Fructose-1,6-bisphosphate after hypoxic ischemic injury is protective to the neonatal rat brain

BRAIN RESEARCH ELSEVIER

Brain Research 741 (1996) 294-299

Research report

Fructose-1,6-bisphosphate after hypoxic ischemic injury is protective to the neonatal rat brain 1 Augusto Sola a,*, Margarita Berrios a R. Ann Sheldon b Donna M. Ferriero ~,b George A. Gregory a,c,d ~ Department of Pediatrics (Neonatology), University of California, San Francisco, CA, USA b Department of Neurology, University of California, San Francisco, CA, USA Department of Anesthesia, Universit3, of California, San Francisco, CA, USA d The Cardiovascular Research Institute, Unirersity of California, San Francisco, CA, USA

Accepted 30 July 1996

Abstract Fructose-l,6-bisphosphate (FBP) has been shown to attenuate central nervous system injury in adult animals. We evaluated whether FBP given after an ischemic-hypoxic insult is protective to the developing brain in a neonatal rat model of hypoxia-ischemia. Postnatal day 7 rat pups were subjected to focal ischemia followed by global hypoxia and then administered either FBP or saline intraperitoneally. A dose of 500 m g / k g or greater of FBP significantly reduced the amount of injury such that 55% of FBP- vs. 17% of saline-treated rats had no injury; 6% of FBP- and 47% of saline-treated rats had severe damage ( P = 0,004). There was less infarcted brain in FBP-treated rats (12 + 11% vs. 37 _+ 32%; P = 0.005); and fewer FBP-treated rats had > 30% ipsilateral cortical injury (12% of FBP- vs. 50% of saline-treated rats; P = 0.002). FBP lowered serum calcium levels during the first 24 h after the insult without significant changes in ionized calcium or osmolarity. These results indicate that FBP treatment administered systemically after hypoxia-ischemia reduces CNS injury in neonatal rats. Keywords: Brain injury; Hypoxia; Ischemia; Development: Therapy: Neonate: hypocalcernia

1. Introduction

Fructose-l,6-bisphosphate (FBP) is a naturally occurring high-energy intermediary metabolite of glycolysis that reduces tissue damage associated with cardiac arrest, myocardial infarction, myocardial ischemia and renal ischemia [14,23,24,26,27]. In vitro studies show that FBP protects astrocytes from hypoxic injury [12], possibly by maintaining ATP concentration by decreasing intracellular ATP depletion [11]. In addition, glutamate uptake and glutamine production appears to be FBP-dependent under hypoxic conditions in astrocytes [15]. Cerebral endothelium incubated with FBP is also protected from hypoxic

* Corresponding author. Division of Neonatology, University of California San Francisco, 533 Parnassus Ave San Francisco, CA 94143-0734, USA. Fax: + 1 (415) 476-9976; E-mail [email protected] i This study was presented in part at the APS/SPR Annual Meeting, Seattle, Washington, 1994.

injury and again preservation of ATP is seen intracellularly [10l. In adult animals, FBP provides significant protection for central nervous system (CNS) injury [4-6,17,34]. In particular in adult rats, FBP reduces infarct volume after reversible middle cerebral artery occlusion and improves functional neurological outcome [17]. However, the effect of this compound in neonatal models of hypoxia-ischemia has not been adequately studied. In one series of studies in neonatal piglets by LeBlanc et al. [19-21], FBP did not ameliorate hypoxic-ischemic brain injury. Since perinatal asphyxia and neonatal hypoxic-ischemic brain injury are significant and unresolved clinical problems, and because FBP has proven to be beneficial in other models of hypoxia-ischemia, it is important to determine if FBP given after hypoxia-ischemia has beneficial effects on the CNS in neonatal animals. The Levine procedure in the rat has been extensively used as a model of hypoxic-ischemic CNS injury in the developing brain [8,22,29,31,35]. Theretore, we used this procedure in neonatal rats to determine

0006-8993/96/$15.00 Copyright © 1996 Elsevier Science B.V. All rights reserved. PI1 S0006- 8 9 9 3 ( 9 6 ) 0 0 9 8 4 - 5

A. Sola et al./ Brain Research 741 (1996)294-299 the efficacy of FBP in preventing CNS injury after hypoxia-ischemia.

2. Materials and methods We used a model of hypoxia-ischemia based on the Levine procedure as modified by Rice et al. for the neonatal rat [8,22,29,31,33]. On postnatal day 7, 158 Sprague-Dawley rat pups from 18 litters were studied. The studies were approved by the University of California San Francisco Committee on Animal Research and were performed with the highest standards of humane care, as set forth in the Guide for the Care and Use of Laboratory Animals, US Department of Health and Human Services, Publication Number 85-23, 1985.

2.1. Hypoxic-ischemic injury At postnatal day 7, 96 animals were anesthetized with 2.5% halothane, 15% N20, balance 02, and the right common carotid artery was exposed and occluded by electrical coagulation (Levine or surgical group). The incision was sutured, and the pups were returned to their dams immediately after the surgery for at least 2 h to recover and feed. The non-surgical control pups (n = 36) were removed from and returned to the dam at the same time as the animals in the surgical group. All these 132 pups were then placed in 1-1 airtight containers partially submerged in a 37°C water bath through which a humidified atmosphere of 8% O z and 92% N 2 was introduced via inlet and outlet tubing. The body temperature of one animal in each container was monitored by external probe throughout the hypoxic period to maintain body temperature between 35-36°C by adjusting the water bath temperature. The 132 pups remained in the hypoxic container for 2.5 h. Four died before the end of this period (3 in the surgical group and one in the non-surgical control group). The 128 pups that survived were randomized for injection of FBP (trisodium salts, Sigma Chemical Co., St. Louis, MO) or saline (0.1 ml i.p.) immediately after removal from the containers. The assignment of dose and FBP- vs. salinetreatment was by block design and was performed randomly and blindly. Both the non-surgical control group (n = 35) and the Levine group (n = 93) were subdivided into 5 subgroups each. In the non-surgical control group, 17 pups received placebo and 18 received one of four FBP doses. In the Levine group, 44 rats received placebo and 49 were treated with FBP at different doses: 250 m g / k g (n = 16), 500 m g / k g (n = 13), 750 m g / k g (n = 10) and 1000 m g / k g (n = 10).

2.2. Preparation and histologic scoring of brains The brains of the rat pups were studied 5 days after the hypoxic-ischemic insult when edema is fully resolved and

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scar formation has been completed [8]. All pups were killed with 50 m g / k g pentobarbital i.p. and were then perfused through the ascending aorta with cold 4% paraformaldehyde in 0.1 M phosphate buffer (pH 7.4). Brains were removed, immersed in the same fixative for 4 h and then transferred to ice-cold 30% sucrose in 0.1 M phosphate buffer. Forebrain sections were cut coronally at 50-~m intervals using a vibratome. Sections were stained with Cresyl violet and then scored from 0 to 2 for degree of damage, as follows: 0, no detectable neuronal cell loss or gliosis; 1, columnar damage in the cortex involving predominantly layers II through IV; and 2, contiguous areas of gliosis with resulting neuronal cell loss including all layers and cystic lesions [1]. Since the damage is diffuse, as long as the grade of injury is present in some location, the highest grade is given. That is, if there is laminar necrosis in frontal regions but cystic infarction parietally, a grade of 2 is assigned.

2.3. Brain infarction The extent of brain infarction was determined by measuring the area of residual cortex with a video image analysis system using NIH Image 1.47. All residual cortex, regardless of damage, is included in the analysis. The cortex of the left and right (contra- and ipsilateral) hemispheres of three contiguous coronal sections from each brain were measured at the level of the anterior hippocampus by tracing the image, measuring the area in rnlTl 2. The mean value of the three measurements for each cortex is obtained and used for comparisons. The measurement of the contralateral cortex (no ischemia) is considered as the reference area. The measurement of the residual area of the cortex of the side where the carotid artery is ligated (ipsilateral cortex) is compared to the measurement of the contralateral cortex and data are expressed as percent of tissue loss or injured in the ipsilateral cortex (contralateral area - residual ipsilateral area divided by contralateral area X 100). The preparation, histological scoring and measurements of tissue loss of the brains were performed by an observer blinded to the group assignment.

2.4. Blood chemistry analysis An additional 30 rat pups were used to evaluate the effects in blood chemistries of FBP. None of the brains of these animals was used for analysis. In 2 litters of pups (n = 14) receiving 500 m g / k g FBP after hypoxiaischemia, we measured plasma calcium, phosphorus, ionized calcium concentration and serum osmolarity at different times after FBP administration. Since the maximum blood volume of these rat pups is 0.8-1.0 ml; each time we obtained the blood samples (0.6 ml) for these biochemical analysis we immediately killed the animals as described above, in order to avoid survival in hypovolemic shock. Therefore, it was impossible for us to measure

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baseline values and compare them to different time points after treatment in the same rat pup. For this blood chemistry, the rats were killed at 1, 2, 24 and 72 h after administration of FBP. For normal control values, 16 animals from 2 litters received an equal volume of saline and the same blood chemistry was obtained at the same time points postsaline. The statistical analysis was performed by Wilcoxon signed rank sum test, Fisher's exact test, and odds ratio with exact 95% confidence intervals where appropriate. Logistic regression analysis was performed to determine if any litter effect was present. Electrolyte data was compared by an unpaired t-test. Differences were considered significant at a P-value less than 0.05.

5O

40 Percent of Rat Pups with a Histological Injury 30 Score of 2 20

10

0

n=

-

-

-

-

-

-

-

F250

S

F500

S

F750

S

FIO00

S

16

14

13

11

10

9

10

10

-

F 250, 500, 750, 1000 = FBP dose (mg/kg) S - Saline placebo control

Fig. 1. Percent of rat pups with severe brain injury in each of the individual FBP-treated groups and their saline-treated controls. FBP 250 mg/kg had no significant beneficial effect.

3. Results No rats died after the hypoxic period was completed and randomization to FBP or placebo had been done. In the non-surgical control group (hypoxia without ischemia; n = 35), the brain of rats treated with saline (n -- 17) showed no histological damage (score of 0) and the measured areas of cortex of the right and left hemispheres of the brain were equal. FBP-treated non-surgical controls (n = 18) also had no evidence of injury in either hemisphere (Table 1). Rat pups subjected to the Levine procedure developed injury to the ipsilateral (right) frontoparietal cortex and lateral caudate as previously described [8,29]. Pups receiving 250 m g / k g of FBP following the Levine procedure (n = 16) had similarly abnormal histological scores and ipsilateral area of injury compared to their saline-treated counterparts (n = 14) (data not shown). Pups treated with 500 m g / k g or greater of FBP after h y p o x i a - i s c h e m i a had significantly less ipsilateral injury than the placebo controls. Fig. 1 shows one example of the different effects of FBP dosing. As shown, between 45 and 50% of rat pups in each of the four saline-placebo control groups had evidence of severe brain injury, with grade 2 histological scoring of their brains. Similar findings occurred in the group of 16 rats treated with 250 m g / k g of FBP (44% with grade 2 histological score) (Fig. 1). In contrast, a

histological brain injury score of 2 was present in only 0 - 1 0 % in each of the individual groups of animals treated with 500, 750 and 1000 m g / k g of FBP. There were no differences in the presence of this injury score of 2 whether the dose given after carotid artery ligation and hypoxia was 500, 750, or 1000 m g / k g of FBP (FBP 750 and 1000 m g / k g vs. 500 m g / k g : P > 0.2; Fig. 1), but each of these groups was different from the one that received a dose of FBP of 250 m g / k g ( P = 0.03-0.04). In addition, the respective P-values for each comparison between individual FBP dosage groups and their saline controls for this histological score of 2 showed no beneficial effect of FBP 250 m g / k g ( P = 0.9), but a significant trend or effect of doses of 500 m g / k g or greater (FBP 500 m g / k g , P = 0.033; 750 m g / k g , P = 0.06; and 1000 m g / k g , P = 0.05). Based on these results, the animals treated with 500 m g / k g or more of FBP (n = 33) are presented together and compared as an entire group to the corresponding saline placebo controls (n = 30) (Table 1). 3.1. H i s t o l o g i c a l s c o r e s

Cellular damage was less severe in animals that received FBP 500 m g / k g or greater after the Levine procedure. A score of 0, with no detectable neuronal cell loss,

Table 1 Histological score and percentage of brain injury in each treatment group Group

Histological score 0 n (%)

Non-surgical control (hypoxia only)

Saline (n = 17) FBP (n = 18)

17 (100) 18 (100)

Hypoxia-ischemia (' Levine')

Saline (n = 30) FBP500-1000mg/kg(n = 33)

5 (16) 18(55)

a Mean _+standard deviation. b p 0.004 for FBP vs. saline histologic severity scores. P = 0.005 for FBP vs. saline percentage brain loss. =

I n (%) 0 0 11 (37) 13(39)

Percentage of brain loss 2 n (%) 0 0 14 (47) b 2(6)

(mean) 0 /I 37 + 32 "'~ 12_+11

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was found in 55% of FBP-treated rats and in 16% of saline-placebo rats. Contiguous areas o f gliosis and neuronal loss through the cortex a n d / o r caudate was observed in only 6% o f FBP-treated rats, while 47% o f saline controls had this degree of injury. The distribution of the histological scores (Table 1) was significantly better in the FBP-treated pups ( P = 0.004).

time point after FBP. As shown, there was a significant depression of serum Ca lasting 24 h after administration with a significant change in ionized calcium only at 1 h after FBP. Serum osmolarity did not change significantly from the control state.

4. Discussion 3.2. Brain infarction

There was a significant difference in the area of brain loss due to brain infarction in the FBP-treated rats. The percentage of brain loss in the ipsilateral cortex was significantly less in the brains of FBP-treated rats, with a mean loss ( _ S . D . ) o f 12% ( + 1 1 % ) compared to 37% ( + 3 2 % ) in the saline controls ( P = 0.005). The extreme values for this percentage o f brain loss varied between 0 and 65% in the brains of FBP-treated rats and between 5 and 96% in the saline control animals. In addition, only 10% of the rats treated with F B P exhibited more than 30% loss o f the ipsilateral cortical area, while 50% of the rats in the saline control group exhibited at least that much loss ( P = 0.002). Finally, the likelihood of developing any form of brain injury by 7-day-old neonatal rat pups exposed to the Levine procedure was significantly decreased after treatment with F B P (odds ratio of 0.26 (confidence interval 0.06-0.90)). 3.3. B l o o d chemistries

Results o f blood chemistries after FBP administration and h y p o x i a - i s c h e m i a performed in 30 additional rats are shown in Table 2. Since the values do not change with time in the saline control group we used the values of the 16 rats as the normal reference for comparison to each

W e have used a standard model for testing therapies for neonatal h y p o x i a - i s c h e m i a [8,29,31] and found that FBP effectively ameliorated brain damage when at least 500 m g / k g was given after the h y p o x i c - i s c h e m i c insult. Animals receiving F B P were injured less frequently and had lower injury severity scores than saline-treated animals. These findings suggest that brain injury in the 7-day-old rat caused by h y p o x i a - i s c h e m i a can be interrupted by intermediaries o f metabolism, such as FBP. The uniqueness of the immature brain and the pathophysiology o f injury in the developing brain have been well recognized [8,29,35]. F o r example, in this same model of 7-day-old rat pups, Ferriero et al. [8] reported that cells containing N A D P H - d are selectively spared in the developing striatum and cortex during hypoxia ischemia and Vanucci et al. [35] showed that 99% o f 2-DG is converted to 2-DG-6 phosphate with increases in cerebral glucose utilization in both hemispheres, suggesting that glucose is metabolized via anaerobic glycolysis to maintain cellular energy production. Our findings in this neonatal model are in keeping with previous data in various adult models. However, our resuits contradict those o f LeBlanc et al. [19-21] who investigated the role of F B P in a neonatal piglet model of h y p o x i a - i s c h e m i a where FBP did not ameliorate the CNS damage. There are several dissimilarities between those

Table 2 Effects of FBP on calcium and phosphorus after hypoxic-ischemic brain injury n

Ca (mg/dl)

P (rag/d1)

Ca I (mmol/1)

Osmolarity (mmol/kg)

4 4 4 4 16

9.35 ± 0.10 9.36 _+0.10 9.30 ± 0.15 9.38 ± 0.11 9.35 _+0.13 (9.2-9.5)

14.10 + 2.6 13.20 ± 2.8 13.70 _+2.9 13.40 ___2.7 13.50 ± 2.85 (10.9-16.9)

1.37 ± 0.15 1.44 + 0.09 1.49 + 0.15 1.36 + 0.12 1.38 ± 0.13 (1.22-1.55)

290.00 ± 6.1 303.00 _+ 10.0 301.00 _+ 3.2 292.00 ± 8.0 294.00 ± 8.9 (284.0-305.0)

5.37 ± 0.25 a 5.80 _+0.10 b 6.20 ± 0.30 c 7.57 ± 0.51

42.33 __+2.02 38.67 ± 1.16 37.33 + 5.03 29.17 4- 2.57

1.13 + 0.03 d 1.30 ± 0.07 1.55 + 0.13 1.57 ± 0.04

288.00 + 3.8 290.00 ± 2.1 301.00 + 3.1 304.33 _+ 5.0

Saline (control)

lh 2h 24 h 72 h Total Normal range Time after FBP 500 m g / kg

lh 2h 24 h 72 h

4 4 3 3

Values are the mean + standard deviation. a p = 0.0001 for FBP vs. saline, calcium at 1 h post-treatment. b p = 0.001 for FBP vs. saline, calcium at 2 h post-treatment. c p = 0.001 for FBP vs. saline, calcium at 24 h post-treatment. d p = 0.02 for FBP vs. saline, ionized calcium at 1 h post-treatment.

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studies and ours that may explain the differences in the results. The model that they employed produced severe global ischemia with reperfusion, while our model is one of focal ischemia without reperfusion. The animals in LeBlanc's studies had both carotid arteries occluded followed by induced hypotension until there was no electrical activity of the brain. In addition, the doses of FBP used by LeBlanc et al. (350 m g / k g or less) were significantly less than those shown to be protective in our study. In fact, we also did not see protection in neonatal rats when providing a low dose (250 m g / k g ) after the ischemia and the hypoxic period. Protective effects of FBP have been shown with doses between 100 and 300 m g / k g only when it is administered before ischemia or reoxygenation [5,33]. In contrast, 350-600 m g / k g of FBP was required to reduce or prevent injury in adult animal models when used after the hypoxic-ischemic event [6,17]. Since our study was designed to determine if this intermediary of metabolism had any central nervous protection in a previously well described neonatal experimental model of hypoxia-ischemia, we did not measure blood concentrations of FBP and cannot comment on pharmacokinetics of FBP after peritoneal administration. Gregory et al. [12] found that astrocyte protection begins at 0.5 m m o l / l of exogenous FBP and was maximum at 3 mmol/1. Intravenous doses of 75 and 150 m g / k g of FBP have been reported to produce serum levels of 0.6 and 4.5 mmol/1, respectively, in newborn piglets [21] and therefore relatively high concentrations would be expected at the doses used in our study. From the present study with intraperitoneal administration, we cannot comment whether an intravenous dose, a continuous infusion, or any other different approach for administration of FBP would produce different effects in the central nervous system. Although it is possible that with peritoneal administration plasma concentrations of FBP are different or more sustained, it has been reported that FBP has a short plasma half-life [23]. Furthermore, the effects of FBP on brain protection are probably not dependent solely on its extracellular concentration. The precise mechanisms of action of FBP are not completely understood. Three hypotheses exist for the mechanisms of FBP protective effects. The first, promoted by Markov et al. [25], attributes FBP's effects to enhancement of anaerobic carbohydrate metabolism following the uptake and utilization of FBP as a high-energy substrate or as a regulator of glycolysis [7,16]. The second hypothesis proposed by Galzigna and by Bernasconi, suggests that FBP increases intracellular pH, either by electrolyte flux or H ÷ flux without FBP entering the cell [1,9,30]. The third hypothesis [ 13,18] proposes that FBP protects by binding Ca 2÷ and reducing intracellular Ca 2÷. Studies have demonstrated reduced uptake of Ca 2+ in myocardial tissue and decreased ionized calcium in cardiac perfusion media in the presence of FBP [13]. However, there are

other possible mechanisms of action, including the maintenance or recovery of high-energy phosphates (e.g., ATP and cAMP) after ischemia [6,7], prevention of oxygen free radical-induced injury [28,32]; activation of glutamine synthetase [ 15] and protection of cerebrovascular endothelial cells from hypoxic injury [10]. Recently, another possible mechanism for protection has been reported: FBP has been shown to reduce Ca 2+ uptake by hypoxic-ischemic astrocytes in culture [2] and by brain slices [34]. In these models, damage produced by hypoxia-ischemia is associated with enhanced calcium uptake into vulnerable regions of the brain. Calcium accumulation in brain was maximal between 0 and 4 h after injury and the uptake in brain correlated directly with plasma Ca levels [34]. Similar results have been reported in adult models of forebrain ischemia [3] and in immature rats [33] with the development of cell necrosis correlating directly with calcium accumulation in the damaged regions. In our study, we have shown a significant decrease in serum calcium concentrations after FBP administration at a time when the delayed neuronal necrosis would be occurring, i.e., during the first day after the insult. The decrease in serum calcium, which may be secondary to the increase in serum phosphorus concentration, could be an additional mechanism of protection, by making less calcium available for uptake by hypoxic-ischemic cells. From the present study, we cannot comment whether the phosphate load has any potential undesired metabolic, renal or organic effects. Preliminary results of toxicity studies of FBP currently being performed do not reveal any renal, hepatic or cerebral toxicity, even at doses four times higher than the ones used in this study. However, if intravenous dosing of FBP is found to produce the same degree of increase in phosphorus concentration as the one found in this study with peritoneal administration, this could become a limiting factor for safe utilization in critically ill infants. In summary, FBP at a dose of at least 500 m g / k g given after a hypoxic-ischemic insult reduces injury to the brain of 7-day-old rats. In addition, we have shown that serum calcium concentrations decrease significantly after FBP administration. Future studies will attempt to determine mechanisms of action and whether the reduction in calcium is one of the important mechanisms by which FBP reduces CNS injury after hypoxia-ischemia in the developing nervous system.

Acknowledgements Supported by N I H / N I N D S Grant NS32533. The authors thank Cory Fergus and Deborah Farrell for their assistance with graphs and with preparation of the manuscript.

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