Nicotinamide reduces infarction up to two hours after the onset of permanent focal cerebral ischemia in Wistar rats

Neuroscience Letters 259 (1999) 21–24

Nicotinamide reduces infarction up to two hours after the onset of permanent focal cerebral ischemia in Wistar rats Issam A. Ayoub a, E. Jian Lee a, Christopher S. Ogilvy a, M. Flint Beal b, Kenneth I. Maynard a ,* a

Neurosurgical Service, Massachusetts General Hospital and Harvard Medical School, Boston, MA 02114, USA b Department of Neurology, Cornell University Medical College, New York, NY 10021, USA Received 13 September 1998; received in revised form 28 October 1998; accepted 28 October 1998

Abstract Ischemia depletes ATP and initiates cascades leading to irreversible tissue injury. Nicotinamide is a precursor of nicotinamide adenine dinucleotide (NAD + ) which increases neuronal ATP concentration and protects against malonate-induced neurotoxicity, trauma and nitric oxide toxicity. We therefore examined whether nicotinamide could protect against stroke, using a model of permanent middle cerebral artery occlusion (MCA) occlusion in Wistar rats. Nicotinamide reduced neuronal infarction in a dosespecific manner. Furthermore, nicotinamide (500 mg/kg) reduced infarcts when administered up to 2 h after the onset of permanent MCA occlusion. The mechanism of action underlying the neuroprotection observed with nicotinamide remains to be clarified. These results are potentially important since nicotinamide is already used clinically, though not in the treatment of stroke.  1999 Elsevier Science Ireland Ltd. All rights reserved

Keywords: Stroke; Energy metabolism; Poly(ADP-ribose) polymerase; Window of opportunity; Poly(ADP-ribose) synthetase; Therapeutic window

Neuronal ischemia begins as an imbalance between energy supply (e.g. reduced during hypoperfusion) and demand (e.g. to maintain ionic homeostasis and for neurotransmission). The consequence of this energy imbalance is the depletion of adenosine triphosphate (ATP) which triggers the onset of numerous ischemia-induced cascades, each of which may lead to irreversible cell injury [1]. There are three ways in which intervention might preserve neuronal viability and limit infarction due to cerebral ischemia [2]. The first option is to improve the energy supply to the tissue ‘at risk’. The second option is to reduce the neuronal energy demands of the tissue [3]. The third, and most investigated option, is to treat the consequences of ischemia to try to protect against the extension of injury, or possibly reverse the injury already caused [1]. We have already shown that the second option works when treatment occurs at the time of the insult [2,3]. In this study we inves* Corresponding author. Neurophysiology Laboratory, Edwards 414, Neurosurgical Service, Massachusetts General Hospital and Harvard Medical School, 55 Fruit Street, Boston, MA 02114, USA. Tel.: +1 617 7245329; fax: +1 617 7263926; e-mail: [email protected]

0304-3940/99/$ - see front matter PII S0304- 3940(98) 00881- 7

tigated whether nicotinamide might reduce infarction in a model of focal cerebral ischemia in rats. Nicotinamide, a soluble B group vitamin, is an essential precursor of nicotinamide adenine dinucleotide (NAD + ) and a poly-ADP-ribose polymerase (PARP) inhibitor [4]. It prevents the depletion of NAD + , protects against the decreased production of ATP and lactate increases, and is neuroprotective against malonate (a mitochondrial complex II inhibitor)-induced lesions in the striatum [5], trauma and nitric oxide exposure in the rat hippocampus [6]. Nicotinamide therefore has the potential to protect against the initial ischemia-induced energy imbalance induced by cerebral ischemia, via boosting neuronal energy reserves to the tissue ‘at risk’ [5]. All procedures performed were approved by the Subcommittee on Research Animal Care of the Massachusetts General Hospital, whose standards meet Federal and State reviewing organizations. Focal cerebral ischemia infarcts were made in male Wistar rats (270–300 g, Charles River Laboratories, Wilmington, MA) which were allowed access to food and water before surgery. Halothane anesthesia (1–2% in 50% N2O/

 1999 Elsevier Science Ireland Ltd. All rights reserved

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I.A. Ayoub et al. / Neuroscience Letters 259 (1999) 21–24

50% O2) was used in free breathing animals whose body temperature were kept stable at 36.5 ± 0.5°C using a heating pad and rectal probe (Yellow Springs Instruments, OH). The right femoral artery was cannulated for measurement of arterial blood gases, glucose, hematocrit and mean arterial pressure. Focal cerebral ischemia was induced using a well established procedure [7,8]. Regional cerebral blood flow measurements were not monitored. The right common carotid artery was exposed at its bifurcation. A 4-0 nylon suture with its tip rounded by heating over a flame and subsequently coated with poly-l-lysine was then advanced 18– 19 mm from the external into the internal carotid artery, until the tip occluded the origin of the middle cerebral artery (MCA). Following closure of the operation sites, the animals were allowed to awaken from the anesthesia. Sacrifice was performed 22–24 h after MCA occlusion under ketamine (44 mg/kg, i.p.) and xylazine (13 mg/kg, i.p.) anesthesia followed by decapitation. The brain was then rapidly removed, cut into 2-mm coronal sections using a rat brain matrix (RBM4000C, ASI Instrument, Warren, MI) and stained according to the standard 2,3,5-triphenyltetrazolium chloride (TTC) method [7,8]. Each slice was drawn using a computerized image analyzer (Bioquant, R and M Biometrics, Nashville, TN) and the infarct areas were calculated to obtain the infarct volumes per brain (in mm3). Infarct volumes were expressed as a percentage of the contralateral hemisphere volume to compensate for edema formation in the ipsilateral hemisphere [8]. All data were expressed as mean ± SEM and analyzed using analysis of variance, followed by Fisher’s Least Significant Difference (protected t) post-hoc tests where necessary. As previously described [7,8], permanent MCA occlusion resulted in large ipsilateral striatal and cerebral cortical infarcts (Fig. 1A). In the first series of experiments, animals which received intraperitoneal injections of nicotinamide at 500 mg/kg, but not at 50 mg/kg or 1000 mg/kg, 1 h before MCA occlusion, had significantly reduced ipsilateral cerebral infarction volumes when compared with saline (vehicle)-injected controls (Fig. 1B). The physiological parameters of the animals were normal and not statistically different from each other (data not shown, but see Table 1 for similar results). In the second set of experiments, animals received nicotinamide (500 mg/kg) or vehicle (saline) at 0.5, 2, 3 or 4 h after MCA occlusion. Since there was no significant difference in infarction volumes amongst the vehicle (saline) injected animals (P = 0.573), these data were pooled in the analysis and compared to the nicotinamide-treated animals. There was a significant reduction in infarction volume when nicotinamide was administered 0.5 and 2 h (see brain slices in Fig. 1A from a (I) saline (vehicle) control and a (II) nicotinamide-treated animal), but not at 3 and 4 h after MCA occlusion (Fig. 1C). The physiological parameters were normal and were not statistically different from each other (Table 1).

Fig. 1. (A) Reduction in infarct volume by intraperitoneal nicotinamide (500 mg/kg). 2,3,5-Triphenytetrazolium chloride (TTC)-stained coronal sections from animals which received (I) a saline (vehicle) injection or (II) nicotinamide (500 mg/kg), 2 h after middle cerebral artery (MCA) occlusion. An infarct is observed (pale region) involving the cerebral cortex and underlying striatum representative of the MCA perfusion region in both sections in (I) and (II), but the infarction volume is much smaller in (II). (B) Nicotinamide at 500 mg/kg (n = 8), but not at 50 mg/kg (n = 8) or 1000 mg/kg (n = 8) injected intraperitoneally 1 h before permanent middle cerebral artery occlusion (MCAo), significantly reduced the volume of infarction relative to vehicle (saline) injected control animals (n = 8). (C) Nicotinamide (500 mg/kg) reduced the infarction volume in animals treated at 0.5 (n = 7) and 2 (n = 6) h, but not at 3 (n = 8) and 4 (n = 6) h after permanent MCAo, compared to pooled vehicle (saline) treated and time-comparable controls (n = 18). In (B,C) infarct volumes were expressed as a percentage of the contralateral (control) hemisphere, and the data are represented as the mean ± SEM. *P , 0.05 which was considered significant. n, number of animals.

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I.A. Ayoub et al. / Neuroscience Letters 259 (1999) 21–24 Table 1

There were no significant differences in the physiologic parameters between the vehicle (saline)-injected control group versus the nicotinamidetreated groups (0.5–4 h) either before (pre-ischemia) or after (post-ischemia) permanent middle cerebral artery occlusion

Pre-ischemia Control 0.5 h 2h 3h 4h Post-ischemia Control 0.5 h 2h 3h 4h

n

pH

pCo2 (mmHg)

18 7 6 8 6

7.50 7.50 7.45 7.52 7.48

± ± ± ± ±

0.01 0.02 0.01 0.01 0.01

30.8 31.4 35.6 31.2 35.1

± ± ± ± ±

0.9 1.7 2.0 0.5 2.1

156.5 149.8 156.3 140.3 170

± ± ± ± ±

9.2 10 12.7 13.5 22.3

38.8 39.8 37.5 39.1 37.8

± ± ± ± ±

0.4 0.4 0.9 0.4 0.4

117 116 119 124 118

± ± ± ± ±

18 7 6 8 6

7.48 7.46 7.47 7.49 7.49

± ± ± ± ±

0.01 0.02 0.01 0.01 0.03

33.0 35.0 31.5 34.2 32.0

± ± ± ± ±

0.8 1.7 1.6 1.7 2.3

146.4 145.2 156.5 148.8 154.8

± ± ± ± ±

7.6 7.7 12.8 12.6 16.8

38.8 39.7 37.5 39.1 38.6

± ± ± ± ±

0.3 0.4 0.7 0.4 0.8

114 114 120 120 114

± ± ± ± ±

p02 (mmHg)

Hct (%)

Gluc (mg/dL)

MABP (mmHg)

HR (Beats/min)

3 4 5 5 6

101 ± 3 97 ± 5 94 ± 1 102 ± 4 99 ± 4

369 337 358 359 350

± ± ± ± ±

11 14 11 10 8

3 5 7 6 6

106 ± 3 99 ± 6 105 ± 4 107 ± 3 107 ± 4

384 350 375 366 372

± ± ± ± ±

9 11 17 9 5

Physiologic data obtained from both control and treated animal groups are represented as the mean ± standard error of the mean. Hct, hematocrit; Glu, blood glucose; MABP, mean arterial blood pressure; HR, heart rate. All animals were maintained at 36.5 ± 0.5°C (rectal temperature). n, number of animals.

We have shown that nicotinamide (500 mg/kg) reduced infarct volumes induced by permanent focal cerebral ischemia in Wistar rats, when given 1 h prior to MCA occlusion, or up to 2 h after the onset of the insult. This reduction cannot be accounted for by changes in arterial blood gases, hemodilution (as measured by blood hematocrit) mean arterial blood pressure, heart rate or differences in core temperature, since these were not significantly different when compared between saline-injected and nicotinamidetreated animals in either the pre- or post-treatment study. A therapeutic window of 2 h compares favorably with glutamate receptor antagonists [9], but not as well as that reported for basic fibroblast growth factor [8]. We believe that perhaps using lower multiple doses of nicotinamide [5], or perhaps with continuous infusion [8], this therapeutic window may be extended and/or the degree of neuroprotection improved. Exactly why the dose-response curve for the reduction in infarction volume caused by MCA occlusion by nicotinamide is U-shaped is not known. Curiously, a similar finding was reported with 3,4-dihydro 5-[4-(1-piperidinyl) butoxyl]-1(2H)-isoquinolinone, a poly-ADP ribose polymerase (PARP) inhibitor [10]. It was recently reported, however, that nicotinamide enhances brain choline levels [11]. An increased release of choline was also reported for 3-aminobenzamide, another PARP inhibitor, though not an NAD + precursor. The increased release of choline may be neuroprotective against ischemic brain injury [12], but it is also associated with neuronal damage due to glutamate or hypoxic toxicity [13]. Consequently the nicotinamideinduced increase release of choline, may underlie the neuroprotection observed at 500 mg/kg as well as its loss at 1000 mg/kg, and PARP inhibition may also be implicated. This study focused on whether or not nicotinamide was protective in an in vivo model of stroke in rats, and not on the mechanism(s) of action by which nicotinamide could be

neuroprotective. It is known, however, that PARP activation contributes to neuronal damage following focal ischemia, and ischemic infarction is reduced in PARP-null mice and in wild-type mice treated with 3-aminobenzamide [10,16,17]. The injurious effects of excessive PARP activation may be due to either the depletion of ATP and/or the augmentation of excitotoxicity mediated by nitric oxide and glutamate [18–20]. Thus the neuroprotective action of nicotinamide seen in our study may be due to its action as a PARP inhibitor [4] counteracting the aforementioned deleterious mechanisms induced by PARP activation. Nicotinamide also increases regional cerebral blood flow (rCBF) and metabolic rate of oxygen [14], and acts as a mild antioxidant [4]. We used nicotinamide primarily because it boosts the amount of ATP in the tissue [5] during ischemia, as it is a precursor to NAD + [4]. We reasoned that it should therefore prevent the depletion of ATP normally used in NAD + synthesis, and therefore lead to neuroprotection by rectifying the ischemia-induced energy imbalance by increasing and/or preserving the tissue ATP content. However, like nicotinamide [14], NAD + is also a vasodilator [15]. Thus improved rCBF may partly contribute to the neuroprotective (direct) effect of nicotinamide and/or (indirectly) through NAD + . An exciting aspect of the present findings is that clinical usage of nicotinamide is well established for the treatment of a variety of disorders, with large daily doses in patients being safe and associated with little or no side-effects [4]. It should therefore be relatively convenient to test this drug in clinical trials for stroke. However, given the outcome of the first series of experiments in this study which showed that neuroprotection was dose-specific, i.e. with 500 mg/kg only, given as a single injection, the dosage and dosing regimen to be used in such an endeavor is crucial. We therefore need to assess whether extended treatment protocols such as repeated administrations at lower doses, following an initial

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loading dose to maintain the therapeutic levels of nicotinamide, will lead to a prolonged and/or improved neuroprotective effect. Further studies are also needed to determine whether nicotinamide is neuroprotective (i.e. reduces infarct volume and improves neurologic outcome) following a longer period of recovery which will allow not only for reduction in damage due to necrosis, but also damage caused by apoptosis [1]. In addition, the mechanism underlying the neuroprotective effect observed here needs to be elucidated.

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The authors thank Dr. Adelbert Ames III and Dr. Eng H. Lo for constructive comments on the manuscript. K.I.M. is an American Heart Association Minority Scientist Development Awardee; C.S.O. is supported by NIH grant #NS01732, and M.F.B. is supported by NIH grants #NS32365 and #NS31579.

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