocrit Ž%.
100"9 7.38"0.02 41"2 141"7 179"9 43"1
98"16 7.38"0.02 40"1 123"7 230"20 46"3
10 min post-ischemia MABP ŽmmHg. pH a PaCO 2 ŽmmHg. PaO 2 ŽmmHg.
125"19 7.31"0.11 43"7 149"24
123"17 7.29"0.10 45"12 154"31
B.M.G. Nellgard ˚ et al.r Brain Research 847 (1999) 262–269
265
observed in the DSP-4 group Ž P s 0.07.. The difference in intra-ischemic microdialysate norepinephrine concentration between groups was significant Ž P s 0.0005.. No difference between Control and DSP-4 groups could be detected at other measurement intervals. Microdialysate epinephrine was under the limit of detection. 3.3. Experiment 3
Fig. 1. Microdialysate from hippocampal CA1 in animals exposed to 8 min of forebrain ischemia. Extracellular brain norepinephrine concentrations increased during ischemia in control animals Žwhite triangles., but not in DSP-4-treated animals Žfilled circles.. At all other time points, a difference between control and DSP-4 animals could not be detected. Microdialysate epinephrine was under the limit of detection. Valuess mean"S.D. w Difference between groups for intra-ischemic microdialysate norepinephrine concentration Ž P s 0.0005..
in both cortex ŽControl: 297 " 59; DSP-4: 16 " 9 ngrg wet weight, P s- 0.0001. and hippocampus ŽControl: 416 " 167; DSP-4: 30 " 24 ngrg wet weight, P s 0.004.. Epinephrine was under the limit of detection in both groups. 3.2. Experiment 2 Physiologic values are reported in Table 1 for rats undergoing hippocampal microdialysis. Pericranial temperature was held between 37.5 " 0.18C throughout the experimental procedure. Baseline microdialysate norepinephrine concentrations were similar between control and DSP-4treated animals ŽFig. 1.. During ischemia, microdialysate norepinephrine increased from baseline in control animals Ž P s 0.001., while no statistically significant change was
Physiologic values were similar between groups in the histologic outcome studies ŽTable 2.. Pericranial temperature was held between 37.5 " 0.18C throughout the experimental procedure. In animals subjected to 8 min of forebrain ischemia, damage in hippocampal CA1 ŽControls 78 " 22; DSP-4s 73 " 28% dead neurons, P s 0.48.; caudoputamen ŽControl s 63 " 20; DSP-4 s 64 " 21% dead neurons, P s 0.91. and neocortex ŽControls 15 " 4; DSP-4s 14 " 6 necrotic neuronsrhigh power field, P s 0.63. was similar between groups ŽFig. 2.. No necrotic neurons were present in the hippocampal CA3 sector or dentate gyrus in either group. Median" interquartile range histologic scores in hippocampal CA4 were not different between groups ŽControls 3 " 1; DSP-4s 3 " 1, P s 0.66.. No differences were observed between groups for plasma epinephrine or norepinephrine prior to, during, or after ischemia ŽFig. 3.. The change in plasma norepinephrine between pre-ischemic and intra-ischemic measurement intervals was not significant in either the control or DSP-4 groups. In contrast, ischemia caused an increase in plasma epinephrine in both groups Ž P s 0.002.. Similar findings were present for animals exposed to 7 min of ischemia. Although overall damage was numerically decreased relative to the 8-min insult, there was no difference between groups for percent dead neurons in hippocampal CA1% ŽControls 57 " 27%; DSP-4s 57 " 34%, P s 0.99. or caudoputamen ŽControls 38 " 25%; DSP-4s 47 " 23%, P s 0.45.. The number of necrotic cortical neuronsrhigh power field in the Control Ž14 " 5.
Table 2 Physiologic values for rats undergoing 7 or 8 min of forebrain ischemia and analysis of histologic outcome ŽExperiment 3.. All values are mean" S.D. Controlr7 min Ž n s 10.
DSP-4r7 min Ž n s 12.
Controlr8 min Ž n s 22.
DSP-4r8 min Ž n s 19.
10 min pre-ischemia MABP ŽmmHg. pH a PaCO 2 ŽmmHg. PaO 2 ŽmmHg. Glucose Žmgrdl. Hematocrit Ž%.
97 " 14 7.32 " 0.05 38 " 2 153 " 22 134 " 36 38 " 2
97 " 12 7.32 " 0.05 37 " 4 155 " 23 137 " 26 39 " 2
88 " 11 7.36 " 0.03 38 " 2 165 " 53 117 " 18 39 " 2
100 " 15 7.36 " 0.04 38 " 3 178 " 45 140 " 23 39 " 2
10 min post-ischemia MABP ŽmmHg. pH a PaCO 2 ŽmmHg. PaO 2 ŽmmHg. Glucose Žmgrdl. Hematocrit Ž%.
105 " 15 7.33 " 0.05 39 " 3 149 " 24 146 " 43 37 " 2
110 " 17 7.35 " 0.05 38 " 2 154 " 31 137 " 40 39 " 2
114 " 11 7.35 " 0.04 40 " 2 163 " 42 110 " 24 39 " 2
124 " 11 7.35 " 0.05 39 " 2 177 " 26 139 " 44 40 " 2
266
B.M.G. Nellgard ˚ et al.r Brain Research 847 (1999) 262–269
Fig. 3. Plasma norepinephrine and epinephrine concentrations in control animals Žopen triangles. and DSP-4-treated animals Žfilled circles. exposed to 8 min of forebrain ischemia. No differences were present between groups at any interval for either catecholamine. Ischemia did not cause a significant change in plasma norepinephrine in either group, but epinephrine was increased in both groups during ischemia. w Difference between pre-ischemic and intra-ischemic values for both the DSP-4-treated and control groups Ž P s 0.002.. Valuess mean"S.D.
and DSP-4 Ž16 " 5, P s 0.36. groups was similar. No necrotic neurons were present in the hippocampal CA3 sector or dentate gyrus in either group. Median " interquartile range histologic scores in hippocampal CA4 were not different between groups ŽControls 3 " 1; DSP4 s 3 " 1, P s 0.29.. Plasma norepinephrine and epinephrine concentrations were similar to those observed in the 8-min group Ždata not shown..
4. Discussion We have demonstrated in the rat that DSP-4 pretreatment: Ž1. markedly reduces brain norepinephrine concentration; Ž2. inhibits intra-ischemic increases in brain extracellular norepinephrine but does not effect plasma catecholamine concentrations; and Ž3. fails to modulate delayed neuronal necrosis resulting from different durations of normothermic forebrain ischemia in the hippocampus, striatum, and neocortex. DSP-4 was chosen for this study because it selectively reduces brain norepinephrine concentration but exerts negligible influence on dopaminergic or serotonergic neurons w13,16,32x. DSP-4 readily penetrates the blood–brain barrier and selectively degrades noradrenergic terminals originating in the locus coeruleus but leaves cell bodies intact w13x. In contrast to reserpine, the effects of DSP-4 on brain are long lasting, while the extracerebral noradrenergic
Fig. 2. Histologic damage was similar in hippocampal CA1 Ž P s 0.48., caudoputamen Ž P s 0.91., and neocortex Ž P s 0.63. in control Ž ns 22. and DSP-4-treated Ž ns19. animals exposed to 8 min of forebrain ischemia. Open circles depict values for individual animals and bars indicate the group mean. HPF s high power field.
B.M.G. Nellgard ˚ et al.r Brain Research 847 (1999) 262–269
effects are transient and normalized after 1 week w11,13x. The regimen of DSP-4 administration utilized in this study was similar to that used by others w1x and resulted in reduction of neocortical and hippocampal norepinephrine concentrations to less than 10% of normal at the time of ischemia induction. The trial of DSP-4-treated vs. control animals exposed to 8-min ischemia showed severe damage in hippocampal CA1 in both groups. However, given the severe CA1 damage observed after 8-min ischemia, there was little apparent opportunity for DSP-4 to exhibit a worsened outcome should that have been a potential effect of norepinephrine depletion. Accordingly, the study was repeated with 7 min of forebrain ischemia. Although the two studies were not performed concurrently, CA1 damage in the control group was reduced Ž7 min s 57 " 34% dead neurons; 8 min s 78 " 22% dead neurons, P s 0.041.. Despite this, no difference between DSP-4-treated and control animals was seen in any structure examined. The failure of DSP-4 to modulate delayed neuronal necrosis in our study is somewhat surprising. There is reason to believe that brain parenchymal norepinephrine plays a role in the eventual histologicrbehavioral outcome from ischemia. In the gerbil, a reduced number of brain a 1-, a 2-, and b-adrenoceptors has been demonstrated as early as 24 h after unilateral carotid artery occlusion w18x. The magnitude of this effect is both anatomically and receptor subtype specific. Consistent with this specificity, a 1-adrenergic receptor binding is reduced by approximately 50% in forebrain homogenates of post-ischemic gerbils while b 1 and b 2 binding undergoes little or no change w21x. In contrast, DSP-4 treatment causes an approximate 50% upregulation of a 1-, a 2-, and b-adrenoceptors within 7 days of treatment w32x. Therefore, if changes in receptor function are important in ischemic brain, it would be expected that an effect of DSP-4 treatment on histologic outcome would have been evident. Others have examined effects of depletion of central norepinephrine on histologic outcome from cerebral ischemia. Blomqvist et al. w2x lesioned the locus coeruleus ascending fibers by bilateral injections of 6-hydroxydopamine 2–3 weeks prior to a cardiac arrest insult in the rat. Although brain norepinephrine was not measured during the ischemic insult, frontal cortex was sampled from recovery animals at 7 days post-ischemia and a near-total depletion of norepinephrine was documented. In lesioned animals, enhanced neuronal necrosis was observed in the dorsal hippocampal CA1 and in neocortex. Furthermore, Nishino et al. w22x utilized DSP-4-treated Mongolian gerbils in a paradigm of global cerebral ischemia and reported that DSP-4 treatment augmented CA3 and CA4, but not CA1 injury. In contrast, our investigation found no effect of norepinephrine depletion on outcome from global ischemia in any of these structures. The studies by both Blomqvist et al. w2x and Nishino et al. w22x were performed prior to widespread appreciation of
267
the effects of brain temperature and plasma glucose on ischemic outcome w3,26x. Both studies monitored rectal temperature only and both groups examined non-fasted animals. Further, the gerbils studied for histologic outcome by Nishino et al. w22x did not undergo physiologic monitoring and were allowed to breathe spontaneously while anesthetized halothane. The effects of locus coeruleus lesioning and DSP-4-treatment on these well-described physiologic determinants of ischemic outcome are unknown. Presumably, the current study, which incorporated strict physiologic regulation during and after ischemia, was devoid of these potential confounding influences. The absence of effect of central norepinephrine depletion on histologic outcome from forebrain ischemia observed in the current experiment has implications for the interpretation of other studies, which have associated either circulating catecholamine concentrations or centrally-acting noradrenergic compounds with ischemic outcome. It has been suggested that peripheral catecholamines might alter outcome by transiting the blood–brain barrier during the ischemic insult w6,14,31x. However, little change in plasma norepinephrine was observed. In contrast, despite a six- to eight-fold intra-ischemic increase in plasma epinephrine, brain epinephrine remained below the limit of detection, which is consistent with prior work, which demonstrated little or no blood–brain barrier leakage resulting from global ischemia w25x. It has also been suggested that drugs such as idazoxan or dexmedetomidine exert effects on ischemic outcome by either antagonizing or agonizing central a 2-adrenoceptors w6,14,31x. This appears unlikely, because the results of the current experiment indicate that near-total depletion of brain norepinephrine has no effect on ischemic outcome. Alternatively, circulating catecholamines, or drugs such as idazoxan and dexmedetomidine, may indirectly influence ischemic outcome by effects on extracranial neurohumoral events, which then influence neurons and glia. Both idazoxan and dexmedetomidine have potent effects on peripheral adrenergic receptors w19,27x. For example, increases in catecholamines induce release of corticosteroids, which in turn can cross the blood–brain barrier and bind to intracellular receptors w12x. Inhibition of corticosterone synthesis has been shown in various models to improve outcome from ischemicrhypoxic insults w15,30x. Furthermore, adrenergic responses to stress can interact with plasma concentrations of interleukin-6 w23x, interleukin-1b w9x, and tumor necrosis factor w4x — factors having potential to influence outcome from brain ischemic insults w20,28,33x. In conclusion, there is a substantial body of evidence that norepinephrine and drugs, which affect the adrenergic nervous system can alter outcome from cerebral ischemic insults. This study examined whether major changes in brain norepinephrine concentration were sufficient to alter histologic damage resulting from severe forebrain ischemia. Pre-ischemic administration of DSP-4 served to
268
B.M.G. Nellgard ˚ et al.r Brain Research 847 (1999) 262–269
deplete brain norepinephrine by 95% and inhibited increases in brain norepinephrine during ischemia, but had no effect on plasma concentrations of norepinephrine or epinephrine. Ischemic intervals of 7 and 8 min caused substantive delayed neuronal necrosis but damage was similar in DSP-4-treated and control rats. These results suggest that central noradrenergic events are unlikely to explain effects of either exogenous catecholamines or noradrenergic compounds on ischemic outcome. Acknowledgements This study was supported by NIH Grant RO1 GM39771-12. Bengt Nellgard ˚ was supported by The Laerdal Foundation for Acute Medicine and Swedish Society of Physicians. G. Burkhard Mackensen was supported by a post-doctoral stipend through the German Academic Exchange Service ŽDAAD.. The authors are grateful to Ann D. Brinkhous for expert technical assistance. References w1x S.S.A. Al-Zahrani, A.S.A. Al-Ruwaitea, M.Y. Ho, C.M. Bradshaw, E. Szabadi, Destruction of central noradrenergic neurones with DSP4 impairs the acquisition of temporal discrimination but does not affect memory for duration in a delayed conditional discrimination task, Psychopharmacology 130 Ž1997. 166–173. w2x P. Blomqvist, O. Lindvall, T. Wieloch, Lesions of the locus coeruleus system aggravate ischemic damage in the rat brain, Neurosci. Lett. 38 Ž1985. 353–358. w3x R. Busto, W.D. Dietrich, M.Y.T. Globus, I. Valdes, ´ P. Scheinberg, M.D. Ginsberg, Small differences in intra-ischemic brain temperature critically determine the extent of neuronal injury, J. Cereb. Blood Flow Metab. 7 Ž1987. 729–738. w4x G. Fantuzzi, E. Di Santo, S. Sacco, F. Benigni, P. Ghezzi, Role of the hypothalamic–pituitary–adrenal axis in the regulation of TNF production in mice, J. Immunol. 155 Ž1995. 3552–3555. w5x T.X. Gionet, D.S. Warner, M. Verhaegen, J.D. Thomas, M.M. Todd, Effects of intra-ischemic blood pressure on outcome from 2-vessel occlusion forebrain ischemia in the rat, Brain Res. 586 Ž1992. 188–194. w6x M.Y. Globus, R. Busto, W.D. Dietrich, E. Martinez, I. Valdes, M.D. Ginsberg, Direct evidence for acute and massive norepinephrine release in the hippocampus during transient ischemia, J. Cereb. Blood Flow Metab. 9 Ž1989. 892–896. w7x I. Gustafson, Y. Miyauchi, T.W. Wieloch, Post-ischemic administration of idazoxan, an a 2 -adrenergic receptor antagonist, decreases neuronal damage in the rat brain, J. Cereb. Blood Flow Metab. 9 Ž1989. 171–174. w8x I. Gustafson, E. Westerberg, T. Wieloch, Protection against ischemia-induced neuronal damage by the a 2-adrenoceptor antagonist idazoxan: influence of time of administration and possible mechanisms of action, J. Cereb. Blood Flow Metab. 10 Ž1990. 885–894. w9x A.R. Gwosdow, Mechanisms of interleukin-1-induced hormone secretion from the rat adrenal gland, Endocr. Res. 21 Ž1995. 25–37. w10x W.E. Hoffman, E. Kochs, C. Werner, R.F. Albrecht, Dexmedetomidine improves neurologic outcome from incomplete cerebral ischemia in the rat, Anesthesiology 75 Ž1991. 328–332. w11x G. Jaim-Etcheverry, L.M. Zieher, DSP-4: a novel compound with neurotoxic effects on noradrenergic neurons of adult and developing rats, Brain Res. 188 Ž1980. 513–523.
w12x M. Joels, ¨ E.R. de Kloet, Control of neuronal excitability by corticosteroid hormones, Trend Neurosci. 15 Ž1992. 25–30. w13x G. Jonsson, H. Hallman, F. Ponzo, S. Ross, DSP4 Ž N-Ž2-chloroethyl.-N-ethyl-2-bromobenzylamine. — a useful denervation tool for central and peripheral noradrenaline neurons, Eur. J. Pharmacol. 72 Ž1981. 173–188. w14x T. Koide, T. Wieloch, B.K. Siesjo, ¨ Circulating catecholamines modulate ischemic brain damage, J. Cereb. Blood Flow Metab. 6 Ž1986. 559–565. w15x H.J. Krugers, R.H.A. Kemper, J. Korf, G.J. Ter Horst, S. Knooema, Metyrapone reduces rat brain damage and seizures after hypoxia– ischemia: an effect independent of modulation of plasma corticosterone levels?, J. Cereb. Blood Flow Metab. 18 Ž1998. 386–390. w16x K.J. Lookingland, D.S. Chapin, D.W. McKay, K.E. Moore, Comparative effects of the neurotoxins N-chloroethyl-N-ethyl-2-bromobenzylamine hydrochloride ŽDSP4. and 6-hydroxydopamine on hypothalamic noradrenergic, dopaminergic and 5-hydroxytryptaminergic neurons in the male rat, Brain Res. 365 Ž1986. 228–234. w17x C. Maier, G.K. Steinberg, G.H. Sun, G.T. Zhi, M. Maze, Neuroprotection by the a 2-adrenoreceptor agonist dexmedetomidine in a focal model of cerebral ischemia, Anesthesiology 79 Ž1993. 306– 312. w18x T. Mizuki, H. Kobasyashi, S. Ueno, Y. Nakashima, A. Kuroiwa, F. Izumi, Differential changes in a- and b-adrenoceptors in the cerebral cortex and hippocampus of the Mongolian gerbil after unilateral brain ischemia, Stroke 26 Ž1995. 2333–2336. w19x G.J. Molderings, J. Likungu, J. Jakschik, M. Gothert, Presynaptic imidazole receptors and non-adrenoceptor w3 Hx-idazoxan binding sites in human cardiovascular tissues, Br. J. Pharmacol. 1997 Ž1997. 43–50. w20x H. Nawashiro, K. Tasaki, C.A. Ruetzler, J.M. Hallenbeck, TNF-alpha pretreatment induces protective effects against focal cerebral ischemia in mice, J. Cereb. Blood Flow Metab. 17 Ž1997. 483–490. w21x K. Nishino, J.N. Davis, Rapid reduction in w3 Hxprazosin binding to gerbil forebrain membranes during bilateral common carotid artery occlusion, J. Cereb. Blood Flow Metab. 9 Ž1989. 358–363. w22x K. Nishino, C.S. Lin, J.K. Morse, J.N. Davis, DSP4 treatment worsens hippocampal pyramidal cell damage after transient ischemia, Neuroscience 43 Ž1991. 361–367. w23x S. Noisakran, W.P. Halford, L. Veress, D.J.J. Carr, Role of the hypothalamic pituitary adrenal axis and IL-6 in stress-induced reactivation of latent herpes simplex virus type I, J. Immunol. 160 Ž1998. 5441–5447. w24x C. Perego, S. Gatti, G.C. Vetrugno, F. Marzatico, S. Algeri, Correlation between electroencephalogram isoelectric time and hippocampal norepinephrine levels, measured by microdialysis, during ischemia in rats, J. Neurochem. 59 Ž1992. 1257–1262. w25x C.K. Petito, W.A. Pulsinelli, G. Jacobson, F. Plum, Edema and vascular permeability in cerebral ischemia: comparison between ischemic neuronal damage and infarction, J. Neuropathol. Exp. Neurol. 41 Ž1982. 423–436. w26x W. Pulsinelli, S. Waldman, D. Rawlinson, F. Plum, Moderate hyperglycemia augments ischemic brain damage: a neuropathologic study in the rat, Neurology 32 Ž1982. 1239–1246. w27x H. Scheinin, R. Aantaa, M. Anttila, P. Hakola, A. Helminen, S. Karhuvaara, Reversal of the sedative and sympatholytic effects of dexmedetomidine with a specific alpha 2-adrenoceptor antagonist atipamezole: a pharmacodynamic and kinetic study in healthy volunteers, Anesthesiology 89 Ž1998. 574–584. w28x G.P. Schielke, G.Y. Yang, B.D. Shivers, A.L. Betz, Reduced ischemic brain injury in interleukin-1b converting enzyme-deficient mice, J. Cereb. Blood Flow Metab. 18 Ž1998. 180–185. w29x M.-L. Smith, G. Bendek, N. Dahlgren, I. Rosen, T. Wieloch, B.K. Siesjo, ¨ Models for studying long-term recovery following forebrain ischemia in the rat: 2. A 2-vessel occlusion model, Acta Neurol. Scand. 69 Ž1984. 385–401. w30x V.L. Smith-Swintosky, L.C. Pettigrew, R.M. Sapolsky, C. Phares,
B.M.G. Nellgard ˚ et al.r Brain Research 847 (1999) 262–269 S.D. Craddock, S.M. Brooke, M.P. Mattson, Metyrapone, an inhibitor of glucocorticoid production, reduces brain injury induced by focal and global ischemia and seizures, J. Cereb. Blood Flow Metab. 16 Ž1996. 585–598. w31x C. Werner, W.E. Hoffman, C. Thomas, D.J. Miletich, R.F. Albrecht, Ganglionic blockade improves neurologic outcome from incomplete ischemia in rats-partial reversal by exogenous catecholamines, Anesthesiology 73 Ž1990. 923–929.
269
w32x C. Wolfman, V. Abo, D. Calvo, J. Medina, F. Dajas, R. Silveria, Recovery of central noradrenergic neurons one year after the administration of the neurotoxin DSP4, Neurochem. Int. 25 Ž1994. 395– 400. w33x M. Yamada, H. Hatanaka, Interleukin-6 protects cultured rat hippocampal neurons against glutamate-induced cell death, Brain Res. 643 Ž1994. 173–180.