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Peripheral and central administration of neuropeptide Y in a rat middle cerebral artery occlusion stroke model reduces cerebral blood flow and increases infarct volume
Brain Research 927 (2002) 138–143 www.elsevier.com / locate / bres
Research report
Peripheral and central administration of neuropeptide Y in a rat middle cerebral artery occlusion stroke model reduces cerebral blood flow and increases infarct volume Shao-Hua Chen, Raymond T.F. Cheung* University Department of Medicine, Queen Mary Hospital, University of Hong Kong, Pokfulam, Hong Kong Accepted 4 December 2001
Abstract Recent studies have shown increased immunoreactivity for neuropeptide Y (NPY) within the perilesional cortex following experimental middle cerebral artery occlusion (MCAO) or focal excitotoxic damage. Downregulation of the NPY Y1 receptor gene using an antisense oligodeoxynucleotide produced a doubling of the infarct volume, implying that NPY may mediate neuroprotection against focal ischemia. The effects of treatment with NPY on infarct volume and hemodynamic parameters were investigated in the present study. Adult male Sprague–Dawley rats were anesthetized with sodium pentobarbital to undergo right-sided endovascular MCAO for 2 h. A single dose of NPY was given via intracarotid injection (10 mg / kg) at the beginning of reperfusion, intracisternal injection (10 or 30 mg / kg) at 30 min of ischemia, or intracerebroventricular (i.c.v.) injection (10 or 70 mg / kg) at 30 min of ischemia. Control groups received the vehicle only via the same route. Body temperature was maintained constant, and hemodynamic parameters were monitored during anesthesia. Laser Doppler flowmetry was used to monitor the regional cerebral blood flow (rCBF) during ischemia and reperfusion in some rats. The rats were decapitated on day 3, and their brains were cut into 2-mm thick coronal slices before reaction with a 2% solution of 2,3,5-triphenyltetrazolium chloride to reveal the infarct. Compared to the respective control groups, NPY treatment via any method of administration increased the relative infarct volume. Suppression of rCBF was observed during reperfusion. These results indicate that peripheral or central administration of NPY impairs reperfusion following experimental MCAO and worsens the outcome of focal cerebral ischemia. 2002 Elsevier Science B.V. All rights reserved. Theme: Disorders of the nervous system Topic: Ischemia Keywords: Cerebral blood flow; Cerebral ischemia; Infarct volume; Hemodynamics; Middle cerebral artery occlusion; Neuropeptide Y
1. Introduction Neuropeptide Y (NPY), a member of the pancreatic polypeptide family of 36-amino-acid peptides, is abundant throughout the peripheral and central nervous systems [4]. NPY has effects on the cardiovascular, gastrointestinal and hypophyseal–adrenocortical systems; it also affects feeding, anxiety, circadian rhythms, reproduction and thermoregulation via G-protein coupled receptors [12]. NPY has been implicated in the pathogenesis of many human
*Corresponding author. Tel.: 1852-28-553-315; fax: 1852-29-741171. E-mail address: [email protected] (R.T.F. Cheung).
diseases, including eating disorders, Huntington’s disease, Alzheimer’s disease, and Parkinson’s disease [15,16,26,31]. Four types of receptors, the Y1, Y2, Y4 and Y5, are expressed in the rat brain, the y6 receptor is inactive in primates and absent in rats, and the putative Y3 receptor has not been cloned. In the rat central nervous system, the Y1 and Y2 receptors predominate over Y4 and Y5 receptors [5,13,22]. Recent studies in the rat have shown that the immunoreactivity for NPY was increased locally within the cerebral cortex around the site of infarct following experimental middle cerebral artery occlusion (MCAO) [2,7,9] and around an excitotoxic damage [8]. In addition, a reduction of the cortical Y1 receptor density by 29% using a well characterized antisense oligodeoxynucleotide [30] prior to
0006-8993 / 02 / $ – see front matter 2002 Elsevier Science B.V. All rights reserved. PII: S0006-8993( 01 )03336-4
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experimental MCAO resulted in doubling of the infarct volume [11]. Taken together, these results were thought to imply a neuroprotective role of NPY in focal cerebral ischemia via potentiation of the neuroprotective effects of norepinephrine, selective inhibition of calcium currents, and / or inhibition of neuronal release of glutamate [11]. Intracerebral or intracarotid injection of exogenous NPY produces profound and prolonged reduction in cerebral perfusion [3,29] that, in turn, may be counter-protective in cerebral ischemia. The present study was conducted to confirm or refute the protective effect of peripherally or centrally administered NPY on infarct volume following MCAO for 2 h and reperfusion for 70 h in the rat. We also examined the hemodynamic effects of exogenous NPY during MCAO and the initial phase of reperfusion. It was thought that the intracerebroventricular (i.c.v.) route would produce the least whereas the intracarotid route the largest reduction in cerebral perfusion.
2. Materials and methods
2.1. Animals Normally fed adult male Sprague–Dawley rats, weighing between 280 and 360 g, were obtained from the Laboratory Animal Unit, the University of Hong Kong. Following transfer to our laboratory, which is adjacent to the Unit, they were maintained under diurnal lighting conditions for about 4 days before experimentation with free access to food and water. All surgical procedures were conducted according to the institutional guidelines and were approved by the University Committee on the Use of Live Animals in Teaching and Research, the University of Hong Kong.
2.2. Drugs Human / rat NPY (cat. no. 7180, Peninsula Lab., San Carlos, CA, USA) was dissolved in normal saline containing 5% of dimethylsulfoxide (DMSO, Lot 108H3442, Sigma, St. Louis, MO, USA) to give the following concentrations: 0.1 mg / ml for the 10 mg / kg groups; 0.2 mg / ml for the 30 mg / kg group; and 0.5 mg / ml for the 70 mg / kg group. Thus, the volume of microinjection varied from 30 to 50 ml. This range of doses accords with previous studies on the hemodynamic effects of peripheral or central administration of NPY [17–19,23,25,28]. A 2% 2,3,5-triphenyltetrazolium chloride (TTC; cat. no. T8877, Sigma) solution was prepared in phosphate buffered saline containing 0.2 M Na 2 HPO 4 and 0.2 M NaH 2 PO 4 (pH 7.4–7.6) [20]. Sodium pentobarbital was obtained from Rhone Merieux (Pinkenba, Queensland, Australia).
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2.3. Experimental groups Peripheral administration was achieved by slow intracarotid injection of a single dose of NPY at 10 mg / kg at about 1 min after onset of reperfusion. Central administration was achieved by either slow intracisternal injection of a single dose of NPY at 10 or 30 mg / kg or slow i.c.v. injection of a single dose of NPY at 10 or 70 mg / kg, both at 30 min of onset of MCAO. In the control groups, the vehicle alone (30 ml) was given via the same routes.
2.4. General surgical procedures The rats were anesthetized with an intraperitoneal injection of sodium pentobarbital at a dose of 60 mg / kg. Additional doses at 20 mg / kg were administered as needed to ensure stable anesthesia. Rectal temperature was maintained between 36.5 and 37.5 8C throughout the experiment by using a rectal thermostat probe and a thermostatically regulated heating pad underneath the animal (FHC, Bowdoinham, ME, USA). The right femoral artery was cannulated with PE-50 polyethylene tubing for monitoring of arterial blood pressure (BP) and heart rate (HR) (Powerlab / 16 Data Acquisition System, AD Instruments, Mountain View, CA, USA). The BP and HR were monitored continuously during MCAO until the first 30 min of reperfusion in all experimental groups except in the groups given intracarotid injection in which the monitoring was continued for the first 2 h of reperfusion.
2.5. Monitoring of regional cerebral blood flow A laser Doppler flowmeter (MBF3D, Moor instruments, Devon, UK) became available during the later stage of the present study. It was used to monitor the regional cerebral blood flow (rCBF). Using a stereotaxic device (SR-6N, Narishige Scientific Instrument, Tokyo, Japan) and a low speed dental drill, a burr hole of 2 mm in diameter was made over the skull at 1 mm posterior and 5 mm lateral to the bregma on the right side. A needle shaped laser probe was placed on the dura away from visible cerebral vessels. Steady state baseline values were recorded before MCAO so that all rCBF data were expressed as percentages of the respective basal values. The rCBF was monitored continuously during focal ischemia until the first 30 min of reperfusion in all experimental groups except in the groups given intracarotid injection in which monitoring of rCBF was continued for the first 2 h of reperfusion.
2.6. Middle cerebral artery occlusion Focal cerebral ischemia was achieved by right-sided endovascular MCAO [21]. Briefly, the right carotid arteries were exposed through a midline cervical incision. The right external carotid artery (ECA) was dissected free and isolated distally by coagulating its branches (Bipolar
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Electric Coagulation, GN60, Aesculap, Tuttlingen, Germany) and placing a distal ligation prior to transection. A piece of 4-o-monofilament nylon suture (Ethicon, JohnsonJohnson, Brussels, Belgium), with its tip rounded by gentle heating, was introduced into the lumen of right ECA stump and gently advanced via the right internal carotid artery (ICA) to embed into the right anterior cerebral artery (ACA) so that the right middle cerebral artery was occluded at its origin. After 2 h of ischemia, the intraluminal suture was withdrawn from the right ACA and right ICA to permit reperfusion. When the laser Doppler flowmeter became available, achievement of focal ischemia and reperfusion was confirmed by the appropriate changes in the rCBF. Wounds were closed using surgical clips. The rats were allowed to recover from the anesthesia before returning to the cages with free access to food and water.
cut into coronal slices of 2 mm in thickness. These slices were reacted with a 2% solution of TTC for 20–30 min to reveal the ischemic infarction. After TTC reaction, the brain slices were fixed with 10% formalin (pH 7.4). TTC reacts with intact mitochondrial respiratory enzymes to generate a bright red color to contrast with the pale color of the infarct. The hemispheric volumes and volume of infarction between the bregma levels of 14 mm (anterior) and 26 mm (posterior) were integrated from the respective calibrated area measurements [10] that were obtained from the digitized images of the TTC-stained brain slices, using a computer-assisted image analysis system ( SIGMASCAN PRO 4.0, SPSS Chicago, IL, USA). The infarct volume was expressed as a percentage of the ipsilateral hemispheric volume.
2.9. Data analysis 2.7. Microinjection In the groups receiving slow intracarotid injection, the right ICA was connected to a Hamilton microsyringe (80630, Hamilton, Reno, NV, USA) via a short PE-50 tubing upon withdrawal of the monofilament suture to permit reperfusion. A single dose of NPY (10 mg / kg) or the vehicle alone was administered over 20 s via the right ICA. At 5 min after the injection, the connection to the right ICA was removed and the ECA stump closed by a surgical suture. In the groups receiving intracisternal injection, a mid-sagittal incision was made to expose the rhombic muscles of the neck after shaving the occipital hairs. A 23-G needle attached to a Hamilton microsyringe via a short PE-50 tubing was inserted into the cisterna magna. Aspiration of a few microliters of cerebrospinal fluid (CSF) into the Hamilton microsyringe verified proper positioning of the needle tip. NPY (10 or 30 mg / kg) or the vehicle alone was injected over 20 s at 30 min after onset of MCAO. The needle was kept in the same position for 5 min before removal. In the groups receiving slow i.c.v. injection, a second burr hole of 1 mm in diameter was made in the skull at 0.5 mm posterior and 1.5 mm lateral to the bregma on the right side. A 23-G needle attached to a Hamilton microsyringe via a short PE-50 tubing was inserted stereotaxically through this second burr hole so that its tip was at 3.4 mm beneath the dural surface. Proper needle placement was verified via withdrawing a few microliters of clear CSF into the Hamilton microsyringe. NPY (10 or 70 mg / kg) or the vehicle alone was administered over 20 s at 30 min after onset of MCAO. The needle was kept in the same position for 5 min before removal, and the burr hole was closed using bone wax.
2.8. Measurement of infarct volume Three days after the MCAO, the rats were deeply re-anesthetized with sodium pentobarbital at a dose of 100 mg / kg. The rats were decapitated, and their brains were
Data are expressed in mean6S.E.M. The mean BP and HR data at different time points of the same experimental group were analyzed using one-way analysis of variance (ANOVA) to reveal any significant change over time with Dunnett post-test to detect any significant difference from the initial values obtained immediately before MCAO. In the groups receiving the intracarotid injection, differences between groups in mean BP, HR, rCBF, and infarct volume were determined using Student’s t-test. In the groups receiving the intracisternal or i.c.v. injection, differences among groups in mean BP, HR, rCBF, and infarct volume were tested using one-way ANOVA with Student–Newman–Keuls post-test. In addition, one sample t-test was applied to the data on rCBF at different time points to detect significant difference from 100% (i.e. a significant change from baseline). A two-tailed P value of 0.05 or less was taken to indicate statistical significance.
3. Results
3.1. Systemic hemodynamic parameters There was no significant change in the mean BP and HR within the same treatment group at different time points when compared to the initial values immediately before MCAO. In addition, there was no significant difference at various time points among different groups of rats received injection of NPY or vehicle via the same route (data not shown).
3.2. Relative regional cerebral blood flow The relative rCBF decreased significantly to less than 30% during MCAO on the same side. Abrupt increase in relative rCBF was seen at the onset of reperfusion (Table 1). Following intracarotid injection of NPY (10 mg / kg) at the commencement of reperfusion, there was a significant
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Table 1 Data on relative rCBF (in per cent) at the following time points in relation to the 2-h middle cerebral artery occlusion (MCAO) and the intracisternal (IC) or intracerebroventricular (i.c.v.) injection in the rat Group (number of rats)
At 5 min of MCAO
At 30 min of MCAO (just before injection)
At 5 min of injection (at 35 min of MCAO)
At 120 min of MCAO
At 5 min after reperfusion
At 30 min after reperfusion
IC NPY at 30 mg / kg (5) IC NPY at 10 mg / kg (5) IC Vehicle only (5)
25.162.3 [,0.0001] 25.363.7 [,0.0001] 23.364.6 [,0.0001]
22.662.7 [,0.0001] 24.964.1 [,0.0001] 23.064.8 [,0.0001]
23.764.4 [,0.0001] 25.461.7 [,0.0001] 24.565.2 [0.0001]
25.663.9 [,0.0001] 26.062.4 [,0.0001] 24.063.1 [,0.0001]
107.3615.0 [0.6678] 94.963.9 [0.2567] 98.966.9 [0.8748]
71.866.2 [0.0102] 90.068.3 [0.2942] 93.665.0 [0.2717]
I.c.v. NPY at 70 mg / kg (8) I.c.v. NPY at 10 mg / kg (8) I.c.v. Vehicle only (7)
23.161.6 [,0.0001] 26.362.5 [,0.0001] 25.161.7 [,0.0001]
20.862.9 [,0.0001] 22.263.0 [,0.0001] 25.762.1 [,0.0001]
16.463.2* [,0.0001] 25.062.5 [,0.0001] 26.162.1 [,0.0001]
16.563.0 [,0.0001] 24.363.1 [,0.0001] 22.862.3 [,0.0001]
124.1625.3 [0.3731] 86.2611.5 [0.2713] 118.8617.5 [0.3252]
62.263.3* [,0.0001] 67.268.7* [0.0069] 107.7610.7 [0.5020]
P values from one sample t-test on the difference from 100% are in the square brackets. *, Significant difference at the same time points among the groups received i.c.v. injection using one-way ANOVA with Student–Newman–Keuls post-test showing significant difference between the i.c.v. vehicle-treated group and the i.c.v. NPY-treated group.
reduction in the relative rCBF to less than 50% beginning at 5 min after injection and persisting throughout the 2-h period of monitoring (data not shown). Intracarotid injection of the vehicle at the commencement of reperfusion did not significantly affect the relative rCBF (data not shown). The difference in the relative rCBF between the two intracarotid groups was significant at all the time points following the injection. Following intracisternal injection of NPY or the vehicle, there was no further drop in the already reduced rCBF during MCAO (Table 1). Intracisternal injection of NPY at 30 but not 10 mg / kg reduced the relative rCBF by about 30% at 30 min after reperfusion, although there was no significant difference among the intracisternal groups at the same time points (Table 1). I.c.v. injection of NPY at 70 but not 10 mg / kg further reduced the relative rCBF even during MCAO; the difference in rCBF among all three i.c.v. groups was significant at 5 min after injection (or at 35 min of MCAO; Table 1). An abrupt increase in relative rCBF was seen at the onset of reperfusion in all three i.c.v. groups, but i.c.v. injection of NPY at either dose significantly reduced the relative rCBF by about 35% at 30 min after reperfusion (Table 1).
3.3. Site and volume of infarct On day 3 of MCAO, the right-sided cerebral infarct was evident as the whitish region after reaction with TTC. In the vehicle-treated control group, the infarct was largest between the brain regions at 2 mm anterior to the bregma and at 2 mm posterior to the bregma. The infarct consistently involved the caudate putamen, external capsule and insular cortex with variable involvement of the overlying primary and secondary somatosensory cortices. In the NPY-treated groups, the infarct was larger with more
extensive involvement of the subcortical structures and the overlying cortices. Computer-assisted image analysis revealed that the relative infarct volumes varied from 15.763.6 to 22.862.3% (Fig. 1) in the vehicle-treated groups, but the difference among the vehicle-treated groups was not significant (one-way ANOVA). When compared to the vehicle-treated group, intracarotid injection of NPY at 10 mg / kg significantly increased the relative infarct volume by about 80% (Fig. 1A). Intracisternal injection of NPY also significantly increased the relative infarct volume by about 50%, and there was no difference between the two doses (Fig. 1B). I.c.v. injection of NPY at 10 mg / kg significantly increased the relative infarct volume by 110% (Fig. 1C).
4. Discussion
4.1. Technical considerations A standard method of inducing focal cerebral ischemia was adopted in the present study. As the study was started with rats in the intracarotid and intracisternal groups when the laser Doppler flowmeter was not available, data on relative rCBF were obtained from some rats of these groups.
4.2. Hemodynamic effects of NPY The present study documents a lack of significant effect of intracarotid, intracisternal or i.c.v. injection of NPY at doses between 10 and 70 mg / kg on BP and HR under the present experimental conditions. Previous studies have reported contradictory results on the effects of central [17,18,23,25,28] or peripheral injection of NPY [19,23].
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NPY [19,23]. Regarding the effects of NPY on HR, most studies reported a decrease or no change [19,23,25], but increased HR has been reported [18]. Use of different animal species, varying doses of NPY, different routes of administration, use of conscious or anesthetized animals, different anesthetic drugs, varying physiological states, and / or under different pathophysiological situations may explain these contradictory observations. Delivery of NPY via any one of the three routes produces significant reduction of rCBF during reperfusion, and the reduction is most dramatic with intracarotid injection. The vasoconstricting effect of NPY is probably masked during MCAO when rCBF is low. Nevertheless, a large dose of NPY may further reduce rCBF during ischemia, as illustrated by the rCBF results of i.c.v. injection of NPY at 70 mg / kg. These results are expected because NPY is a potent vasoconstrictor of the cerebral arteries in most animal species [1,3,14,27]. Allen et al. first demonstrated the presence of NPY in the human circle of Willis and showed that intracarotid administration of 50 pmol to 2 nmol of NPY in the rat reduced mean cortical cerebral blood flow by 40–98% for at least 2 h [3]. Between 20 and 55 min after intracerebral microinjection of NPY at 2–200 pmol into the striatum of conscious rats, the rCBF of the ipsilateral entorhinal cortex, amygdala, and caudate nucleus was found to reduce by 30–45%, but the regional glucose utilization was unaffected [29]. Thus, NPY has been implicated to play an important role in cerebrovascular regulation under physiological and pathological conditions.
4.3. Worsening of infarction following injection of NPY
Fig. 1. Bar graphs showing the infarct volume (mean6S.E.M.) between the bregma level 14 and the bregma level 26 expressed as a percentage of the corresponding right hemispheric volume following right-sided middle cerebral artery occlusion for 2 h and intracarotid injection of NPY at 10 mg / kg or of the vehicle alone in (A), intracisternal injection of NPY at 10 or 30 mg / kg or of the vehicle alone in (B), or intracerebroventricular injection of NPY at 10 or 70 mg / kg or of the vehicle alone in (C). *, Significant difference between the NPY-treated group and the respective vehicle-treated control group with P,0.05 using Student’s t test or one-way analysis of variance and the Student–Newman–Keuls post-test.
Some studies reported a decreased BP with central administration of NPY [17,25], whereas others reported no change [23,25] or an increase [18]. In contrast, a sustained increase in BP is seen after peripheral administration of
Peripheral or central administration of NPY at the doses of the present study exacerbates rather than ameliorates ischemic damage during experimental MCAO. Nevertheless, a 29% reduction in the density of NPY Y1 receptors using a well characterized antisense oligodeoxynucleotide was found to exacerbate ischemic infarction [11]. One possible explanation is that hemodynamic parameters were not recorded in the previous study [11]. The method of MCAO using craniectomy and direct coagulation may be associated with significant hemodynamic disturbances from augmented sympathetic discharges [6]. The antisense oligodeoxynucleotide will suppress the Y1 receptor density throughout the body and, in turn, may affect the systemic and / or cerebral vasomotor tone as well as cerebral perfusion. Another plausible explanation is that downregulation of NPY Y1 receptors due to antisense treatment may lead to a compensatory increase in the endogenous secretion of NPY (see discussion in [24]). When the local concentration of NPY is further increased during focal cerebral ischemia [7], the observed worsening of infarction volume could have been caused by excessive increase in the concentration of free NPY rather than the reduction in NPY Y1 receptor density.
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5. Conclusions In conclusion, peripheral or central administration of NPY at doses between 10 and 70 mg / kg does not significantly affect BP and HR during MCAO. Cerebral perfusion is adversely affected, resulting in a larger cerebral infarct. The present results do not support any neuroprotective effect of NPY during focal ischemia. Further studies with specific receptor agonists or antagonists are useful to clarify the role of NPY and its receptor types in cerebral ischemia.
Acknowledgements This study was supported by a grant (10202658) from the Committee of Research and Conference Grants, The University of Hong Kong, Hong Kong.
References [1] P.W. Abel, C. Han, Effects of neuropeptide Y on contraction, relaxation, and membrane potential of rabbit cerebral arteries, J. Cardiovasc. Pharmacol. 13 (1989) 52–63. [2] G.V. Allen, R.T. Cheung, D.F. Cechetto, Neurochemical changes following occlusion of the middle cerebral artery in rats, Neuroscience 68 (1995) 1037–1050. [3] J.M. Allen, F. Schon, N. Todd, J.C. Yeats, H.A. Crockard, S.R. Bloom, Presence of neuropeptide Y in human circle of Willis and its possible role in cerebral vasospasm, Lancet 2 (1984) 550–552. [4] Y.S. Allen, T.E. Adrian, J.M. Allen, K. Tatemoto, T.J. Crow, S.R. Bloom, J.M. Polak, Neuropeptide Y distribution in the rat brain, Science 221 (1983) 877–879. [5] A.G. Blomqvist, H. Herzog, Y-Receptor subtypes—how many more?, Trends Neurosci. 20 (1997) 294–298. [6] D.F. Cechetto, J.X. Wilson, K.E. Smith, D. Wolski, M.D. Silver, V.C. Hachinski, Autonomic and myocardial changes in middle cerebral artery occlusion: stroke models in the rat, Brain Res. 502 (1989) 296–305. [7] R.T. Cheung, T. Diab, D.F. Cechetto, Time-course of neuropeptide changes in periischemic zone and amygdala following focal ischemia in rats, J. Comp. Neurol. 360 (1995) 101–120. [8] R.T. Cheung, D.F. Cechetto, Neuropeptide changes following excitotoxic lesion of the insular cortex in rats, J. Comp. Neurol. 362 (1995) 535–550. [9] R.T. Cheung, D.F. Cechetto, Colchicine affects cortical and amygdalar neurochemical changes differentially after middle cerebral artery occlusion in rats, J. Comp. Neurol. 387 (1997) 27–41. [10] R.T. Cheung, V.C. Hachinski, D.F. Cechetto, Cardiovascular response to stress after middle cerebral artery occlusion in rats, Brain Res. 747 (1997) 181–188. [11] R.T. Cheung, D.F. Cechetto, Neuropeptide Y-Y1 receptor antisense oligodeoxynucleotide increases the infarct volume after middle cerebral artery occlusion in rats, Neuroscience 98 (2000) 771–777. [12] W.F. Colmers, C. Wahlestedt (Eds.), The Biology of Neuropeptide Y and Related Peptides, Humana Press, New Jersey, 1993. [13] Y. Dumont, J.C. Martel, A. Fournier, S. St-Pierre, R. Quirion, Neuropeptide Y and neuropeptide Y receptor subtypes in brain and peripheral tissues, Prog. Neurobiol. 38 (1992) 125–167.
143
[14] L. Edvinsson, P. Emson, J. McCulloch, K. Tatemoto, R. Uddman, Neuropeptide Y: immunocytochemical localization to and effect upon feline pial arteries and veins in vitro and in situ, Acta Physiol. Scand. 122 (1984) 155–163. [15] C.F. Elias, C. Aschkenasi, C. Lee, J. Kelly, R.S. Ahima, C. Bjorbaek, J.S. Flier, C.B. Saper, J.K. Elmquist, Leptin differentially regulates NPY and POMC neurons projecting to the lateral hypothalamic area, Neuron 23 (1999) 775–786. [16] G. Figueredo-Cardenas, C.L. Harris, K.D. Anderson, A. Reiner, Relative resistance of striatal neurons containing calbindin or parvalbumin to quinolinic acid-mediated excitotoxicity compared to other striatal neuron types, Exp. Neurol. 149 (1998) 356–372. [17] R. Grimaldi, I. Zini, G. Biagini, G. Toffano, L.F. Agnati, Effects of centrally administered clonidine and neuropeptide Y on arterial blood pressure in the rat after transient forebrain ischemia, J. Auton. Nerv. Syst. 30 (1990) S67–S69. [18] Y. Hu, J.C. Dunbar, Intracerebroventricular administration of NPY increases sympathetic tone selectively in vascular beds, Brain Res. Bull. 44 (1997) 97–103. [19] Y. Hu, J.C. Dunbar, Regional vascular and cardiac responses to systemic neuropeptide-Y in normal and diabetic rats, Peptides 18 (1997) 809–815. [20] K. Isayama, L.H. Pitts, M.C. Nishimura, Evaluation of 2,3,5-triphenyltetrazolium chloride staining to delineate rat brain infarcts, Stroke 22 (1991) 1394–1398. [21] E.Z. Longa, P.R. Weinstein, S. Carlson, R. Cummin, Reversible middle cerebral artery occlusion without craniectomy in rats, Stroke 20 (1989) 84–91. [22] M.C. Michel, A. Beck-Sickinger, H. Cox, H.N. Doods, H. Herzog, D. Larhammar, R. Quirion, T. Schwartz, T. Westfall, XVI. International Union of Pharmacology recommendations for the nomenclature of neuropeptide Y, peptide YY, and pancreatic polypeptide receptors, Pharmacol. Rev. 50 (1998) 143–150. [23] M.A. Petty, R. Dietrich, R.E. Lang, The cardiovascular effects of neuropeptide Y (NPY), Clin. Exp. Hypertens. 6 (1984) 1889–1892. [24] A.O. Schaffhauser, S. Whitebread, R. Haener, K.G. Hofbauer, A. Stricker-Krongrad, Neuropeptide Y Y1 receptor antisense oligodeoxynucleotides enhance food intake in energy-deprived rats, Reg. Pept. 75–76 (1988) 417–423. [25] N.A. Scott, V. Webb, J.H. Boublik, J. Rivier, M.R. Brown, The cardiovascular actions of centrally administered neuropeptide Y, Reg. Pept. 25 (1989) 247–258. [26] S.L. Stoddard, G.M. Tyce, J.E. Ahlskog, A.R. Zinsmeister, D.K. Nelson, S.W. Carmichael, Decreased levels of [Met] enkephalin, neuropeptide Y, substance P, and vasoactive intestinal peptide in parkinsonian adrenal medulla, Exp. Neurol. 114 (1991) 23–27. [27] Y. Suzuki, M. Shibuya, I. Ikegaki, S. Satoh, M. Takayasu, T. Asano, Effects of neuropeptide Y on canine cerebral circulation, Eur. J. Pharmacol. 146 (1988) 271–277. [28] Y. Suzuki, S. Satoh, I. Ikegaki, T. Okada, M. Shibuya, K. Sugita, T. Asano, Effects of neuropeptide Y and calcitonin gene-related peptide on local cerebral blood flow in rat striatum, J. Cereb. Blood Flow Metab. 9 (1989) 268–270. [29] U.I. Tuor, P.A. Kelly, L. Edvinsson, J. McCulloch, Neuropeptide Y and the cerebral circulation, J. Cereb. Blood Flow Metab. 10 (1990) 591–601. [30] C. Wahlestedt, E.M. Pich, G.F. Koob, F. Yee, M. Heilig, Modulation of anxiety and neuropeptide Y–Y1 receptors by antisense oligodeoxynucleotides, Science 259 (1993) 528–531. [31] G. Weng, D. Feinstein, D. Reis, C. Wahlestedt, Neuropeptide Y receptor gene regulation in mouse adrenocortical Y-1 cells, Reg. Pept. 63 (1996) 53–56.
Research report
Peripheral and central administration of neuropeptide Y in a rat middle cerebral artery occlusion stroke model reduces cerebral blood flow and increases infarct volume Shao-Hua Chen, Raymond T.F. Cheung* University Department of Medicine, Queen Mary Hospital, University of Hong Kong, Pokfulam, Hong Kong Accepted 4 December 2001
Abstract Recent studies have shown increased immunoreactivity for neuropeptide Y (NPY) within the perilesional cortex following experimental middle cerebral artery occlusion (MCAO) or focal excitotoxic damage. Downregulation of the NPY Y1 receptor gene using an antisense oligodeoxynucleotide produced a doubling of the infarct volume, implying that NPY may mediate neuroprotection against focal ischemia. The effects of treatment with NPY on infarct volume and hemodynamic parameters were investigated in the present study. Adult male Sprague–Dawley rats were anesthetized with sodium pentobarbital to undergo right-sided endovascular MCAO for 2 h. A single dose of NPY was given via intracarotid injection (10 mg / kg) at the beginning of reperfusion, intracisternal injection (10 or 30 mg / kg) at 30 min of ischemia, or intracerebroventricular (i.c.v.) injection (10 or 70 mg / kg) at 30 min of ischemia. Control groups received the vehicle only via the same route. Body temperature was maintained constant, and hemodynamic parameters were monitored during anesthesia. Laser Doppler flowmetry was used to monitor the regional cerebral blood flow (rCBF) during ischemia and reperfusion in some rats. The rats were decapitated on day 3, and their brains were cut into 2-mm thick coronal slices before reaction with a 2% solution of 2,3,5-triphenyltetrazolium chloride to reveal the infarct. Compared to the respective control groups, NPY treatment via any method of administration increased the relative infarct volume. Suppression of rCBF was observed during reperfusion. These results indicate that peripheral or central administration of NPY impairs reperfusion following experimental MCAO and worsens the outcome of focal cerebral ischemia. 2002 Elsevier Science B.V. All rights reserved. Theme: Disorders of the nervous system Topic: Ischemia Keywords: Cerebral blood flow; Cerebral ischemia; Infarct volume; Hemodynamics; Middle cerebral artery occlusion; Neuropeptide Y
1. Introduction Neuropeptide Y (NPY), a member of the pancreatic polypeptide family of 36-amino-acid peptides, is abundant throughout the peripheral and central nervous systems [4]. NPY has effects on the cardiovascular, gastrointestinal and hypophyseal–adrenocortical systems; it also affects feeding, anxiety, circadian rhythms, reproduction and thermoregulation via G-protein coupled receptors [12]. NPY has been implicated in the pathogenesis of many human
*Corresponding author. Tel.: 1852-28-553-315; fax: 1852-29-741171. E-mail address: [email protected] (R.T.F. Cheung).
diseases, including eating disorders, Huntington’s disease, Alzheimer’s disease, and Parkinson’s disease [15,16,26,31]. Four types of receptors, the Y1, Y2, Y4 and Y5, are expressed in the rat brain, the y6 receptor is inactive in primates and absent in rats, and the putative Y3 receptor has not been cloned. In the rat central nervous system, the Y1 and Y2 receptors predominate over Y4 and Y5 receptors [5,13,22]. Recent studies in the rat have shown that the immunoreactivity for NPY was increased locally within the cerebral cortex around the site of infarct following experimental middle cerebral artery occlusion (MCAO) [2,7,9] and around an excitotoxic damage [8]. In addition, a reduction of the cortical Y1 receptor density by 29% using a well characterized antisense oligodeoxynucleotide [30] prior to
0006-8993 / 02 / $ – see front matter 2002 Elsevier Science B.V. All rights reserved. PII: S0006-8993( 01 )03336-4
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experimental MCAO resulted in doubling of the infarct volume [11]. Taken together, these results were thought to imply a neuroprotective role of NPY in focal cerebral ischemia via potentiation of the neuroprotective effects of norepinephrine, selective inhibition of calcium currents, and / or inhibition of neuronal release of glutamate [11]. Intracerebral or intracarotid injection of exogenous NPY produces profound and prolonged reduction in cerebral perfusion [3,29] that, in turn, may be counter-protective in cerebral ischemia. The present study was conducted to confirm or refute the protective effect of peripherally or centrally administered NPY on infarct volume following MCAO for 2 h and reperfusion for 70 h in the rat. We also examined the hemodynamic effects of exogenous NPY during MCAO and the initial phase of reperfusion. It was thought that the intracerebroventricular (i.c.v.) route would produce the least whereas the intracarotid route the largest reduction in cerebral perfusion.
2. Materials and methods
2.1. Animals Normally fed adult male Sprague–Dawley rats, weighing between 280 and 360 g, were obtained from the Laboratory Animal Unit, the University of Hong Kong. Following transfer to our laboratory, which is adjacent to the Unit, they were maintained under diurnal lighting conditions for about 4 days before experimentation with free access to food and water. All surgical procedures were conducted according to the institutional guidelines and were approved by the University Committee on the Use of Live Animals in Teaching and Research, the University of Hong Kong.
2.2. Drugs Human / rat NPY (cat. no. 7180, Peninsula Lab., San Carlos, CA, USA) was dissolved in normal saline containing 5% of dimethylsulfoxide (DMSO, Lot 108H3442, Sigma, St. Louis, MO, USA) to give the following concentrations: 0.1 mg / ml for the 10 mg / kg groups; 0.2 mg / ml for the 30 mg / kg group; and 0.5 mg / ml for the 70 mg / kg group. Thus, the volume of microinjection varied from 30 to 50 ml. This range of doses accords with previous studies on the hemodynamic effects of peripheral or central administration of NPY [17–19,23,25,28]. A 2% 2,3,5-triphenyltetrazolium chloride (TTC; cat. no. T8877, Sigma) solution was prepared in phosphate buffered saline containing 0.2 M Na 2 HPO 4 and 0.2 M NaH 2 PO 4 (pH 7.4–7.6) [20]. Sodium pentobarbital was obtained from Rhone Merieux (Pinkenba, Queensland, Australia).
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2.3. Experimental groups Peripheral administration was achieved by slow intracarotid injection of a single dose of NPY at 10 mg / kg at about 1 min after onset of reperfusion. Central administration was achieved by either slow intracisternal injection of a single dose of NPY at 10 or 30 mg / kg or slow i.c.v. injection of a single dose of NPY at 10 or 70 mg / kg, both at 30 min of onset of MCAO. In the control groups, the vehicle alone (30 ml) was given via the same routes.
2.4. General surgical procedures The rats were anesthetized with an intraperitoneal injection of sodium pentobarbital at a dose of 60 mg / kg. Additional doses at 20 mg / kg were administered as needed to ensure stable anesthesia. Rectal temperature was maintained between 36.5 and 37.5 8C throughout the experiment by using a rectal thermostat probe and a thermostatically regulated heating pad underneath the animal (FHC, Bowdoinham, ME, USA). The right femoral artery was cannulated with PE-50 polyethylene tubing for monitoring of arterial blood pressure (BP) and heart rate (HR) (Powerlab / 16 Data Acquisition System, AD Instruments, Mountain View, CA, USA). The BP and HR were monitored continuously during MCAO until the first 30 min of reperfusion in all experimental groups except in the groups given intracarotid injection in which the monitoring was continued for the first 2 h of reperfusion.
2.5. Monitoring of regional cerebral blood flow A laser Doppler flowmeter (MBF3D, Moor instruments, Devon, UK) became available during the later stage of the present study. It was used to monitor the regional cerebral blood flow (rCBF). Using a stereotaxic device (SR-6N, Narishige Scientific Instrument, Tokyo, Japan) and a low speed dental drill, a burr hole of 2 mm in diameter was made over the skull at 1 mm posterior and 5 mm lateral to the bregma on the right side. A needle shaped laser probe was placed on the dura away from visible cerebral vessels. Steady state baseline values were recorded before MCAO so that all rCBF data were expressed as percentages of the respective basal values. The rCBF was monitored continuously during focal ischemia until the first 30 min of reperfusion in all experimental groups except in the groups given intracarotid injection in which monitoring of rCBF was continued for the first 2 h of reperfusion.
2.6. Middle cerebral artery occlusion Focal cerebral ischemia was achieved by right-sided endovascular MCAO [21]. Briefly, the right carotid arteries were exposed through a midline cervical incision. The right external carotid artery (ECA) was dissected free and isolated distally by coagulating its branches (Bipolar
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Electric Coagulation, GN60, Aesculap, Tuttlingen, Germany) and placing a distal ligation prior to transection. A piece of 4-o-monofilament nylon suture (Ethicon, JohnsonJohnson, Brussels, Belgium), with its tip rounded by gentle heating, was introduced into the lumen of right ECA stump and gently advanced via the right internal carotid artery (ICA) to embed into the right anterior cerebral artery (ACA) so that the right middle cerebral artery was occluded at its origin. After 2 h of ischemia, the intraluminal suture was withdrawn from the right ACA and right ICA to permit reperfusion. When the laser Doppler flowmeter became available, achievement of focal ischemia and reperfusion was confirmed by the appropriate changes in the rCBF. Wounds were closed using surgical clips. The rats were allowed to recover from the anesthesia before returning to the cages with free access to food and water.
cut into coronal slices of 2 mm in thickness. These slices were reacted with a 2% solution of TTC for 20–30 min to reveal the ischemic infarction. After TTC reaction, the brain slices were fixed with 10% formalin (pH 7.4). TTC reacts with intact mitochondrial respiratory enzymes to generate a bright red color to contrast with the pale color of the infarct. The hemispheric volumes and volume of infarction between the bregma levels of 14 mm (anterior) and 26 mm (posterior) were integrated from the respective calibrated area measurements [10] that were obtained from the digitized images of the TTC-stained brain slices, using a computer-assisted image analysis system ( SIGMASCAN PRO 4.0, SPSS Chicago, IL, USA). The infarct volume was expressed as a percentage of the ipsilateral hemispheric volume.
2.9. Data analysis 2.7. Microinjection In the groups receiving slow intracarotid injection, the right ICA was connected to a Hamilton microsyringe (80630, Hamilton, Reno, NV, USA) via a short PE-50 tubing upon withdrawal of the monofilament suture to permit reperfusion. A single dose of NPY (10 mg / kg) or the vehicle alone was administered over 20 s via the right ICA. At 5 min after the injection, the connection to the right ICA was removed and the ECA stump closed by a surgical suture. In the groups receiving intracisternal injection, a mid-sagittal incision was made to expose the rhombic muscles of the neck after shaving the occipital hairs. A 23-G needle attached to a Hamilton microsyringe via a short PE-50 tubing was inserted into the cisterna magna. Aspiration of a few microliters of cerebrospinal fluid (CSF) into the Hamilton microsyringe verified proper positioning of the needle tip. NPY (10 or 30 mg / kg) or the vehicle alone was injected over 20 s at 30 min after onset of MCAO. The needle was kept in the same position for 5 min before removal. In the groups receiving slow i.c.v. injection, a second burr hole of 1 mm in diameter was made in the skull at 0.5 mm posterior and 1.5 mm lateral to the bregma on the right side. A 23-G needle attached to a Hamilton microsyringe via a short PE-50 tubing was inserted stereotaxically through this second burr hole so that its tip was at 3.4 mm beneath the dural surface. Proper needle placement was verified via withdrawing a few microliters of clear CSF into the Hamilton microsyringe. NPY (10 or 70 mg / kg) or the vehicle alone was administered over 20 s at 30 min after onset of MCAO. The needle was kept in the same position for 5 min before removal, and the burr hole was closed using bone wax.
2.8. Measurement of infarct volume Three days after the MCAO, the rats were deeply re-anesthetized with sodium pentobarbital at a dose of 100 mg / kg. The rats were decapitated, and their brains were
Data are expressed in mean6S.E.M. The mean BP and HR data at different time points of the same experimental group were analyzed using one-way analysis of variance (ANOVA) to reveal any significant change over time with Dunnett post-test to detect any significant difference from the initial values obtained immediately before MCAO. In the groups receiving the intracarotid injection, differences between groups in mean BP, HR, rCBF, and infarct volume were determined using Student’s t-test. In the groups receiving the intracisternal or i.c.v. injection, differences among groups in mean BP, HR, rCBF, and infarct volume were tested using one-way ANOVA with Student–Newman–Keuls post-test. In addition, one sample t-test was applied to the data on rCBF at different time points to detect significant difference from 100% (i.e. a significant change from baseline). A two-tailed P value of 0.05 or less was taken to indicate statistical significance.
3. Results
3.1. Systemic hemodynamic parameters There was no significant change in the mean BP and HR within the same treatment group at different time points when compared to the initial values immediately before MCAO. In addition, there was no significant difference at various time points among different groups of rats received injection of NPY or vehicle via the same route (data not shown).
3.2. Relative regional cerebral blood flow The relative rCBF decreased significantly to less than 30% during MCAO on the same side. Abrupt increase in relative rCBF was seen at the onset of reperfusion (Table 1). Following intracarotid injection of NPY (10 mg / kg) at the commencement of reperfusion, there was a significant
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Table 1 Data on relative rCBF (in per cent) at the following time points in relation to the 2-h middle cerebral artery occlusion (MCAO) and the intracisternal (IC) or intracerebroventricular (i.c.v.) injection in the rat Group (number of rats)
At 5 min of MCAO
At 30 min of MCAO (just before injection)
At 5 min of injection (at 35 min of MCAO)
At 120 min of MCAO
At 5 min after reperfusion
At 30 min after reperfusion
IC NPY at 30 mg / kg (5) IC NPY at 10 mg / kg (5) IC Vehicle only (5)
25.162.3 [,0.0001] 25.363.7 [,0.0001] 23.364.6 [,0.0001]
22.662.7 [,0.0001] 24.964.1 [,0.0001] 23.064.8 [,0.0001]
23.764.4 [,0.0001] 25.461.7 [,0.0001] 24.565.2 [0.0001]
25.663.9 [,0.0001] 26.062.4 [,0.0001] 24.063.1 [,0.0001]
107.3615.0 [0.6678] 94.963.9 [0.2567] 98.966.9 [0.8748]
71.866.2 [0.0102] 90.068.3 [0.2942] 93.665.0 [0.2717]
I.c.v. NPY at 70 mg / kg (8) I.c.v. NPY at 10 mg / kg (8) I.c.v. Vehicle only (7)
23.161.6 [,0.0001] 26.362.5 [,0.0001] 25.161.7 [,0.0001]
20.862.9 [,0.0001] 22.263.0 [,0.0001] 25.762.1 [,0.0001]
16.463.2* [,0.0001] 25.062.5 [,0.0001] 26.162.1 [,0.0001]
16.563.0 [,0.0001] 24.363.1 [,0.0001] 22.862.3 [,0.0001]
124.1625.3 [0.3731] 86.2611.5 [0.2713] 118.8617.5 [0.3252]
62.263.3* [,0.0001] 67.268.7* [0.0069] 107.7610.7 [0.5020]
P values from one sample t-test on the difference from 100% are in the square brackets. *, Significant difference at the same time points among the groups received i.c.v. injection using one-way ANOVA with Student–Newman–Keuls post-test showing significant difference between the i.c.v. vehicle-treated group and the i.c.v. NPY-treated group.
reduction in the relative rCBF to less than 50% beginning at 5 min after injection and persisting throughout the 2-h period of monitoring (data not shown). Intracarotid injection of the vehicle at the commencement of reperfusion did not significantly affect the relative rCBF (data not shown). The difference in the relative rCBF between the two intracarotid groups was significant at all the time points following the injection. Following intracisternal injection of NPY or the vehicle, there was no further drop in the already reduced rCBF during MCAO (Table 1). Intracisternal injection of NPY at 30 but not 10 mg / kg reduced the relative rCBF by about 30% at 30 min after reperfusion, although there was no significant difference among the intracisternal groups at the same time points (Table 1). I.c.v. injection of NPY at 70 but not 10 mg / kg further reduced the relative rCBF even during MCAO; the difference in rCBF among all three i.c.v. groups was significant at 5 min after injection (or at 35 min of MCAO; Table 1). An abrupt increase in relative rCBF was seen at the onset of reperfusion in all three i.c.v. groups, but i.c.v. injection of NPY at either dose significantly reduced the relative rCBF by about 35% at 30 min after reperfusion (Table 1).
3.3. Site and volume of infarct On day 3 of MCAO, the right-sided cerebral infarct was evident as the whitish region after reaction with TTC. In the vehicle-treated control group, the infarct was largest between the brain regions at 2 mm anterior to the bregma and at 2 mm posterior to the bregma. The infarct consistently involved the caudate putamen, external capsule and insular cortex with variable involvement of the overlying primary and secondary somatosensory cortices. In the NPY-treated groups, the infarct was larger with more
extensive involvement of the subcortical structures and the overlying cortices. Computer-assisted image analysis revealed that the relative infarct volumes varied from 15.763.6 to 22.862.3% (Fig. 1) in the vehicle-treated groups, but the difference among the vehicle-treated groups was not significant (one-way ANOVA). When compared to the vehicle-treated group, intracarotid injection of NPY at 10 mg / kg significantly increased the relative infarct volume by about 80% (Fig. 1A). Intracisternal injection of NPY also significantly increased the relative infarct volume by about 50%, and there was no difference between the two doses (Fig. 1B). I.c.v. injection of NPY at 10 mg / kg significantly increased the relative infarct volume by 110% (Fig. 1C).
4. Discussion
4.1. Technical considerations A standard method of inducing focal cerebral ischemia was adopted in the present study. As the study was started with rats in the intracarotid and intracisternal groups when the laser Doppler flowmeter was not available, data on relative rCBF were obtained from some rats of these groups.
4.2. Hemodynamic effects of NPY The present study documents a lack of significant effect of intracarotid, intracisternal or i.c.v. injection of NPY at doses between 10 and 70 mg / kg on BP and HR under the present experimental conditions. Previous studies have reported contradictory results on the effects of central [17,18,23,25,28] or peripheral injection of NPY [19,23].
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NPY [19,23]. Regarding the effects of NPY on HR, most studies reported a decrease or no change [19,23,25], but increased HR has been reported [18]. Use of different animal species, varying doses of NPY, different routes of administration, use of conscious or anesthetized animals, different anesthetic drugs, varying physiological states, and / or under different pathophysiological situations may explain these contradictory observations. Delivery of NPY via any one of the three routes produces significant reduction of rCBF during reperfusion, and the reduction is most dramatic with intracarotid injection. The vasoconstricting effect of NPY is probably masked during MCAO when rCBF is low. Nevertheless, a large dose of NPY may further reduce rCBF during ischemia, as illustrated by the rCBF results of i.c.v. injection of NPY at 70 mg / kg. These results are expected because NPY is a potent vasoconstrictor of the cerebral arteries in most animal species [1,3,14,27]. Allen et al. first demonstrated the presence of NPY in the human circle of Willis and showed that intracarotid administration of 50 pmol to 2 nmol of NPY in the rat reduced mean cortical cerebral blood flow by 40–98% for at least 2 h [3]. Between 20 and 55 min after intracerebral microinjection of NPY at 2–200 pmol into the striatum of conscious rats, the rCBF of the ipsilateral entorhinal cortex, amygdala, and caudate nucleus was found to reduce by 30–45%, but the regional glucose utilization was unaffected [29]. Thus, NPY has been implicated to play an important role in cerebrovascular regulation under physiological and pathological conditions.
4.3. Worsening of infarction following injection of NPY
Fig. 1. Bar graphs showing the infarct volume (mean6S.E.M.) between the bregma level 14 and the bregma level 26 expressed as a percentage of the corresponding right hemispheric volume following right-sided middle cerebral artery occlusion for 2 h and intracarotid injection of NPY at 10 mg / kg or of the vehicle alone in (A), intracisternal injection of NPY at 10 or 30 mg / kg or of the vehicle alone in (B), or intracerebroventricular injection of NPY at 10 or 70 mg / kg or of the vehicle alone in (C). *, Significant difference between the NPY-treated group and the respective vehicle-treated control group with P,0.05 using Student’s t test or one-way analysis of variance and the Student–Newman–Keuls post-test.
Some studies reported a decreased BP with central administration of NPY [17,25], whereas others reported no change [23,25] or an increase [18]. In contrast, a sustained increase in BP is seen after peripheral administration of
Peripheral or central administration of NPY at the doses of the present study exacerbates rather than ameliorates ischemic damage during experimental MCAO. Nevertheless, a 29% reduction in the density of NPY Y1 receptors using a well characterized antisense oligodeoxynucleotide was found to exacerbate ischemic infarction [11]. One possible explanation is that hemodynamic parameters were not recorded in the previous study [11]. The method of MCAO using craniectomy and direct coagulation may be associated with significant hemodynamic disturbances from augmented sympathetic discharges [6]. The antisense oligodeoxynucleotide will suppress the Y1 receptor density throughout the body and, in turn, may affect the systemic and / or cerebral vasomotor tone as well as cerebral perfusion. Another plausible explanation is that downregulation of NPY Y1 receptors due to antisense treatment may lead to a compensatory increase in the endogenous secretion of NPY (see discussion in [24]). When the local concentration of NPY is further increased during focal cerebral ischemia [7], the observed worsening of infarction volume could have been caused by excessive increase in the concentration of free NPY rather than the reduction in NPY Y1 receptor density.
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5. Conclusions In conclusion, peripheral or central administration of NPY at doses between 10 and 70 mg / kg does not significantly affect BP and HR during MCAO. Cerebral perfusion is adversely affected, resulting in a larger cerebral infarct. The present results do not support any neuroprotective effect of NPY during focal ischemia. Further studies with specific receptor agonists or antagonists are useful to clarify the role of NPY and its receptor types in cerebral ischemia.
Acknowledgements This study was supported by a grant (10202658) from the Committee of Research and Conference Grants, The University of Hong Kong, Hong Kong.
References [1] P.W. Abel, C. Han, Effects of neuropeptide Y on contraction, relaxation, and membrane potential of rabbit cerebral arteries, J. Cardiovasc. Pharmacol. 13 (1989) 52–63. [2] G.V. Allen, R.T. Cheung, D.F. Cechetto, Neurochemical changes following occlusion of the middle cerebral artery in rats, Neuroscience 68 (1995) 1037–1050. [3] J.M. Allen, F. Schon, N. Todd, J.C. Yeats, H.A. Crockard, S.R. Bloom, Presence of neuropeptide Y in human circle of Willis and its possible role in cerebral vasospasm, Lancet 2 (1984) 550–552. [4] Y.S. Allen, T.E. Adrian, J.M. Allen, K. Tatemoto, T.J. Crow, S.R. Bloom, J.M. Polak, Neuropeptide Y distribution in the rat brain, Science 221 (1983) 877–879. [5] A.G. Blomqvist, H. Herzog, Y-Receptor subtypes—how many more?, Trends Neurosci. 20 (1997) 294–298. [6] D.F. Cechetto, J.X. Wilson, K.E. Smith, D. Wolski, M.D. Silver, V.C. Hachinski, Autonomic and myocardial changes in middle cerebral artery occlusion: stroke models in the rat, Brain Res. 502 (1989) 296–305. [7] R.T. Cheung, T. Diab, D.F. Cechetto, Time-course of neuropeptide changes in periischemic zone and amygdala following focal ischemia in rats, J. Comp. Neurol. 360 (1995) 101–120. [8] R.T. Cheung, D.F. Cechetto, Neuropeptide changes following excitotoxic lesion of the insular cortex in rats, J. Comp. Neurol. 362 (1995) 535–550. [9] R.T. Cheung, D.F. Cechetto, Colchicine affects cortical and amygdalar neurochemical changes differentially after middle cerebral artery occlusion in rats, J. Comp. Neurol. 387 (1997) 27–41. [10] R.T. Cheung, V.C. Hachinski, D.F. Cechetto, Cardiovascular response to stress after middle cerebral artery occlusion in rats, Brain Res. 747 (1997) 181–188. [11] R.T. Cheung, D.F. Cechetto, Neuropeptide Y-Y1 receptor antisense oligodeoxynucleotide increases the infarct volume after middle cerebral artery occlusion in rats, Neuroscience 98 (2000) 771–777. [12] W.F. Colmers, C. Wahlestedt (Eds.), The Biology of Neuropeptide Y and Related Peptides, Humana Press, New Jersey, 1993. [13] Y. Dumont, J.C. Martel, A. Fournier, S. St-Pierre, R. Quirion, Neuropeptide Y and neuropeptide Y receptor subtypes in brain and peripheral tissues, Prog. Neurobiol. 38 (1992) 125–167.
143
[14] L. Edvinsson, P. Emson, J. McCulloch, K. Tatemoto, R. Uddman, Neuropeptide Y: immunocytochemical localization to and effect upon feline pial arteries and veins in vitro and in situ, Acta Physiol. Scand. 122 (1984) 155–163. [15] C.F. Elias, C. Aschkenasi, C. Lee, J. Kelly, R.S. Ahima, C. Bjorbaek, J.S. Flier, C.B. Saper, J.K. Elmquist, Leptin differentially regulates NPY and POMC neurons projecting to the lateral hypothalamic area, Neuron 23 (1999) 775–786. [16] G. Figueredo-Cardenas, C.L. Harris, K.D. Anderson, A. Reiner, Relative resistance of striatal neurons containing calbindin or parvalbumin to quinolinic acid-mediated excitotoxicity compared to other striatal neuron types, Exp. Neurol. 149 (1998) 356–372. [17] R. Grimaldi, I. Zini, G. Biagini, G. Toffano, L.F. Agnati, Effects of centrally administered clonidine and neuropeptide Y on arterial blood pressure in the rat after transient forebrain ischemia, J. Auton. Nerv. Syst. 30 (1990) S67–S69. [18] Y. Hu, J.C. Dunbar, Intracerebroventricular administration of NPY increases sympathetic tone selectively in vascular beds, Brain Res. Bull. 44 (1997) 97–103. [19] Y. Hu, J.C. Dunbar, Regional vascular and cardiac responses to systemic neuropeptide-Y in normal and diabetic rats, Peptides 18 (1997) 809–815. [20] K. Isayama, L.H. Pitts, M.C. Nishimura, Evaluation of 2,3,5-triphenyltetrazolium chloride staining to delineate rat brain infarcts, Stroke 22 (1991) 1394–1398. [21] E.Z. Longa, P.R. Weinstein, S. Carlson, R. Cummin, Reversible middle cerebral artery occlusion without craniectomy in rats, Stroke 20 (1989) 84–91. [22] M.C. Michel, A. Beck-Sickinger, H. Cox, H.N. Doods, H. Herzog, D. Larhammar, R. Quirion, T. Schwartz, T. Westfall, XVI. International Union of Pharmacology recommendations for the nomenclature of neuropeptide Y, peptide YY, and pancreatic polypeptide receptors, Pharmacol. Rev. 50 (1998) 143–150. [23] M.A. Petty, R. Dietrich, R.E. Lang, The cardiovascular effects of neuropeptide Y (NPY), Clin. Exp. Hypertens. 6 (1984) 1889–1892. [24] A.O. Schaffhauser, S. Whitebread, R. Haener, K.G. Hofbauer, A. Stricker-Krongrad, Neuropeptide Y Y1 receptor antisense oligodeoxynucleotides enhance food intake in energy-deprived rats, Reg. Pept. 75–76 (1988) 417–423. [25] N.A. Scott, V. Webb, J.H. Boublik, J. Rivier, M.R. Brown, The cardiovascular actions of centrally administered neuropeptide Y, Reg. Pept. 25 (1989) 247–258. [26] S.L. Stoddard, G.M. Tyce, J.E. Ahlskog, A.R. Zinsmeister, D.K. Nelson, S.W. Carmichael, Decreased levels of [Met] enkephalin, neuropeptide Y, substance P, and vasoactive intestinal peptide in parkinsonian adrenal medulla, Exp. Neurol. 114 (1991) 23–27. [27] Y. Suzuki, M. Shibuya, I. Ikegaki, S. Satoh, M. Takayasu, T. Asano, Effects of neuropeptide Y on canine cerebral circulation, Eur. J. Pharmacol. 146 (1988) 271–277. [28] Y. Suzuki, S. Satoh, I. Ikegaki, T. Okada, M. Shibuya, K. Sugita, T. Asano, Effects of neuropeptide Y and calcitonin gene-related peptide on local cerebral blood flow in rat striatum, J. Cereb. Blood Flow Metab. 9 (1989) 268–270. [29] U.I. Tuor, P.A. Kelly, L. Edvinsson, J. McCulloch, Neuropeptide Y and the cerebral circulation, J. Cereb. Blood Flow Metab. 10 (1990) 591–601. [30] C. Wahlestedt, E.M. Pich, G.F. Koob, F. Yee, M. Heilig, Modulation of anxiety and neuropeptide Y–Y1 receptors by antisense oligodeoxynucleotides, Science 259 (1993) 528–531. [31] G. Weng, D. Feinstein, D. Reis, C. Wahlestedt, Neuropeptide Y receptor gene regulation in mouse adrenocortical Y-1 cells, Reg. Pept. 63 (1996) 53–56.