effects of nitrobenzylthioinosine on adenosine levels and neuronal injury in rat forebrain ischemia

NEUROSCIENCE RESEARCH COMMUNICATIONS, VOL. 30, NO. 2 83

EFFECTS OF NITROBENZYLTHIOINOSINE ON ADENOSINE LEVELS AND

NEURONAL INJURY IN RAT FOREBRAIN ISCHEMIA

YiWei Zhang, P. Nicholas Shepel, James Peeling, Jonathan D. Geiger and Fiona E. Parkinson*

Department of Pharmacology and Therapeutics, 753 McDermot Avenue, University of Manitoba, Winnipeg, Canada, R3E OT6

@ccepted January I7, 2002)

Summary Nitrobenzylthioinosine (NBMPR) can potentiate the actions of adenosine through inhibition of adenosine influx mediated by the equilibrative nucleoside transporter subtype 1 (ENTl). As adenosine can decrease ischemic neuronal injury, we tested the hypothesis that peripheral administration of the pro-drug NBMPR-phosphate (NBMPR-P) can increase brain adenosine levels and reduce ischemia-induced loss of hippocampal CA1 neurons. Pre-ischemic, but not post-ischemic, peripheral administration of NBMPR-P significantly (P = 0.03) increased neuronal survival. Mechanistically, NBMPR-induced neuroprotection was associated with significant (P = 0.03) increases in adenosine levels relative to saline-treated controls. Hypothermia was tested for but did not account for the beneficial effects of NBMPR. Together, these data suggest that selective inhibition of ENTl adenosine transporters can increase post-ischemic levels of adenosine and reduce ischemic neuronal death.

Key words: cerebral ischemia; nucleoside transport; ENT 1; adenosine

Introduction

Ischemic and hypoxic events increase levels of adenosine by decreasing rates of ATP

synthesis and enhancing rates of ATP hydrolysis. Previously, we demonstrated that forebrain

ischemia increased levels of adenosine by up to loo-fold with levels returning to baseline within 30

min of reperfusion (1). Several studies have demonstrated that endogenous adenosine interacts with

adenosine receptors to attenuate neuronal injury (2, 3). This neuroprotection is primarily attributed to

adenosine A, receptors, activation of which leads to hyperpolarization of neurons, inhibition of

neuronal release of glutamate, and attenuation of glutamate receptor activity (4).

0 2002 Wiley-Liss, Inc. DO1 10.1002/n rc. 10020

84 NEUROSCIENCE RESEARCH COMMUNICATIONS, VOL. 30, NO. 2

Nucleoside transport processes regulate the trans-membrane movements of adenosine and,

thus, can regulate extracellular levels and receptor-mediated effects of adenosine. At least three

subtypes of nucleoside transporters mediate adenosine transport in rat cells: equilibrative (Na’-

independent) transporters ENTl and ENT2 and the Na’-coupled symporter CNT2 (5).

Nitrobenzylthioinosine (NBMPR) is a potent and selective inhibitor of the ENTl subtype of

transporter (5). The pharmacological effects of NBMPR can be tested using the phosphorylated pro-

drug form of NBMPR, nitrobenzylthioinosine S-monophosphate (NBMPR-P), which is water-

soluble and readily dephosphorylated in vivo (6, 7). In a previous study (1) we demonstrated that

pre-ischemic intracerebroventricular injections of NBMPR-P reduced ischemic neuronal death in the

CA1 region of the hippocampus. In the present study we tested, first, whether peripheral

administration of NBMPR-P could reduce ischemic neuronal death, second, whether pre- and post-

ischemic treatment with NBMPR-P have similar beneficial effects, and third, whether the beneficial

effects were due to increases in post-ischemic adenosine levels. .

Materiak and Methods Forebrain Ischemia

All experimental procedures were performed in adherence to the guidelines of the Canadian Council on Animal Care and were approved by the University of Manitoba Animal Protocol Management and Review Committee. Reversible forebrain ischemia was achieved in male Sprague Dawley rats (275 - 325 g) as described (1, 8, 9). Briefly, a total of 72 rats were fasted overnight then administered anesthetic (1.5% halothane and 50% 0, in N,O) via ventilator-y support throughout the period of surgical preparation and ischemia. Brain temperature was monitored using a tympanic membrane probe and was maintained between 36.5 - 37.0 OC through the use of a heated water pad. The tail artery was exposed and cannulated to obtain samples for blood gases, hematocrit and blood glucose measurements and for blood pressure monitoring and control. Both carotid arteries were exposed through a neck incision and ischemia was induced, 30 minutes after injection, by occluding the carotid arteries and aspirating sufficient blood (- 5 ml) into a syringe containing 30 I.U. heparin to produce a controlled hypotension (45 & 5 mm Hg). Blood flow through the carotid arteries was restored after 6 min and the heparinized blood was re-infused. Post-ischemic rectal temperatures were monitored for at least 4 hours.

Rats were given intraperitoneal (i.p.) injections of NBMPR-P (Alberta Nucleoside Therapeutics, Edmonton, AB, Canada), 10 mg/kg, or an equivalent volume of saline either 30 min prior to initiation of ischemia or 30 min post-reperfusion.

Qualified animal care technicians monitored 40 rats for a 7 day recovery period. Rats exhibiting signs of ill health (poor eye and fur appearance, weight loss, lack of food and water consumption, inactivity) were euthanized. In total, 6 rats died or were euthanized during the 7 day period following surgery. Of our four treatment groups, per cent survival was 86% (12/14) for pre- ischemic saline injections, 1 OO”/o (1202) for pre-ischemic NBMPR-P injections, 63% (5/8) for post- ischemic saline injections and 83% (5/6) for post-ischemic NBMPR-P injections. After a recovery


period of 7 days, rats were perfusion-fixed with buffered formaldehyde. Brains were then removed, fixed for a further 12 hours, paraffin embedded, sectioned and stained with hematoxylin and eosin. Sections at approximately -3.8 mm from the bregma (10) were examined and the numbers of live neurons in the CA1 regions of hippocampus of the left and right hemispheres were determined.

Adenosine measurements To determine the amount of adenosine in specific brain regions, 32 rats were sacrificed by

focused microwave (Cober Electronics, Stamford, CT) irradiation 7 or 30 min post-reperfusion as described previously (1, 11). Immediately after microwave irradiation, brains were removed and placed on an ice-cold surface. From both sides of the brain, a sagittal slice was cut at 4.0 mm lateral to the midline. From each sagittal slice, the hippocampus was removed and then cut coronally at the anterior extent of the hippocampal fissure, to give the CA2/3 hippocampal subregion, and then at the posterior extent of the dentate gyrus. The hippocampus was then sliced along the hippocampal fissure between the two coronal cuts to obtain the CA1 (dorsal) and CA3/dentate gyrus (ventral) subregions. From the same sagittal slices, striatal and frontal cortex regions were also dissected. Dissected regions from both sides of each brain were combined for adenosine analysis, which was performed as described previously (11).

Results

Forebrain ischemia resulted in loss of pyramidal neurons in the CA1 region of the

hippocampus. In rats injected with saline 30 min prior to ischemia and evaluated 7 days post-

ischemia, 44.5 + 7.8% (mean + SEM, n = 12) of the CA1 neurons had pyknotic nuclei and

eosinophilic cytoplasm. This was signficantly greater (P < 0.05, t-test) than the 24.0 + 5.2% (n = 12)

of neurons in hippocampus from rats that had been treated with pre-ischemic injections (i.p.) of

NBMPR-P (Figure 1A). The number of apparently healthy neurons in the CA1 region of the

hippocampus was also significantly different between the two groups: 130.3 f: 19.1 neurons/mm for

saline treated and 182.5 + 13.4 neurons/mm for NBMPR-P treated rats. The NBMPR-P injection was

associated with a slight decrease in body temperature in the early post-reperfusion period although

this did not reach statistical significance (Figure 1B).

In contrast, we found no evidence for improved survival of CA1 neurons following post-

ischemic injections (i.p.) of NBMPR-P (Figure 1C). However, NBMPR-P did affect body

temperature, with statistically significant decreases detected at 30 min (0.9OC) and at 60 min (0.8”C)

post-injection (Figure 1 D).

Adenosine levels were measured in cortex, striatum and hippocampal subfields at 7 or 30 min

post-reperfusion to determine the extent to which pre-ischemic injections (i.p.) of NBMPR-P affected

post-ischemic brain adenosine levels (Table 1). As reported previously, (1, 12), adenosine levels

decreased rapidly post-reperfusion, a factor that contributed to the variability of these measurements.


A

P c 0.05

lYll3 Saline NBMPR-P

B D

1 Saline

7 NBMPR-P

Post-reperfusion (hours) Post-reperfusion (hours)

Figure 1. Effects of pre- and post-ischemic i.p. injections of saline or NBMPR-P on post- reperfusion body temperature and neuronal survival. Rats were injected with 10 mg/kg NBMPR- P (circles) or an equivalent volume (-0.5 ml) of saline (squares) 30 min prior to (A, B; n = 12) or 30 min following (C, D; n = 5) initiation of forebrain ischemia. A, C. Both live and dead hippocampal neurons in the CA1 subfield were counted 7 days post-ischemia. Both the injections and the histological evaluations were performed by an individual who was unaware of the experimental conditions. Bars represent means + SEM. B, D. Rectal temperatures were monitored for 4 hours after reperfusion. Symbols represent means + SEM. *P < 0.05; **P < 0.0 1; unpaired t-tests.

In this study, adenosine levels were 6-fold (striatum) to 27-fold (frontal cortex) higher at 7 min than

30 min post-reperfusion. With only one exception (striatum, 30 min) adenosine levels were greater in

NBMPR-P treated rats at 7 or 30 min post-reperfusion. The adenosine levels at 7 min post-

reperfusion were analyzed by repeated measures 2-way ANOVA. To satisfy the assumption of

equivalence of variation, the data were log-transformed. This analysis indicated that NBMPR-P

treatment caused a statistically significant (P = 0.03) increase in adenosine levels. No effect of brain

region was detected, indicating that NBMPR-P treatment affected adenosine levels similarly in all

brain regions tested.

Discussion

Nucleoside transport inhibitors are predicted to exhibit site- and event-specific effects (13).

During normal physiological conditions adenosine levels are low and inhibition of adenosine uptake


Table 1. Adenosine levels (pmol/mg protein) in selected brain regions following global forebrain ischemia. Rats were given intraperitoneal injections of saline or NBMPR-P (10 mg/kg) 30 min prior to onset of forebrain ischemia. Ischemia was maintained for 6 min. Seven or 30 min after the onset of reperfusion, rats were killed by focused microwave irradiation (10 kW, 1.9 set). Brains were dissected and analyzed for adenosine content in frontal cortex, striatum, and the hippocampal subfields CA1 , CA2/CA3 and CA3/dentate gyrus (DG). Values are means & SEM; n =lO (7 min) or 6 (30 min). A repeated measures 2 way ANOVA was performed on the 7 min values after log transformation and a significant (P = 0.03) effect of NBMPR-P treatment was identified.

Brain Region

Frontal cortex

S triatum

Hippocampus CA1

CA213

CA3/DG

Post-reperfusion Iniection (min) Saline NBMPR-P

7 30 7 30

7 30 7 30 7 30

838 Ifr 184 1158+289 31+3 49 + 8

651 + 125 871 + 142 105 + 32 95 + 22

1017+ 179 1164+ 143 80 Ik 22 119+46

774 + 171 863 + 69 80+ 12 120+38

744 + 162 1039+ 174 88 k 15 105 + 30

is predicted to have little effect on adenosine’s levels or receptor-mediated effects. In contrast, during

conditions that promote adenosine formation, such as hypoxia or ischemia, transport inhibitors are

predicted to enhance adenosine levels and to potentiate adenosine receptor-mediated effects. In the

present study, we found that NBMPR significantly increased survival of CA1 neurons if it was

administered prior to the ischemic event. This treatment regimen also increased adenosine levels in

cortex, striatum and hippocampus, supporting the hypothesis that enhanced adenosine concentrations

are required for the pharmacological effects of NBMPR,

NBMPR and NBMPR-P cross the blood-brain barrier weakly. In a previous study, we

demonstrated that 30 min after i.p. injections of 10 mg/kg NBMPR-P plasma levels of 5 PM and CSF

levels of 24 nM NBMPR were measured (7). In contrast, 30 min after i.c.v. injections of 50 nmol

NBMPR-P, 1 pM NBMPR was detected in CSF (1). In light of the low permeability of brain to

NBMPR, the neuroprotective effects detected in the present study are intriguing. One possible

explanation is that ischemia increased the permeability of the blood-brain barrier (14) and enhanced

the permeability of the brain to NBMPR. Alternatively, it is possible that NBMPR may act at the


blood-brain barrier to reduce fluxes of adenosine from the brain to the blood. Previously, it was

found that intravenous administration of dipyridamole, a brain impermeant inhibitor of ENTl and

ENT2, also enhanced adenosine levels in brain (15).

Decreasing body temperature is an effective neuroprotective strategy; previously it was

reported that decreases of 1 .O - 2.O”C prolonged for at least 24 hours can protect hippocampal CA1

neurons from ischemic injury (16). In the present study, pre-ischemic injections of NBMPR-P

decreased body temperature by 0.2 - 0.4”C for up to 4 hours post-ischemia. These temperature

effects were not significantly different from control animals and are, thus, unlikely to explain the

enhanced neuronal survival that was observed. A statistically significant decrease in body

temperature was recorded following post-ischemic injections of NBMPR-P; this treatment did not

affect neuronal survival indicating that this degree of hypothermia was not sufficient to produce

neuroprotection.

Our findings indicate that the effects of NBMPR are mediated by adenosine: NBMPR is a

potent inhibitor of ENTl transporters, it increased post-ischemic adenosine levels, it was effective

when administered prior to the ischemia-induced increases in adenosine level, and it was ineffective

when administered following the return of adenosine to basal levels. Studies to assess the role of

other nucleoside transporter subtypes, including ENT2 and CNT2, in regulating the neuroprotective

effects of adenosine are warranted.

The authors thank Drs. Dale Corbett and Don Smyth for helpful comments and Mrs. Cheang for statistical consultations. This work was supported by the Heart and Stroke Foundation of Manitoba and by the Canadian Institutes of Health Research (CIHR) of Canada. FEP is an Investigator of CIHR and the Manitoba Health Research Council.

References [l] Parkinson FE, Zhang YW, Shepel PN, Greenway SC, Peeling J, Geiger JD. Effects of

nitrobenzylthioinosine on neuronal injury, adenosine levels, and adenosine receptor activity in rat forebrain ischemia. J Neurochem, 2000;75:795-802.

[2] Rudolphi KA, Schubert P, Parkinson FE, Fredholm BB. Adenosine and brain ischemia. Cerebrovasc Brain Metab Rev, 1992;4:346-369.

[3] Sweeney MI. Neuroprotective effects of adenosine in cerebral ischemia: window of opportunity. Neurosci Biobehav Rev, 1997;2 1:207-2 17.

[4] von Lubitz DK. Adenosine and cerebral ischemia: therapeutic future or death of a brave concept? Eur J Pharmacol, 1999:371:85-102.

[5] Cass CE, Young JD, Baldwin SA. Recent advances in the molecular biology of nucleoside transporters of mammalian cells. Biochem Cell Biol, 1998;76:761-770.


Gati WP, Paterson AR. Measurement of nitrobenzylthioinosine in plasma and erythrocytes: a pharmacokinetic study in mice. Cancer Chemother Pharmacol, 1997;40:342-346. Anderson CM, Sitar DS, Parkinson FE. Ability of nitrobenzylthioinosine to cross the blood-brain barrier in rats. Neurosci Lett, 1996;2 19: 19 l-l 94. Sutherland GR, Peeling J, Lesiuk HJ, Brownstone RM, Rydzy M, Saunders JK, Geiger JD. The effects of caffeine on ischemic neuronal injury as determined by magnetic resonance imaging and histopathology. Neuroscience, 199 1;42: 17 l-l 82. Delaney SM, Sutherland GR, Peeling J, Geiger JD. Failure of 2’-deoxycoformycin to protect against transient forebrain ischemia in rat. Neurosci Lett, 1993; 149:3 l-34. In: Paxinos G, Watson C, editors. The Rat Brain in Stereotaxic Co-ordinates. Sydney:Academic

Press, 1986. Delaney SM, Geiger JD. Brain regional levels of adenosine and adenosine nucleotides in rats killed by high-energy focused microwave irradiation. J Neurosci Methods, 1996;64: 15 1- 156. Phillis JW, Smith Barbour M, O’Regan MH. Changes in extracellular amino acid neurotransmitters and purines during and following ischemias of different durations in the rat cerebral cortex. Neurochem Int, 1996;29: 115 120. Geiger JD, Parkinson FE, Kowaluk EA. Regulators of endogenous adenosine levels as therapeutic agents. In: Jacobson&A, Jarvis,MF, editors. Purinergic Approaches in Experimental Therapeutics. New York: Wiley-Liss; 1997. p 55-84. Preston E, Foster DO. Evidence for pore-like opening of the blood-brain barrier following forebrain ischemia in rats. Brain Res, 1997;761:4-10.

[ 151 Phillis JW, O’Regan MH, Walter GA. Effects of two nucleoside transport inhibitors, dipyridamole and soluflazine, on purine release from the rat cerebral cortex. Brain Res, 1989;48 1:309-3 16.

[ 161 Nurse S, Corbett D. Neuroprotection after several days of mild, drug-induced hypothermia. J Cereb Blood Flow Metab, 1996; 16:474-480.

effects of nitrobenzylthioinosine on adenosine levels and neuronal injury in rat forebrain ischemia

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