Mechanisms of perinatal brain damage and protective possibilities

Drug Discovery Today: Disease Mechanisms

DRUG DISCOVERY

TODAY

Vol. 3, No. 4 2006

Editors-in-Chief Toren Finkel – National Heart, Lung and Blood Institute, National Institutes of Health, USA Charles Lowenstein – The John Hopkins School of Medicine, Baltimore, USA

DISEASE Perinatal disorders MECHANISMS

Mechanisms of perinatal brain damage and protective possibilities H. Hagberg1,2,*, C.I. Rousset2, X. Wang2, C. Mallard2 1 2

Perinatal Center, Department of Obstetrics and Gynecology, East Hospital, 41685 Go¨teborg, Sweden Perinatal Center, Department of Physiology and Sahlgrenska Academy, Go¨teborg, Sweden

Randomized trials show that hypothermic treatment after birth attenuates neuromotor disability/death offering evidence that neuroprotective treatment is

Section Editor: Vineta Fellman – Division of Pediatrics, Lund University, Lund, Sweden

a clinical possibility in neonates. To develop more effective protective strategies for term and preterm infants, research has focused on understanding the mechanisms underlying brain lesions in grey and white

immature brain, provide a summary of selected therapeutic agents that are already in clinical use and that could be considered for clinical trials and present a few examples of interventions that hold promise for the future.

matter. The key mechanisms will be reviewed and some agents for neuroprotection that are already in clinical use and novel potential interventions will be highlighted. Introduction Research on the pathophysiology of perinatal brain injury has resulted in information about the mechanisms behind the heightened central nervous system (CNS) vulnerability in extreme prematurity, infection/inflammation and hypoxiaischemia (HI). Such data is critical for development of novel neuroprotective strategies that, hopefully, could contribute to reduce serious disability in children and adults. Recent data in both animals and human infants have shown that brain injury evolves over a period of hours to days following the insult and several treatments administered only after the insult can substantially reduce damage in animals. Hence, recently published clinical trials, based on experimental work in animal models, demonstrated that post-insult hypothermia can produce some reduction in death or disability in term infants [1,2]. The objective of this review is to provide an update of the most crucial hypoxic-ischemic injury mechanisms in the very *Corresponding author: H. Hagberg ([email protected]) 1740-6765/$ ß 2006 Elsevier Ltd. All rights reserved.

DOI: 10.1016/j.ddmec.2006.11.003

Brief summary of pathophysiological mechanisms in the immature brain Cerebral HI of sufficient severity to deplete tissue energy reserves (primary insult) is often followed by transient but complete restoration of glucose utilization, ATP, and phosphocreatine upon reperfusion [3,4]. Thereafter, a secondary decrease of high-energy phosphates occurs in parallel with a decrease in tissue utilization of glucose and development of cell injury [3–5]. Such a biphasic response to HI opens up a therapeutic window following HI before the secondary phase of tissue deterioration and indeed successful interventions after the insult in animals and now recently in human asphyxiated newborns [1,2] support this concept. The mechanisms leading to secondary brain injury are still partly unknown but excitatory amino acids (EAAs), intracellular calcium, reactive oxygen species (ROS), mitochondrial impairment, inflammation and apoptotic processes all seem to be involved [6–9] (Fig. 1).

Excitotoxicity and mitochondrial impairment HI induces an increase of EAAs (glutamate, aspartate) in the extracellular space in grey [10] and white [11] matter leading to activation of N-methyl-D-aspartate (NMDA) 397

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Figure 1. Summary of pathophysiological mechanisms in the grey and white matter. Abbreviations: AIF, apoptosis inducing factor; Akt,; AMPA, alphaamino-3-hydroxy-5-methyl-4-isoxazole propionic acid; Bax, Bcl-2 associated x protein; Bcl-2, B-cell lymphoma/leukemia 2; Cyt, cytochrome; EAAs, excitatory amino acids; GSH, glutathione, reduced form; ICAD, inhibitor of caspase activated DNase; IL, interleukin; JNK, c-jun C-terminal kinase; KA, kainic acid; MOMP, mitochondrial outer membrane permeabilization; NMDA, N-methyl-D-aspartate; NO, nitric oxide; PI3K, phosphatidylinositol 3-kinase; PKB, protein kinase B; ROS, reactive oxygen species; TLR, Toll like receptor; TNF, tumor necrosis factor; VDCC, voltage dependent calcium channels.

and alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA)/kainic acid (KA) receptors on neurons and oligodendroglial precursors [12–14] (Fig. 1). Activation of receptor-operated as well as voltage-dependent channels will evoke intracellular accumulation of Ca2+, which in turn will trigger activation of proteases and lipases that will contribute in the injury cascade. EAAs also initiate activation of nitric oxide synthase (NOS) leading to the production of nitric oxide (NO), which together with other ROS (superoxide and hydroxyl radicals) produce peroxynitrate and may have harmful effects on DNA, proteins and cellular membranes. In addition, intracellular Ca2+ overflow, ROS and NO will exert adverse effects on mitochondria that play a key role in the cellular cascade leading to cell death. Mitochondrial impairment could have serious consequences, for example lead to respiratory failure, production 398

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of ROS and release of pro-apoptotic proteins through mitochondrial outer membrane permeabilization (MOMP) [15,16] (Fig. 1).

Apoptotic mechanisms It is now generally accepted that HI in the immature brain leads to cell death with a morphologically mixed necrotic-apoptotic phenotype [17]. Recent biochemical studies suggest, however, that both caspase dependent and caspase independent apoptotic mechanisms participate in the injurious process [9,18,19]. There is some evidence that the extrinsic pathway is activated (either by Fas ligand or TNF-a) after HI leading to activation of caspase-8 and caspase-3 [20]. In addition, the intrinsic (mitochondrial) pathway is activated through MOMP leading to release of cytochrome C, assembly of the apoptosome, activation of caspase-9 and subsequently activation of caspase-3

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(see [9] and references therein). Marked activation of caspase-3 results in cleavage of multiple proteins in the cell including inhibitor of caspase activated DNase (ICAD) leading to DNA fragmentation and cell death. In addition, other proapoptotic proteins like apoptosis inducing factor (AIF) [19], SMAC/ Daiblo [21], and HtrA2/Omi [21] translocate from the mitochondria to a nuclear localization. AIF trigger DNA damage in the nucleus and contribute to HI brain injury [22]. It is still uncertain what triggers MOMP but there are data suggesting that an increased pro- versus anti-apoptotic Bcl-2 protein family balance is important [23,24] leading to translocation of Bax to mitochondria [25], which could be partly triggered by a combination of failing trophic factor support (decrease of Akt) and activation of c-jun N-terminal kinase 3.

ROS ROS are produced in the electron transport chain in mitochondria, in prostaglandin metabolism, by xanthine oxidase and by NADPH-oxidase in inflammatory cells. Superoxide can generate hydrogen peroxide through the action of superoxide dismutase (SOD) that, owing to deficient expression of glutathione peroxidase and catalase, is not degraded but generates hydroxyl radicals in a reaction promoted by free iron (Fe2+). Superoxide, hydroxyl radicals and peroxynitrate (above) may all be injurious to cells as they can cause protein nitrosylation, DNA damage and lipid peroxidation (see [8,26] and references therein).

Inflammation HI induces activation of microglial cells and/or macrophages in the brain starting as early as six hours after the insult [27]. Neutrophils accumulate in post-capillary venules in early reperfusion and T-cells and NK-cells infiltrate in injured areas of the brain during the delayed recovery phase [28,29]. Several pro-inflammatory cytokines (IL-1a, IL-1b, IL-18, TNF-a, IL-6) and chemokines (MIP-1a, MIP-1b, Rantes, MCP-1, MIP2, gro, IP-10, MCP-3) are often produced by microglia but sometimes also by astrocytes (see [30,31] and references therein). These proteins are crucial for attracting and activating inflammatory cells and for coordination of the immune response. The initial phase of inflammation probably serves the purpose to scan the region of injury for microbes to combat and limit a possible infectious process to the benefit of the host but at the cost of additional brain injury [32]. The pro-inflammatory phase thereafter shifts to an anti-inflammatory response and later to a repair phase. There are several studies showing that interventions that attenuate the proinflammatory phase confer neuroprotection.

Mechanisms of injury in white matter The very immature white matter in preterm infants is highly susceptible to various insults including HI. Also brain damage resulting from severe asphyxia in term infants often involves

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the white matter [8]. This means that neuroprotective treatment has to be able to reduce white as well as grey matter damage and even though several factors mentioned above like ROS [33–35] and apoptosis [36,37] may be crucial for development of injury also in the white matter, there may be mechanisms that differ. The late oligodendrocyte progenitors have been shown to be selectively vulnerable to HI [8,33], which may be related to their sensitivity to ROS [33], expression of Ca2+ permeable AMPA/KA (GluR4) receptors in the somata [12] or NMDA receptors in the processes [14]. Even if the axons appear to be less sensitive to HI [38], the interplay between axonal and oligodendrocyte injury remains to be elucidated.

Experimental interventions using agents that are in clinical use Anti-excitotoxic drugs In Table 1, experimental data is presented that support a protective effect of three anti-excitotoxic drugs, topiramate, xenon and BW1003C87. Topiramate is an anti-convulsant that acts as an AMPA/KA receptor antagonist, inhibitor of voltage dependent Na+ channels and enhancer of GABAmediated chloride fluxes. Topiramate is in use as an antiepileptic drug for adults and children over the age of three years. Experimental data suggest that white matter injury is reduced by topiramate through blockage of AMPA/KA receptors present on pre-oligodendrocytes [12]. Recently, some support has emerged that AMPA receptor inhibition may account also for its neuroprotective ability in grey matter [39,40]. Considering the clinical benefit of hypothermia [1,2] it is interesting to note that topiramate appears to act synergistically with delayed hypothermia (Table 1) [41], and generally, to combine hypothermia and pharmacologic agents is an attractive approach to widen the therapeutic window. Xenon is believed to mainly act as an NMDA receptor antagonist [42] but activation of the two-pore domain K channel [43] or blockage of calcium- and/or calmodulin-dependent protein kinase-II [44] could also contribute to its neuroprotective efficacy. It is important to emphasize that xenon, for unknown reasons, does not seem to cause widespread apoptosis [45] as shown for other NMDA-receptor antagonists [46], which may be related to its effect on the dopamine system [42]. Xenon is very expensive and unless cost-efficient methods of delivery are developed this may severely hamper its clinical use. Lamotrigine (a pre-synpatic Na+ channel and glutamate release inhibitor) represents another anti-epileptic that is registered for use in humans and that has been shown to be protective in adult animals [47]. There are no experimental data on lamotrigine as a neuroprotectant in immature animals but it’s structurally and functionally similar Na+ channel inhibitor, BW1003C87, has been shown to be effective but suffering from a narrow therapeutic window (Table 1). www.drugdiscoverytoday.com

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Proposed mechanism of action

Animal model and treatment protocol

Outcome

Use in humans

Refs

Topiramate (10, 30 or 50 mg/kg i.p.) over 48 h, immediately after hypoxia

AMPAa/KAb receptor antagonist

Rat, HIc (unilateral carotid ligation and 6% O2 for 1 h) at PNDd 7; Subcortical white matter injury and neurological function were evaluated

Only the 30 mg/kg dose reduced white matter injury (MBPe and ISELf staining). At PND21 the treated rats had an improved motor ability to climb an inclined plane

Yes, FDAg approved for use as an anti-epileptic

[12]

Topiramate (20, 50 or 100 mg/kg i.p. or 50 mg/kg orally) pre- and post-HI

AMPA/KA receptor antagonist

Rat, HI unilateral carotid ligation and 8% O2 for 2 h at PND7. Evaluation of brain injury and cognitive function

Pre- and post-treatment with 50 or 100 mg/kg or 20, 50 or 100 mg/kg after HI reduced infarction in grey and white matter. Pre- and post-orally or i.p. was more efficient than post-HI treatment. Treatment starting 2 h after HI was not effective. Pre-treatment (50 mg/kg) improved ability in the Morris water maze at 8 weeks of age

Topiramate in low dose (20 mg/kg i.v. 1 h post-HI + 10 mg/kg  day for 2 days) high dose (50 mg/kg + 20 mg/kg  day for 2 days) compared with vehicle

AMPA/KA receptor antagonist, inhibits voltage dependent Na channels, enhances GABAh-mediated chloride flux

Piglet (2–5 days) HI (bilat cartotid ligation, hypotension, reduction of FiO2 until reduction of ECoGi activity by 85%). Neurology evaluated at 20, 28, 44, 52 h and brain injury at 68 h after HI

Brain injury was reduced in cerebral cortex, hippocampus and striatum in the high dose group and in temporoparietal cortex in the low dose group. Neurological deficits and seizure activity tended to be reduced in the high dose group but the results were not statistically significant. The number of apoptotic cells (TUNELj pos) was higher in the frontal white matter in the high dose topiramate

Yes

[78]

Topiramate (30 mg/kg 15 min post-HI + hypothermia 28–29 8C during 3 h starting 3 h after HI

AMPA receptor antagonist

Rat hypoxia-ischemia (unilateral carotid ligation + 8% oxygen for 90 min). Vibrissae stimulation test and histopathology at 1 and 4 weeks after HI

Topiramate or hypothermia alone did not provide protection. Combined hypothermia and topiramate reduced injury and improved sensorimotor function substantially at PND15 and 35

Yes, see Topiramate above and hypothermia has been used in newborns

[41]

Topiramate (1–90 mg/kg i.p. preand post-insult)

AMPA receptor antagonist

Mouse PND5, Ibotenate and AMPA agonist (S-bromowillardiine). Brain injury, apoptosis and cellular response were evaluated

Topiramate (10–90 mg(kg) reduced S-bromowillardiine but not ibotenate toxicity in a dose dependent manner in cortical plate and white matter. Pre- as well as post-treatment was effective. The number of apoptotic cells (caspase-3, TUNEL positive cells) microglial and astroglial cells were reduced and myelin and pre-oligodendrocytes were preserved by 30 mg/kg of topiramate

Yes

[39]

Xenon (0–85% during or up to 24 h after HI alone or in combination with delayed hypothermia)

NMDAk receptor antagonist, activator av 2-pore domain K+ channel

Rat HI (unilateral carotid ligation and 8% O2 for 90 min) at PND7 with recovery up to 30 days after HI with neurological functional and histological evaluation

Hypothermia and xenon provided protection if administered separately, for example 70% of xenon for 90 min at 6 h after HI reduced injury. Combination of hypothermia (35 8C) and xenon (20%) for 90 min starting 4 h after HI attenuated injury from 33 to 14% and restored neurological function to normal if evaluated 30 days after HI

Yes, has been used in humans for anesthesia in higher concentrations than needed for protection

[45]

[40]

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Compound

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Table 1. Anti-excitotoxic agents

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[80]

Anti-ROS treatment

c

b

a

Alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate receptor. Kainic acid. Hypoxia-ischemia. d Postnatal day. e Myelin basic protein. f In situ end-labeling. g Food and drug Administration. h Gamma-aminobutyric acid. i Electrocorticography. j Terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling. k N-methyl-D-aspartate. l Excitatory amino acids.

No, but structurally similar to lamotrigine that is registered for use as an anti-epileptic Pre-treatment reduced brain injury by 46% but post-treatment in doses of 10, 15 or 30 mg/kg was not effective Rat HI (unilateral carotid ligation + 7.7% oxygen for 1 h 40 min). Brain was evaluated by brain weight, infarct volume and morphological score Blocker of voltage dependent Na channels and inhibits the release EAAsl BW1003C87 (before or after HI in doses of 10-30 mg/kg)

The neuropathology score was reduced in cerebral cortex, white matter, hippocampus and the basal ganglia NMDA receptor antagonist, inhibits release of glutamate and dopamine Xenon (50% for 3 h directly after HI)

Rat HI (unilateral carotid ligation and 8% O2 for 90 min) at PND7. Histopathologic evaluation of brain injury 7 days after HI

Yes

[79]

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Free radicals, including ROS, are important in the pathogenesis of perinatal brain injury and as shown in Table 2, several antoxidants and/or free radical scavengers, such as melatonin, N-acetylcysteine (NAC), edaravone and deferoxamine show promise as neuroprotective agents in the immature brain. Melatonin is an endogenous hormone that is secreted by the pineal gland and that plays an important role in circadian rhythm regulation and has successfully been used clinically to treat sleep disorders in handicapped children [48]. Furthermore, melatonin reduces oxidative stress in association with neonatal septicaemia [49] and decreases malondialdehyde and nitrite/nitrate levels in the blood of asphyxiated newborns [50]. Experimental studies have shown that melatonin, even when given after an excitotoxic insult in neonatal mice or following intrauterine asphyxia in sheep can reduce, particularly white matter, injury [51,52]. The doses given in the fetal studies were higher (20 mg/kg, i.v. infusion for 6 h) compared with the effective doses used in neonatal mice (0.005–5 mg/kg), but resulted in no significant effects on fetal morbidity, suggesting that melatonin has a safe and broad effective dose range. Melatonin may also be a particularly interesting drug for clinical use in the perinatal setting as it crosses the placenta and blood brain barrier, which is supported by studies showing protective effects of maternally administered melatonin on fetal brain injury [53]. NAC, which is an acetylated sulphur-containing amino acid, crosses the placenta [54] and blood brain barrier [55]. Furthermore, its profile as a GSH precursor, anti-oxidant, anti-apoptotic as well as an anti-inflammatory agent makes it an interesting substance, acting at multiple neuroprotective sites. NAC is in clinical use for paracetamol intoxication and has been given in high doses as an antidote also to pregnant women [56]. Interestingly, experimental studies show that NAC is neuroprotective, in several different experimental settings, such as fetal LPS exposure [57] and excitotoxicity in neonatal animals [51]. Furthermore, it appears safe in combination with hypothermia and also improves functional outcome in rats subjected to HI [58]. Edaravone (3-methyl-1-phenyl-2-pyrazolin-5-one) is a novel free radical scavenger, which appears to increase eNOS, while decreasing nNOS and iNOS. It is also a potent scavenger of hydroxyl radicals inhibiting not only hydroxyl radicals but also iron-induced peroxidative injuries [59]. Both treatment before and after experimental HI appear to be beneficial; however, short-term treatment after HI appears to be optimal, whereas treatment for longer periods may be less effective [60,61]. At present edaravone is approved for clinical use for treatment of stroke in Japan and some protective effects have been reported [62]. Unbound iron contributes to hypoxic-ischemic brain damage by catalyzing the formation of free radicals www.drugdiscoverytoday.com

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Compound

Proposed mechanism of action

Animal model and treatment protocol

Outcome

Use in humans

Refs

Melatonin 0–5 mg/kg, i.p. 0, 4 or 8 h after ibotenate

Antioxidant

Excitotoxicity by intracerebral ibotenate injection (10 mg) in PNDa5 mice. Brain injury evaluated at PND10 and PND20.

Reduced white matter injury at doses of 0.005–5 mg/kg. The protection in the white matter was apparent from 48 h to 2 weeks after ibotenate. No effect on grey matter injury

For children with sleep disorders

[51]

Melatonin Maternal administration (1 mg bolus, then 1 mg/h for 2 h, starting before occlusion)

Antioxidant

Umbilical cord occlusion in near term fetal sheep; hydroxyl radical formation by microdialysis in grey matter. Injury evaluation immunohistochemically by 4-hydroxynonenal (lipid peroxiadtion) and DNA/RNA fragmentation (8-hydroxydeoxyguanosine immunoreactivity

Reduced hydroxyl radical and lipid peroxidation in the fetal brain after melatonin, but no effect on DNA/RNA fragmentation

[53]

Melatonin Fetal administration, 20 mg/kg/h, for 6 h, i.v, starting after occlusion

Antioxidant, anti-inflammatory, anti-apoptotic

Umbilical cord occlusion in preterm fetal sheep. Brain injury evaluation in grey and white matter 4 days after occlusion. 8-Isoprostane and GSH in serum, 2–96 h after occlusion

Reduced number of activated microglia and TUNELb positive cells in white matter and attenuated levels of isoprostanes in fetal blood

[52]

N-acetyl cysteine + hypothermia Systemic hypothermia (30 8C) for 2 h after HI. NACc (50 mg/kg, i.p.), daily until sacrifice (48 h for 4 weeks).

Antioxidant, increases glutathione levels

HId (carotid artery ligation + 8% O2, 2 h) in PND7 rats. Infarct volume was evaluated after 48 h of reperfusion; ipsilateral brain hemisphere volume and white matter injury at 2 or 4 weeks. Negative geotaxis and cliff aversion tests at 24 h and 1 week

Neonatal reflexes were improved at days 1 and 7 and brain volumes and myelin preserved at 2 and 4 weeks after HI

N-acetyl cysteine Maternal pre-treatment (2 h) with NAC (50 mg/kg, i.p.)

LPS (i.p.) to pregnant rats at E18. Brain tissues collected for RNA and IHCe on E18, E19, E20 and PND9, 16, 23 and 30. Grey and white matter and expression of cytokine mRNA evaluated

Reduced cytokine activation, death of oligodendrocyte progenitors, fCD11b/OX42-positive microglia cells and diminished myelin loss

[57]

N-acetyl cysteine 0–250 mg/kg, i.p. immediately after ibotenate

Excitotoxicity by ibotenate (10 ug) in PND5 mice. White and grey matter evaluated at PND10

NAC doses of 25 and 250 mg/kg reduced cortical plate and white matter injury

[51]

PND7 rats subjected to HI by carotid artery ligation + 8% O2 (2 h). Neuroprotective effect evaluated by behavioral test and histological analysis

Two day treatment improved learning and memory capability and morphological recovery. Five day treatment showed morphological improvement but no behavioral improvement. 10 day treatment did not show either morphological or behavior improvement

Free radical scavenger, increases eNOS (beneficial NOSg for rescuing ischemic stroke) and decreases nNOS and iNOS (detrimental NOS); scavenger of hydroxyl radicals

Approved in Japan as a neuroprotectant for the treatment of stroke

[58]

[60]

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Edaravone (3-methyl-1-phenyl2-pyrazolin-5-one) 9 mg/kg, i.p. after HI every 24 h for 2, 5 or 10 consecutive days

Antidote for paracetamol (acetaminophen) intoxication

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402

Table 2. Anti-ROS treatment

PND7 rats with HI (carotid artery ligation + 8% O2, 2 h). Tissue collection for western blot and IHC at 0, 24, 48 h and 7 days after HI

Reduced brain injury, including inhibition of cytochrome c release from mitochondria to cytosol and caspase-3 activation in cortex and hippocampus between 24 and 168 h post-HI

Newborn piglets subjected to 1 h HI by occluding both carotid arteries and reduced FiO2 until the PCr/Pi ratios had decreased to at least 30% of baseline values. Outcome measured at 24 h after HI by T2-weighted MRI, caspase-3 activation and histology

Treatment improved the energy status and prevented an increase in water T2 values. There was no effect on caspase-3 activity, histologic outcome, or TUNEL-labeling

Deferoxamine 10 min after HI, pups received 100 mg/kg deferoxamine mesylate, s.c.

PND7 mice with HI (carotid artery ligation + 8% O2, 30 min). Neuropathology 7 days after HI

Treatment resulted in reduced brain injury (brain injury score)

[64]

Deferoxamine 5 mins after HI administration of 100 mg/kg deferoxamine mesylate, s.c.

PND7 rats with HI (carotid artery ligation + hypoxia 8% O2, 2.25 h). Edema was assessed at 42 h and neuropathlogy at 30 days after HI

Deferoxamine reduced water content at 42 h after HI and atrophy at 30 days

[63]

Deferoxamine 12.5 mg/kg/d, upon reperfusion and reoxygenation

Chelator of nonprotein-bound iron and anti-oxidant

[61]

Used in humans for iron chelation therapy

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Edaravone (3-methyl-1phenyl-2-pyrazolin-5-one) 3 mg/kg, i.p. immediately before hypoxic exposure and subsequently, every 12 h until the animals were killed

[65]

a

Postnatal day. Terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling. c N-acetyl cystein. d Hypoxia-ischemia. e Immunohistochemistry. f Cluster of differentiation. g Nitric oxide synthase. b

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Table 3. Experimental neuroprotective interventions in HI models Intervention

Mechanism of action

Animal model and outcome

Refs

BAFa, hypothermia or BAF + hypothermia immediately before insult

Pan-caspase inhibitor

Reduced injury in the PND7 rat model of HIb. Protection was stronger with hypothermia + BAF than either alone

[68]

BAF(3 h post-HI)

Pan-caspase inhibitor

Provided substantial decrease of injury in cortex, hippocampus, and striatum in response to HI in PND7 rats

[18]

BAF (2 h and 12 h post-HI)

Pan-caspase inhibitor

Did not confer tissue protection in PND7 rats subjected to HI

[19]

BAF(5 min before and 9 h after insult)

Pan-caspase inhibitor

Did not confer tissue protection in PND7 rats subjected to focal ischemia

[81]

BAF (immediately before HI)

Pan-caspase inhibitor

Reduction of injury in PND7 Rat model of HI

[67]

TAT-Bcl-xL (after HI)

Anti-apoptotic protein

Reduction of injury in PND7 Rat model of HI

[67]

M826 (immediately before HI)

Selective, reversible caspase-3 inhibitor

Reduction of injury in PND7 rat model of HI

[82]

Transgenic XIAPc over-expression in mice before HI

Enhancement of an endogenous caspase inhibitor

Reduction of injury in PND9 mice subjected to HI

[21]

AIFd deficiency (Harlquin mice) and/or Q-VD-OPhe in mice subjected to HI

Downregulation of AIF and/or a broad-spectrum caspase inhibitor

Both AIF depletion and caspase inhibition reduced injury and the two interventions had additive protective effect in response to HI

[22]

JNKf3 KO and wild-type mice subjected to HI

Depletion of a pro-apoptotic protein acting upstream from the intrinsic pathway

Reduction of brain injury in PND7 mice accompanied by a shift in the pro- versus anti-apoptotic Bcl-2 family balance and upregulation of the Akt pathway

[69]

IL-1ra (i.c. before or after HI)

Antagonist of the IL-1 receptor I

Pre-treatment: Reduction of brain damage 14 days after HI; rat model of HI

[83]

IL-1ra (100 mg/kg i.p. before or after HI)

Antagonist of the IL-1 receptor I

Pre- and post-treatment reduced brain injury

[84]

IL-18 knockout and wild-type mice subjected to HI

Depletion of a pro-inflammatory cytokine

Reduction of total tissue volume loss and white matter injury

[85,86]

C1q knockout and wild-type mice subjected to HI mice

Depletion of a complement protein involved in the innate immune response

Reduction of infarct volume after HI and improvement in neurofunctional deficit

[87]

Aminoguanidine (i.p administration once before HI and 3 times daily after)

iNOS inhibition

Reduction of infarct volume in PND10 rats. Rat model of HI

[88]

Caspase-1 knockout mice and wild-type mice subjected to HI

Depletion of the enzyme that activates IL-1 and IL-18

Damage after moderate HI insult was attenuated

[89]

Anti-apoptotic

Anti-inflammatory

a

Bocaspartyl-(OMe)-fluoromethyl-ketone. Hypoxia-ischemia. c X-linked inhibitor of apoptosis. d Apoptosis inducing factor. e Quinoline-Val-Asp(OMe)-CH2-PH. f c-Jun N-terminal kinase. b

(Fig. 1). The immature brain has high iron levels and limited antioxidant defenses, making the iron chelator deferoxamine an interesting candidate for neuroprotection in newborns. Deferoxamine is also used clinically as a chelator of non protein-bound iron and anti-oxidant. Initial experimental studies in both neonatal rats and mice showed reduced edema and long-term improvement in brain atrophy following deferoxamine treatment of hypoxic-ischemic injury 404

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[63,64]. Deferoxamine treatment of neonatal piglets subjected to HI showed less promise, however, these animals were only examined in the short term (24 h) [65].

Novel experimental neuroprotective interventions Table 3 summarizes several interventions supporting the view that anti-apoptotic and anti-inflammatory (or rather interventions that modify the immune response to HI) mechan-

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isms are important. Interlekin-1 receptor antagonist (IL-1ra) was evaluated in a recently published stroke trial in humans [66] but has not been used in neonates and the other interventions listed have not been used in humans at all. Most data support that either enhancement of endogenous antiapoptotic systems [21,67] or broad-spectrum caspase inhibitors [18,22,68] are efficient neuroprotectants; especially agents such as QVD-O-ph that block both caspases acting upstream from MOMP, like caspase-2, and those acting downstream in the apoptotic cascade. Such a concept is also supported by the strong protection afforded by JNK3 inhibition [69], enhancement of Bcl-xL [23] or Bax deficiency [24] all regulating mitochondrial permeabilization. JNK inhibitors are of particular interest as they have been shown to possess a broad therapeutic window [70]. Alternatively, both the caspase dependent and caspase nondependent apoptotic pathways could be inhibited as supported by recent results showing that AIF depletion acts synergistically with QVDO-Ph [22]. Several studies support the attenuation of the early proinflammatory cascade by interaction with IL-1 family receptors (IL-1ra, IL-18 deficiency, caspase-1 deficiency), inhibition of iNOS, blockade of the complement cascade (C1q deficiency) or interference with MAP kinases such as p38 provide protection (Table 3). There are also data showing that PAFantagonists [71], minocyline [72] and thiorphan [73] (the two latter are registered for clinical use) could be protective but where conflicting results have been obtained [74] and/or the mechanisms of action are uncertain.

Concluding remarks It is important to stress that this brief review by no means is a complete list of potential pharmacological agents that could be used for neuroprotection in the setting of the immature brain. For example, drugs such as amiloride [75], erythropoietin [76], and allopurinol [77] are all registered for use in humans and have been suggested to be promising candidates. Secondly, there is a general lack of information for most of the interventions mentioned to what extent the treatment is effective also with a delay after HI (‘post-HI therapeutic window’). Please observe that in the clinical studies hypothermia was initiated at 3–4 h after birth and pharmacological agents also have to be efficient if given with a delay. Some studies imply, however, that combination of pharmacological therapy and hypothermia may be a way to prolong the therapeutic window and to increase the neuroprotective efficacy [41,45]. Thirdly, many of the experimental studies have proven efficacy only with regard to histopathology in the short and not in the long-term and neurological function has not been evaluated. Most studies are performed only in rodents and additional data is required in large animal models (piglets and or fetal sheep) before their clinical potential can be assessed.

Drug Discovery Today: Disease Mechanisms | Perinatal disorders

Acknowledgements This work was supported by the Swedish Medical Research Council 09455 (HH), K2004-33X-14185-03A (CM) and Swedish governmental grants to scientists working in health care (ALFGBG-2863) (HH).

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