Magnesium pre-treatment reduces neuronal apoptosis in newborn rats in hypoxia–ischemia

Brain Research 955 (2002) 133–137 www.elsevier.com / locate / brainres

Research report

Magnesium pre-treatment reduces neuronal apoptosis in newborn rats in hypoxia–ischemia a, b c *, Zafer Turkyilmaz ¨ ¨ ¨ Canan Turkyilmaz , Yildiz Atalay a , Figen Soylemezoglu , ¨ Bulent Celasun d a

¨ , 06300, Ankara, Turkey Department of Pediatrics, Gazi University Hospital, Kizlarpinari Cad 31 /10, Kec¸ioren b Department of Pediatric Surgery, Gazi University Hospital, Ankara, Turkey c Department of Pathology, Hacettepe University Hospital, Ankara, Turkey d ¨ Military Medical Academy, Ankara, Turkey Department of Pathology, Gulhane Accepted 5 August 2002

Abstract Hypoxic–ischemic brain damage has significant mortality and morbidity in newborns. Although the role of magnesium in neonatal hypoxic–ischemic brain injury related to N-methyl-D-aspartate receptors has been widely studied; the effects of magnesium on neuronal apoptosis have not been known exactly in hypoxia–ischemia. The aim of this study was to investigate the effects of magnesium on neuronal apoptosis in the 7-day-old rat hypoxia–ischemia model. Seven-day-old rats were administered magnesium sulfate (group 1; n59) or saline (group 2; n59) intraperitoneally before hypoxia–ischemia. Additionally 18 seven-day-old rats were given magnesium sulfate (group 3; n59) or saline (group 4; n59) after hypoxic–ischemic insult. Neuronal apoptosis was investigated by the dUDP-biotin nick end-labeling (TUNEL) method following 3-day recovery in all subjects. In evaluating TUNEL-positive cells, we firstly calculated the areas (mm 2 ) of brain regions, hippocampus, striatum, cortex, in right and left hemispheres in subjects by IMAGE analysis. The numerical density was calculated as the number of cells per square millimeter by counting all TUNEL-positive cells. Afterwards, the ratio of right side numeric density to sum of right and left side numeric densities (right Apoptosis Index) was calculated for every brain region in rats receiving magnesium and compared to vehicle groups. The right Apoptosis Index of the hippocampus in magnesium pre-treated rats (mean6S.D.; 36.6622.1) was significantly lower than vehicle (61.0616.0; P,0.05); whereas right apoptosis indices were not changed by magnesium pre-treatment in striatum and cortex. Additionally, magnesium sulfate administration following hypoxic–ischemic insult also had no effect on right apoptosis indices in all three brain regions. It is concluded that magnesium might have a role in preventing neuronal apoptosis due to neonatal hypoxic–ischemic brain injury.  2002 Elsevier Science B.V. All rights reserved. Theme: Disorders of the nervous system Topic: Ischemia Keywords: Apoptosis; 7-Day-old rat; Hypoxia–ischemia; Magnesium; Regional

1. Introduction Despite the advances in obstetrics and neonatology, perinatal hypoxic–ischemic brain injury (HIBI) is still a significant cause of death and neurological impairment in newborn infants [37,40]. N-Methyl-D-aspartate (NMDA) receptor mediated cascade results in increased calcium *Corresponding author. Tel.: 190-312-358-5700; fax: 190-312-3170743. ¨ E-mail address: [email protected] (C. Turkyilmaz).

entry and triggers neuronal injury in hypoxia–ischemia (HI) [12,25,29]. This NMDA-related mechanism eventually leads to the activation of caspases and results in apoptosis [4,5]. The activity of NMDA receptor ion channel complex can be modulated by Mg as a noncompetitive antagonist [10,23,25,29,37]. Magnesium (Mg) is also suggested to regulate apoptosis by controlling the nuclear membrane proteins in brain [32]. Apoptosis is a distinct form of neuronal death which may play an important role in developing HIBI [1,9,19,24]. Apoptosis differs from necrosis by being programmed, delayed,

0006-8993 / 02 / $ – see front matter  2002 Elsevier Science B.V. All rights reserved. PII: S0006-8993( 02 )03395-4

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genetically controlled, having different morphological characteristics, and requiring energy [1,3,5]. Recently, the role of apoptosis has been investigated in HI animal models [3,31]. The TUNEL method has been widely used in detecting DNA fragmentation as a marker of apoptosis [2,13,25,27,31]. The role of Mg on neuronal apoptosis in the 7-day-old rat HI model has not been throughly studied. The aim of this study was to investigate the effects of Mg when administered before or following HI in reducing neuronal apoptosis and assessing any distributional differences between different brain regions in the 7-day-old rat HI model.

2. Material and methods

2.1. Subjects Thirty-six Wistar rat pups were enrolled into the study. They were divided into four groups. In pre-treatment groups, nine subjects were administered magnesium sulfate (200 mg / kg) (group I) and nine subjects, as vehicle, were administered 0.9% NaCl (group II) in the same volume intraperitoneally just before HI. In post-treatment groups, nine subjects were given magnesium sulfate at the same dose (group III) and nine subjects were administered 0.9% NaCl (group IV) just after HI. Additionally, four normoxic and nontreated rat pups were also studied as control. All experimental protocols were pre-approved by the Animal Ethical Committee of Gazi University Hospital.

2.2. Surgical procedure The modified Levine–Rice procedure [11,15,38], the most often used model of neonatal HI, was used. The right carotid artery was ligated in all subjects that were anaesthetized by placing them in a jar with cotton wood containing methoxyflurane. Additional doses were administered if required. Rat pups rested with the dam for 2 h after surgical procedure. Then they were exposed to 8% oxygen with 92% nitrogen in a 500-ml air-tight jar in 37 8C water bath for 60 min. Finally, the pups were allowed to recover for 72 h with their mothers. They were sacrificed under deep pentobarbital anaesthesia (100 mg / kg, intraperitoneally) and brains were removed for pathological examination.

2.3. Image analysis Cut surface areas of hippocampus, striatum, cortex of the right and left hemispheres were calculated as square millimeters by image analysis performed with a Nikon Labophot 2A microscope, Pentium 233 MMX,128 MB RAM, IBM compatible PC, Matrox Millenium VGA video

card and frame grabber. Zeiss Vision KS400 version 3.0 was used for macro.

2.4. Pathological examination In coronal brain sections, apoptosis was identified through DNA terminal deoxynucleotidyl transferase-mediated dUDP-biotin nick end-labeling assay using a commercially available kit (ApopTagPlus in Situ Apoptosis Detection Kit, Oncor Inc, Gaithersburg, MD, USA). All brain sections were examined under a light microscope with 320. In evaluating TUNEL-positive cells, we first calculated the surface areas (mm 2 ) of hippocampus, striatum, and cortex of the right and left hemispheres in all subjects by image analysis. The numeric density was calculated as the number of cells per square millimeter by counting all TUNEL-positive cells in every region. Afterwards, right side apoptosis indices expressed as percent [(numeric density of right hemisphere / numeric densities of right and left hemispheres)3100] was determined for hippocampus, striatum and cortex in rats receiving Mg before HI and compared to vehicle. Apoptosis indices (AI) were also calculated for every brain region in subjects receiving post-treatment Mg and compared to vehicle groups. Apoptotic cells have regularly shaped, round condensed or fragmented chromatin, cytoplasmic shrinkage and apoptotic bodies. When evaluating TUNEL positivity, a few cells containing diffusely stained or irregularly fragmented chromatin or breakdown of nuclear and plasma membranes or swollen cytoplasms were observed. These cells were accepted as necrotic cells and excluded from calculations.

2.5. Data analysis All values were expressed as mean6S.D. ANOVA and post-hoc tests in SPSS statistical analysis system were used in statistical evaluations. A P-value less than 0.05 was considered significant.

3. Results The right side AI in hippocampus was significantly decreased in Mg pre-treatment group (P,0.05), when compared to vehicle. There were no significant differences of right AIs of striatum and cortex between Mg-pretreated and vehicle groups. (Table 1). There were also no significant differences between the right side AIs of Mg posttreatment and vehicle group in all three regions (P.0.05). The mean of AIs of an additional four normoxic and nontreated control subjects was found to be below 5% in all three regions, but a statistical comparison was not performed.

¨ et al. / Brain Research 955 (2002) 133–137 C. Turkyilmaz Table 1 Right side apoptosis indices (mean6S.D.) of the magnesium pre- and post-treatment and vehicle groups in hippocampus, striatum and cortex Group

Mg pre-treatment (n59) (200 mg / kg) Vehicle pretreatment (n59) Mg post-treatment (n59) Vehicle posttreatment (n59)

Apoptosis Index Right hippocampus

Right striatum

Right cortex

36.6622.1*

55.7617.1

46.3617.1

61.0616.0*

57.5628.0

58.3623.0

46.968.6

48.8619.6

50.3623.5

60.0614.0

59.1626.0

58.0619.0

*P,0.05.

4. Discussion We found that Mg which was given prior to HI reduced apoptosis detected by TUNEL positivity in hippocampus in neonatal rat model. HIBI is a significant cause of mortality and morbidity in newborns. A large number of experimental studies gave rise to a better understanding and management of perinatal HI. The 7-day-old rat model of HI is one of the most frequently used methods for investigating different treatment modalities [11,15,18,38]. Neuronal apoptosis is a distinct mode of cell death that differs from necrosis by being delayed, programmed, genetically controlled, and having morphological characteristics [3]. It has been shown that neuronal apoptosis besides necrosis had an important role in HIBI [1,9,13,19,31]. Although increased calcium influx induced by NMDA receptors is known to be a major component of HIBI in newborns, the relationship between NMDA receptor activation and neuronal apoptosis has not been throughly investigated. The potential neuroprotective role of Mg via modulation of NMDA receptor has been investigated in animal and human studies [6,10,14,21,30,34–36]. NMDA-mediated Ca influx causes massive disruption of cellular, nuclear, and mitochondrial functions as well as triggering neuronal apoptosis. Calcium entry may activate caspases besides other cell degrading enzymes and result in apoptosis [3–5]. Magnesium can prevent Ca influx through the NMDA receptor as a noncompetitive antagonist [10,20,25,29,37]. Magnesium is also suggested to regulate apoptosis by controlling the nuclear membrane proteins in brain [32]. On the other hand, there is a connection between NMDA-mediated events and nitric oxide (NO) pathway and free oxygen radical generation [8,12,25,29]. NO contributes in producing a series of toxic species and radicals causing apoptosis [39]. Other possible antiapoptotic roles of Mg include the inhibition of nitric oxide synthase activation in certain brain regions. Some pharmacological agents which block the NMDA receptor have been introduced to prevent or treat neuronal apoptosis. However, antiapoptotic effects of

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Mg administered before hypoxic–ischemic insult in the 7-day-old rat model of HI have not been studied extensively. In the present study, Mg administered intraperitoneally to 7-day-old rat pups before HI caused a decrease in neuronal apoptosis in the hippocampus. Magnesium as a NMDA receptor antagonist may block all pathways, at the first step, which eventually results in neuronal apoptosis. Down-regulation of NMDA receptors is another possible explanation for the antiapoptotic effect of Mg. Thus, the antiapoptotic effects of Mg seem to be related to blockage of the NMDA receptors. Therefore, Mg pre-treatment may reduce neuronal apoptosis due to HI in 7-day-old rat pups. However, Mg post-treatment did not decrease apoptosis in three brain regions: the hippocampus, striatum, and cortex in this study. One explanation for the lack of antiapoptotic effect of Mg post-treatment may be the continuity of NMDA-mediated cascade, when once triggered and resulting in inevitable apoptosis. So Mg posttreatment may not stop Ca influx once the receptor is activated. Although Mg treatment following hypoxia has been recently reported by Malik et al. [22] to decrease apoptosis in fetal guinea pigs, their study design and experimental model were different from our study. Moreover, the amount of apoptosis and differences in efficacy of treatment modalities may vary between species (e.g. rat vs. guinea pigs) and between maturity levels of brains (e.g. fetus vs. neonates). It has been found that Mg pre-treatment was effective in reducing neuronal apoptosis in the hippocampus whereas it had no effect in striatum and cortex. This may be explained by the relative vulnerability of neurons to HI in hippocampus. Thus, blockage of this cascade by Mg may be more remarkable in reducing apoptosis. Furthermore, this regional difference in neuronal vulnerability to HI may be due to alterations in density and distribution of NMDA receptors in the brain. Certain neurons in different brain regions like the hippocampus may have more NMDA receptors. So blocking of these receptors by Mg may be more prominent in the hippocampus. Despite the contradictory results in literature, the hippocampus seems to be one of the most HI vulnerable and sensitive regions of the brain [28,41]. There are only a few reports about selective sensitivity of brain regions to apoptosis [26]. Although the mechanism of selective vulnerability to HI is not fully understood, some metabolic factors, the presence of trophic and growth factors and synaptic connectivity may play a role. Different suicide programmes, and their activators and inhibitors in these regions may affect the selective antiapoptotic effect in HI in neonates. As a result, this regional vulnerability may lead to regional differences in the responses of the treatment which may give rise to new approaches in the selection of combined treatment modalities in the future. Although some researchers expressed limited confidence in TUNEL because of shortcomings in sensitivity and

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specificity because TUNEL is blamed for staining necrotic cells [7], several authors had used TUNEL positivity in order to detect apoptotic cells in tissue staining [2,13,16,17,27,31]. We did not have any technical difficulty in staining and detecting apoptotic cells and, a few cells having diffusely stained or irregularly fragmented chromatin or breakdown of nuclear and plasma membranes or swollen cytoplasms were accepted as necrotic cells and excluded from calculations. So we suggested that TUNEL was acceptable in describing apoptosis in the 7-day-old rat model of HI. Mean right AIs of normoxic rat pups was observed to be extremely low. The presence of apoptotic cells in normoxic subjects shows that apoptosis is a process which normally occurs in the developing brain. However, they were homogeneously distributed in both hemispheres and considered to have little importance for the results of this study. The 7-day-old rat HI model with unilateral artery ligation was used in this study. Pathological changes occurred in this model while both hypoxia and ischemia were created. This model also provided the contralateral hemisphere as an internal control while ipsilateral HI was created [33,37]. For these reasons, the treatment groups were only compared to vehicles in the present study. In conclusion, neuronal apoptosis due to hypoxic–ischemic injury may likely be prevented by pre-treatment of Mg in the 7-day-old rat model. Our results suggested that Mg might be used in preventing HIBI in newborns. From the clinical point of view, maternal use of Mg (before HI) may reduce HIBI by decreasing neuronal apoptosis. Further studies of the antiapoptotic role of magnesium are needed for a better understanding of the mechanism and of using Mg as a prophylactic modality in newborns suffering from perinatal asphyxia.

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