Na+/H+ Exchangers and Intracellular pH in Perinatal Brain Injury
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- Uria-Avellanal, C. & Robertson, N.J. Transl. Stroke Res. (2014) 5: 79. doi:10.1007/s12975-013-0322-x
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Encephalopathy consequent on perinatal hypoxia–ischemia occurs in 1–3 per 1,000 term births in the UK and frequently leads to serious and tragic consequences that devastate lives and families, with huge financial burdens for society. Although the recent introduction of cooling represents a significant advance, only 40 % survive with normal neurodevelopmental function. There is thus a significant unmet need for novel, safe, and effective therapies to optimize brain protection following brain injury around birth. The Na+/H+ exchanger (NHE) is a membrane protein present in many mammalian cell types. It is involved in regulating intracellular pH and cell volume. NHE1 is the most abundant isoform in the central nervous system and plays a role in cerebral damage after hypoxia–ischemia. Excessive NHE activation during hypoxia–ischemia leads to intracellular Na+ overload, which subsequently promotes Ca2+ entry via reversal of the Na+/Ca2+ exchanger. Increased cytosolic Ca2+ then triggers the neurotoxic cascade. Activation of NHE also leads to rapid normalization of pHi and an alkaline shift in pHi. This rapid recovery of brain intracellular pH has been termed pH paradox as, rather than causing cells to recover, this rapid return to normal and overshoot to alkaline values is deleterious to cell survival. Brain pHi changes are closely involved in the control of cell death after injury: an alkalosis enhances excitability while a mild acidosis has the opposite effect. We have observed a brain alkalosis in 78 babies with neonatal encephalopathy serially studied using phosphorus-31 magnetic resonance spectroscopy during the first year after birth (151 studies throughout the year including 56 studies of 50 infants during the first 2 weeks after birth). An alkaline brain pHi was associated with severely impaired outcome; the degree of brain alkalosis was related to the severity of brain injury on MRI and brain lactate concentration; and a persistence of an alkaline brain pHi was associated with cerebral atrophy on MRI. Experimental animal models of hypoxia–ischemia show that NHE inhibitors are neuroprotective. Here, we review the published data on brain pHi in neonatal encephalopathy and the experimental studies of NHE inhibition and neuroprotection following hypoxia–ischemia.
KeywordsBrain pHi NHE blockade Neonatal encephalopathy Brain alkalosis Birth asphxyia Neuroprotection Hypoxia–ischemia
Encephalopathy consequent on perinatal hypoxia–ischemia occurs in 1–3 per 1,000 term births in the UK and frequently leads to serious and tragic consequences that devastate lives and families, with huge financial burdens for society . Although the recent introduction of cooling represents a significant advance, only around 40 % survive with normal neurodevelopmental function . There is thus a significant unmet need for novel, safe, and effective therapies to optimize brain protection following brain injury around birth.
In this review, we discuss both preclinical and clinical research studies that have led to a better understanding of the pathophysiology and metabolic pathways following an acute hypoxic–ischemic perinatal event. Magnetic resonance spectroscopy (MRS) has been an important tool to understand the evolution of energy failure and brain intracellular pH (pHi) after hypoxia–ischemia . Phosphorus-31 (31P) MRS has demonstrated an alkaline shift in brain pHi ; those infants with worse cerebral injury on magnetic resonance imaging (MRI) have a more alkaline brain pHi on 31P MRS . It is likely that the rapid shift in brain pHi to alkaline values following hypoxia–ischemia plays an important role in cell death.
The Na+/H+ exchangers (NHE) are a family of ion membrane transport proteins involved in maintaining a normal pHi and cell volume in many mammalian cell types, by extruding protons in exchange for sodium influx into cells in an electroneutral manner. Excessive activation of NHE is likely to lead to the alkaline shifts in pHi and increased cell death via Na+ overload, which promote intracellular Ca2+ entry. Here, we also review the role of NHE in hypoxia–ischemia, the evidence of neuroprotection with NHE blockade in animal models, and the important association of alkaline brain pHi and seizures.
Acute Hypoxic–Ischemic Perinatal Brain Injury in the Term Infant
Perinatal hypoxic–ischemic brain injury in the term baby is a significant problem throughout the world. Hypoxia–ischemia may occur acutely or chronically and is most commonly associated with maternal factors (hypotension, severe hypoxia), cord factors (prolapse, occlusion), placental factors (insufficiency and abruption), and uterine factors (rupture). Neonatal postnatal events such as shock, respiratory, or cardiac arrest can also lead to hypoxic–ischemic injury. Neonatal encephalopathy (NE) is the clinical manifestation of the ensuing disordered brain function and occurs in 1–3 per 1,000 live term births in the UK and other developed world countries . The encephalopathy typically presents as respiratory difficulties, depression of tone and reflexes, subnormal level of consciousness, and often seizures. Serious consequences follow moderate to severe NE; these include death in 10–15 %, cerebral palsy in 15 %, and other significant cognitive, developmental, and behavioral problems in 40 % of survivors . The financial and human costs to the family and society are thus very high.
Following two decades of laboratory studies [7, 8], clinical trials , and recent endorsement from regulatory bodies , therapeutic hypothermia is now the standard clinical care for moderate to severe NE in the UK and developed world. Meta-analysis [2, 10] of three large pragmatic trials [2, 11, 12, 13] show that therapeutic hypothermia reduces death or disability at 18 months with a risk ratio of 0.81 (95 % CI 0.71–0.93) and a number needed to treat of 6–7. Importantly, data are emerging, which confirm that the favorable outcome of cooled babies at age 18 months is associated with favorable outcome at age 7–8 years .
However, hypothermia is only partially effective after neonatal hypoxia–ischemia, and 40 % of all treated infants still suffer severe neurodevelopmental disability or death despite treatment. Additional neuroprotective agents are needed either to augment hypothermic neuroprotection  or to use alone in settings where hypothermia has not been established as standard care.
Pattern of Brain Injury on Conventional MRI
Understanding the fundamental mechanisms underlying brain injury in the newborn brain at term has rapidly advanced in recent years. Using MRI, the patterns of hypoxic–ischemic brain injury that have been described in pathology studies in the past can now be demonstrated in superb detail and resolution in vivo in human neonates [16, 17]. Using MRS, the timing of the evolution of energy failure and changes in brain pHi have been fundamental in suggesting new avenues for neuroprotection.
Predominant basal ganglia–thalamus pattern (BGT) is the most frequent pattern in “acute asphyxia,” affecting bilaterally the central grey nuclei (ventrolateral thalami and posterior putamina) and perirolandic cortex (Fig. 1 middle row). The hippocampus and brainstem can also be involved. This pattern of injury is most often seen following an acute sentinel event, e.g., uterine rupture, placental abruption, or prolapsed cord . The basal ganglia and thalami are susceptible to acute perinatal hypoxic–ischemic injury due to their high metabolic rate and increased concentration of NMDA receptors . Survivors face a range of functional impairments, which include cerebral palsy, feeding problems, speech and language problems, visual and hearing impairment, later seizures, behavioral difficulties, and cognitive impairment. Cerebral palsy affects three quarters of infants with NE with BGT lesions, and the severity of the BGT lesions is the best predictor of motor problems. A normal signal intensity of the posterior limb of the internal capsule within the neonatal period following neonatal encephalopathy is a good predictor of the ability to walk at 2 years .
Watershed predominant pattern of injury typically follows “prolonged partial asphyxia.” The areas involved are the vascular watershed zones (anterior–middle cerebral artery and posterior–middle cerebral artery), affecting white matter and in more severely affected infants the overlying cortex (Fig. 1, bottom row). The lesions can be uni- or bilateral, posterior and/or anterior. Conventional MRI can detect the loss of the cortical ribbon [24, 25]. These lesions may become cystic, atrophy, or develop gliosis . Common associated etiological factors are hypotension, infection, and hypoglycemia . Infants with a watershed pattern of injury have predominantly cognitive impairments often without functional motor deficits. Cognitive deficits include memory impairments, visual–motor or visual–perceptive dysfunction, or increased hyperactivity, sometimes in the absence of functional motor problems [28, 29, 30, 31, 32].
Cerebral Energy Metabolism Following Perinatal Hypoxia–Ischemia
Brain pHi Changes in Babies with Neonatal Encephalopathy
GA at birth (weeks)
Age when studies
Mean brain pHi
Hope et al. 
Median 40 (28–40)
Mean 76 h (16 h–97 days)
7.14 ± 0.10
Hamilton et al. 
Median 32 (28–42)
5 (1–61) days GA + PNA median 35 weeks (28–42)
6.98 ± 0.34 (28 weeks)
Boesch et al. 
GA + PNA median 43 weeks (33 weeks–6 years)
7.08 (SD 0.1)
8 were studied at 40 weeks
Azzopardi et al. 
30 (data from 23)
Median 33 (24–42)
GA + PNA median 34 weeks (26–42)
7.1—no change with maturation
AGA 28 weeks 7.14 (±0.28)
42 weeks 7.09 (±0.28)
SGA 28 weeks 6.97 (±0.24)
42 weeks 7.19 (±0.24)
Laptook et al. 1989 
10 examinations within the first 2 weeks after birth
7.02 ± 0.08
Van der Knapp et al. 
Mean 71 months (1 months–16 years)
7.04 (95 % CI 6.96–7.12)
Volume localized (ISIS)
No significant change
Buchli et al. 
2–28 days GA + PNA mean 42 weeks (39–44)
Volume localized (ISIS)
Martin et al. 
Median 40 (36.3–42.1)
Median 4.3 days
Volume localized (ISIS)
Robertson et al., unpublished data
Median 39 (38–40)
GA + PNA median 44 weeks (41–64)
Volume localized (ISIS)
GA at birth (weeks)
Age when studies
Mean brain pHi
Cady et al. 
7 (3 birth asphyxia)
42 h–26 days
7.2 (SD 0.1)a
One infant studies on 5 occasions (on day 26: brain pHi = 7.4)
Hope et al. 1984 
8 h–27 days (obtained in 3–6 times in each infant)
7.17 ± 0.1 at time of lowest PCr/Pi (mean age 113 h: 16 h–9 days)
Hamilton et al. 
27 Echodensities seen on USS (13 birth asphyxia)
Median 40 (27–42)
Median 3 days (8 h–23 days)
“Tended to be raised.” Values from 19 of the infants were above the regression line for normal infants; 6 were above the 95 % CI
Laptook et al. 
1.5, 8, 15 days, 9 months
38 ± 2
Within first 2 weeks after birth
7.14 (±0.12) Significantly different from the normal infants when other 31P metabolites showed no difference
Azzopardi et al. 
61 (all infants)
3 days (0–23)
7.14 ± 0.27 (n = 61)
40 infants with asphyxia
3 days (1–10)
7.16 ± 0.19 (n = 36)
Martin et al. 
Median 40 (36.9–41.9)
Median 3 days (NNS 1—severe)
Volume localized (ISIS)
Median 5.5 days (NNS 2—moderate)
Median 3 days (NNS 3—mild)
Robertson et al. 
Median 39.5 (36–42)
77 examinations: 25 within 2 weeks age 16 between 2 and 4 weeks
At time of lowest PCr/Pi (within first 2 weeks of age)
Volume localized (CSI)
25 between 4 and 30 weeks
7.23 (±0.07)—abnormal outcome group
11 when >30 weeks old
7.08 (±0.04)—normal outcome group
Robertson et al. 
39.5 (SD 1.6) (36–42)
151 examinations within the first year after birth. 50 infants studies within 2 weeks of age
At time of lowest PCr/Pi (within first 2 weeks of age)
Volume localized (ISIS)
35 (+ 43 presented above)
7.28 (±0.15)—23 infants with severe outcome or died
7.11 (±0.09)—normal outcome group
In vivo data suggest that the return of pHi to normal or alkaline values may be deleterious to cells that have undergone hypoxia–ischemia. This rebound alkalosis has been termed pH paradox and has been described in several cell types [46, 47, 48]. Possible mechanisms leading to pH-dependent injury include activation of phospholipases and proteases, which have an alkaline pKa, onset of the mitochondrial permeability transition pore, leading to uncoupling of oxidative phosphorylation and aggravation of ATP depletion , and an exacerbation of excitotoxic neuronal injury due to an increased NMDA activation at alkaline pHi [50, 51]. An understanding of one of the main transporter groups leading to brain alkalosis after hypoxia–ischemia (the NHEs) is therefore important for future neuroprotection strategies.
The NHEs are a family of ion integrate membrane transport proteins involved in maintaining pHi and cell volume in many mammalian cell types [52, 53], by extruding protons from, and taking up sodium ions into cells in an electroneutral manner (1:1 stoichiometry) (Fig. 5).
To date at least ten NHE isoforms (NHE1 to NHE10) have been identified in mammals . NHE10 was recently described as an osteoclast-specific member of NHE family, regulating osteoclasts differentiation and survival . NHE1–5 are expressed on the plasma membranes in various types. NHE6–9 reside on intracellular organellar membranes of the endosomal-trans Golgi network [56, 57]. NHE isoforms have similar membrane topologies, with an N-terminal membrane domain consisting of 12 predicted transmembrane segments and a more divergent C-terminal cytoplasmic domain [45, 57]. The NHE1 isoform has been studied closely and is present at the cell surface of most cells, especially plasma membranes. It is the most abundant isoform in the CNS [58, 59]. NHE1 plays a crucial role in protecting cells from internal acidification, acting together with bicarbonate-transporting systems and restoring cell volume to steady-state levels .
Intracellular acidosis is the major stimulus that regulates NHE1 activity. As the H+ concentration of the cytosol rises, a rapid increase in the activity of the transporter occurs. Near-maximal velocity is achieved in approximately one pH unit (Hill coefficient >1), thereby minimizing exposure of the cytoplasm to excess acidification [61, 62].
In addition to responding to intracellular acidification, various signals can alter the NHE1 internal pH sensitivity, such as hormones, mitogens, and physical stimuli (e.g., mechanical stretch and hyperosmolarity), modulating its state of phosphorylation [45, 53, 63, 64, 65, 66, 67]. The exchange activity is more active at alkaline pH values due to a shift of the set point by phosphorylation of the residues. It is also regulated at the transcriptional level, supporting the control at mRNA levels and at protein production level [68, 69]. An alkaline cytoplasmic pH (pHc) is thought to provide a permissive environment for the progression of diverse cellular processes, including changes in cell shape , adhesion , migration [72, 73], chemotaxis [74, 75], and proliferation [76, 77, 78].
Effect of Hypoxia–Ischemia on NHE
Reductions in blood flow with ischemia and reduction in oxygen supply with hypoxia decrease the supply of oxygen required to maintain tissue ATP levels, especially in excitable organs such as the heart and brain that have a high demand for energy. As ATP stores are depleted, lactate, pyruvate, and protons accumulate owing to anaerobic metabolism of glycogen stores. The accompanying cytoplasmic acidification causes hyperactivation of plasma membrane NHE1 and the consequent accumulation of intracellular Na+. The Na+ overload reverses the mode of operation of Na+/Ca2+ exchange, driving excess Ca2+ into the cell. The resulting elevation in intracellular Ca2+ concentration, and subsequent activation of proteases, phospholipases, and formation of oxygen- and nitrogen-free radicals, precipitates a cascade of deleterious effects, including altered membrane excitability and contractility, generation of toxic free radicals, cellular hypertrophy, apoptosis, and necrosis—events that result in cell death [79, 80]. Activation of NHE also leads to the rapid normalization of pHi during reperfusion after hypoxia ischemia [45, 54, 81, 82].
Removal of external Na+ or reducing the external pH attenuates the postanoxia alkalization . When ATP levels fall to 35 % of control in isolated CA1 neurons during chemical anoxia, NHE activity is reduced by 44 %. Activation of NHE function, Na+ pump function and the Na+ driving for NHE are all dependent upon ATP energy levels, ATP being required for phosphorylation of the protein.
NH Exchanger Blockade and Neuroprotection in Neonatal Models
Model, study design
In vitro studies
Vornov et al. 
Ex vivo Rodent
17-day rat fetuses
10–12 cell culture
–Neuronal tissue culture model of ischemia (18–19-day culture) from embryonic 17-day rat fetuses
–20 min ischemia with metabolic inhibition (KCN + 2-DG)
–Injury: LDH liberation
–Group 1: ischemic conditions vs. prolonged ischemia (30 min)
–Group 2: incubation with NHE inhibitors at normal pHe (dimethylamiloride and harmaline) slowed pHi recovery
–Profound protective effects: ↓ pHe during 1st hour recovery.
→suggesting protective effects due to intracellular acidosis
–1st demonstration of protective effects of blocking NHE in cerebral ischemia model (during recovery); worst injury if pHi normalizes fast
→acidosis protects: suppressing pH-sensitive mechanisms of injury or blocking Na entry (NHE)
Matsumoto et al. 
Ex vivo rodent
Culture of cortical neurons
–Hypercapnia (5 % CO2) for 10–14 days, then cortical neurons cultured on glass-based dishes
–Assess glutamate-induced neuronal death; neurons morphological change; Ca2+i concentration and pHi
–Some given SM-20220 20 min preglutamate exposure
or MK-801 (NMDA receptor antagonist)
–SM-20220: ↓ glutamate-induced neuronal death over 6 h, inhibited postglutamate exposure: acute cellular swelling, persistent ↑ [Ca2+]i and intracellular acidification
→Neuroprotection: inhibit persistent ↑[Ca2+]i and acidification in excitotoxicity
Robertson et al. 
Ex vivo rodent
14- and 7-day models of rat pups
–Progressive energy decline after HI insult in rat brain slice neonatal model; P31and H1 MRS 350 μm slice
–7-day rat pups brain slices perfused in KHB: (1) at 37 °C; (2) at 32 °C, and 14-day slices perfused for 8 h in similar solutions and then NHE blocker
–14-day pups brain slices perfused for 8 h:
(1) at 37 °C in KHB
(2) at 32 °C in KHB
(3) at 37 °C in HEPES buffer,
(4) amiloride at 37 °C in HEPES
–No gestational age effect on energy decline between 7- and 14-day model
–Brain slice model underwent secondary energy failure
At 5 h: alkaline pHi, ↓ PCr/Pi and ↑ Lac/NAA, and ↓ NTP/PME, at 37°C
–Changes delayed with hypothermia (32o C) or amiloride (pHi acidified and preserved NTP/PME, at baseline and at 5 h)
Kersh et al. 
Ex vivo rodent
3–15-day rat both sex
–Hypercapnia (15 % CO2)
–NH4Cl-induced acidification in brainstem neurons from chemosensitive regions of neonatal rats (brainstem slices from RTNn, NTSn, and LCn)
–pHi recovery mediated by different pH-regulating transporters in neurons from different chemosensitive regions (NHE1 in RTNn; NHE1 and 3 in NTSn; NBC in LCn)
–Recovery suppressed by hypercapnia in all neurons (maintained acidic pH)
Liu et al. 
Ex vivo rodent
1–3-day neonatal mice
–Isolation of mixed primary glial cultures in mice
–Activation of microglia after lipopolysaccharide or oxygen and glucose deprivation and reoxygenation
–Group 1: untreated
–Group 2: HOE 642
–HOE 642 abolished pHi regulation in microglia basal conditions
–Activation of microglia accelerated pHi regulation (↑ pHi, ↑ Na+i and Ca2+i, and production of superoxide anion (SOA) and cytokines (CK))
–HOE 642 abolished pHi regulation, ↓ production SOA, CK and iNOS
–Hypothesis: NHE1 to maintain microglial pHi homeostasis (NADPH oxidase and “respiratory” burst)
In vivo studies
Ferimer et al. 
13 Wistar rats
Cardiac arrest (KCl) in rats followed by resuscitation 7 min later in untreated vs. MIA
–MIA delays normalization of brain pHi after cardiac arrest in rats
–MIA: ↓ cardiac pH in rats postarrest +15 min reperfusion
–MIA doesn’t change pHi from nonischemic value.
Phillis et al. 
21 Sprague–Dawley rats
–Ischemia: 20 min occlusion CA (group 3 30 min), with EEG (flat). Then 40 min reperfusion
–Cortical superfusate (bilaterally every 10 min): free fatty acids (FFA), lactate, and glucose levels
–Group 1 (n = 9): aCSF (control)
–Group 2 (n = 6): EIPA topical (cortex) 35 min pre- and during ischemia
–Group 3 (n = 6): 30 min ischemia
–NHE inhibition prevented activation phospholipases (suppress ↑ FFA during reperfusion)
–EIPA: lactate levels significantly lower by end of experiment
Pilitsis et al. 
24 Sprague–Dawley rats
–Cerebral ischaemia (20 min CA occlusion)
–Measurement of phospholipase activation by efflux of FFA in the ischemic/reperfused rat cerebral cortex
–Group 1: SM-20220 topical (cortex) pre- and during ischemia (n = 13)
–Group 2: control (ischemia) (n = 11)
–↓significantly ischemia-evoked efflux of FFAs: importance NHEs in eliciting FFA efflux
–Inhibition may be essential for neuroprotection in ischemia–reperfusion injury
Kendall et al. 
47 mice 7-day (adult C57/Bl6 female and males bred in-house)
–HI: 2 h left CA occlusion followed by moderate (30 min) or severe (1 h) hypoxia (8 % O2)
–Outcome at 48 h: viable tissue in injured hemisphere (severe HI) or injury score and TUNEL stain (moderate)
–Group 1: MIA intraperitoneal
–Group 2: 0.9 % saline equivalent volume
Given 8 hourly starting 30 min before HI
–MIA neuroprotective when commenced before HI (no weight difference)
–Severe insult: significant neuroprotective (↑forebrain tissue survival)
–Moderate insult: ↓ damage hippocampus
–MIA ↓ neutrophil count and hence brain swelling after HI
Rocha et al. 
3–4-month mice male Swiss-Webster
–Metabolic stress and dopaminergic damage in mice caused by malonate (mitochondrial inhibitor)
–Dialysate levels of DA and metabolites baseline (1 h prior to drug delivery) and afterwards, every 20 min
–Group 1: HOE-642 dialized intracerebral (striatum) 20-min periods, separated by drug washout ≥1 h
–Group 2: EIPA
–Group 3: control (only malonate)
–HOE-642 pretreatment: ↓malonate-induced DA overflow and ↓ striatal DA content, without ↓ intensity metabolic stress or subsequent DAergic axonal damage
–Absence NHE1 on nigrostriatal DAergic neurons suggests HOE-642 effects on striatal DA overflow via NHE1 on other cell types or via multiple NHE isoforms
Hwang et al. 
6 m Mongolian gerbils
–HI by 5 min bilateral occlusion common CA
–Assess delayed neuronal death and immunohistochemistry for NHE1 (at 30 min, 3 h, 12 h and 1, 2, 3, 4, and 5 days following surgery)
–Locomotor activity monitored for 10 days post-HI
–Group 1: normal (sham: same surgical procedure but NO ischemia)
–Group 2: vehicle (saline given)
–Group 3: EIPA OD for 3–9 days after ischemic sugery, starting 30 min postischemic surgery
–↑NHE protein level in CA1 region from 2 days post-HI; activation NHE1 in CA1 glial cells from 2 to 3 days post-HI; in CA1 pyramidal neurons and glial cells(astrocytes) from 4 days
–EIPA potently protected CA1 pyramidal neurons from ischemic injury, and ↓ activation of astrocytes and microglia in ischemic CA1 region
–Hypothesis: role of NHE1 in delayed death
NHE inhibitors protect neurons from ischemic damage
Shi et al. 
–NHE1+/− heterozygous mice
–Wild-type mice SV129/Black Swiss
–NHE1+/− and +/+ litter mate males
–Transient focal cerebral ischaemia and reperfusion (I/R) by 60 min occlusion left MCA
–Activated microglial cells identified by expression of 2 microglial marker proteins (CD11b and Iba1) and by transformation of morphology
–Group 1: vehicle control (equivalent volume of saline intraperitoneal)
–Group 2: HOE 642 intraperitoneal at 30 min prior to the onset of reperfusion, and then daily up to 1–7 days during reperfusion
–Immediate ↑ microglial activation ipsilateral to ischemia in NHE1+/+ brains at 1 h I/1 h R (gradually ↓ during 6–24 h)
Sharp ↑ microglial activation peri-infarct and ↑ proinflammatory CK 3 days after I/R
–HOE 642 or NHE1+/− mice: less microglia activation, lNADPH oxidase activation, ↓ proinflammatory response at 3–7 days post-I/R
Blocking NHE1 significantly ↓ microglial phagocytosis in vitro
–↑↑ NHE1 protein expression in activated microglia and astrocytes
NHE1 inhibition ↓ microglial proinflammatory activation following I/R
Ferrazzano et al. 
44 wild-type controls (NHE1+/−), NHE1 genetic knockdown mice (NHE1+/−)
–Transient focal cerebral ischemia by 30–60 min occlusion of left MCA induced in wild-type controls (NHE1+/+), NHE1 genetic knockdown mice (NHE1+/−), and NHE1+/+ mice treated with HOE-642
–Brain MRI (diffusion DWI and T2 weighted)
–Group 1: HOE 642 30 min pre- or 1 h postreperfusion intraperitoneally. Then at 24 and 48 h after reperfusion
–Group 2: control (saline as vehicle)
–Significant protection in NHE1+/− mice
↓injury in DWI 1 h postreperfusion in NHE1+/−; and smaller infarct in T2 at 72 h vs NHE1+/+mice
–HOE642 prereperfusion or during early reperfusion: ↓ ischemic damage (remains protective given during early reperfusion!)
→Therapeutic potential for inhibition NHE1 in cerebral ischemia
Cengiz et al. 
46 C57BL/6J mice
–30 min unilateral ligation of the left common CA, plus exposure to hypoxia (8 % O2 for 55 min)
–Assessment of morphology, neurodegenerationand motor and spatial learning abilities at 4–8 weeks of age after HI
–Group 1 (n = 13): HOE 642 intraperitoneal: 5 min pre-HI, 24 and 48 h post
–Group 2 (n = 10): control (saline) pre/posttreatment
–Group 3 (n = 13): HOE-642 posttreatment (10 min, 24 and 48 h post-HI)
–Group 4 (n = 10): control (saline) post
Inhibition of NHE1: neuroprotective in neonatal HI brain injury
–Control brains 72 h post-HI: neurodegeneration in several areas brain; NHE1 upregulated in specific astrocytes; and motor-learning deficit seen at 4 weeks age
–HOE 642: better preserved morphologic hippocampal structures; less neurodegeneration in acute stage HI;
and improved striatum-dependent motor and spatial learning at 8 weeks of age after HI
→NHE1-mediated disruption of ionic homeostasis contributes to striatal and CA1 pyramidal neuronal injury after neonatal HI
Helmy et al. 
159 Male Wistar rat pups
–60 min of asphyxia by hypoxia 9 %, or hypercapnia 20 %, or both combined. Then normal restoration of room air or graded re-establishment of normocapnia (half CO2 levels every 30 min)
–Monitoring with EEG recording and pH-sensitive microelectrodes
Some in each group: MIA intraperitoneally 30 min preasphyxia
–Group 1 (60 min hypoxia 9 % then 21 %)
–Group 2 (60 min hypercapnia 20 %)
–Group 3 (asphyxia: CO2 20 % + O2 9 %)
–Group 4 (asphyxia like group 2 and then graded re-establishment of normocapnia)
–Group 5: controls (room air only)
–Recovery from asphyxia followed by large seizure burden and ↑ brain pH
–Graded restoration of normocapnia after asphyxia strongly suppresses alkaline shift in brain pH and seizure burden
–MIA pre-insult: virtually blocked seizures
Helmy et al. 
Male Wistar rat pups
–60 min of asphyxia by hypoxia 9 % and hypercapnia 20 %. Then normal restoration or graded re-establishment of normocapnia (half CO2 levels every 30 min)
–Monitoring with EEG recording, pH-sensitive microelectrodes and histology
5 pups in each group: MIA intraperitoneally 30 min pre-HI
A few: amiloride intraperitoneally 30 min preasphyxia
–Group 1 (asphyxia CO2 20 % + O2 9 %, then room air)
–Group 2 (asphyxia like group 1 and then graded restoration normocapnia)
–Neocortical neurons in vivo: biphasic pH changes acid–alkaline response
–Graded restoration normocapnia: strongly suppress alkaline overshoot
–Parallel ↑ pHe and pHi post-HI: net loss acid equivalents from brain tissue not attributable to BBB disruption (lack of ↑Na fluorescein extravasation into brain and EEG characteristics of BBB)
–MIA: abolition net efflux acid equivalents from brain, and suppression seizure (sz) activity
–Post-asphyxia sz: due to brain alkalosis (NHE-dependent net extrusion acid across BBB)
–BBB-mediated pH regulation: new approach prevention and therapy neonatal sz
Robertson et al. 
18 white male
<24 h old
–Transient global cerebral HI (bilateral occlusion common CA)
31P and 1H MRS before, during and up to 48 h after HI. Tissue injury at 48 h
–iv MIA 10 min post-HI and 8 hourly
–MIA starting 10 min after severe HI: neuroprotection: ↓ brain Lac/NAA, cell death and microglial activation
There are two major classes of pharmacological NHE inhibitors. One class includes amiloride and its derivatives by double substitution of the nitrogen of the 5-amino group: dimethylamiloride (DMA), N-(N-ethyl-N-isopropyl)-amiloride (EIPA), N-methyl-isobutyl-amiloride (MIA), and 5-(N,N-hexamethylene)-amiloride. They are nonselective inhibitors of the NHE system in various cell types, and EIPA is highly potent. Another class of inhibitors includes the derivatives of the benzylguanidines such as HOE 694, HOE 642 (cariporide mesilate or 4-isopropyl-3-methylsulfonylbenzoyl-guanidine methanesulfonate), eniporide, and BIIB-513, which selectively inhibited NHE1. The replacement of the pyrazine ring of amiloride by a pyridine ring or by a phenyl increased the potency and the NHE selectivity. In the last two decades, several bicyclic guanidines were prepared: zoniporide, MS-31038, SM-20220, SM-20550, SMP-300, KBR9032, BMS-284640, T-162559, TY-12533, S-3226, or SL-591227 . Of these, HOE 642 is extremely potent, being 105 times more specific for NHE1 vs. NHE3 , and zoniporide is very potent too and 150-fold selective for NHE1 vs. the other isoforms. S1611 and S3226 are two selective NHE3 inhibitors. Harmaline is a nonamiloride NHE5 inhibitor. The inhibitory potency of NHE inhibitors towards the different NHE isoforms is described in detail in a review by Masereel et al., .
During in vitro cerebral ischemia, NHE inhibitors (DMA, harmaline , SM-20220 , amiloride , and HOE-642 [85, 87, 90]) had a protective effect, in terms of delayed pHi normalization both in neuronal cells and in astrocytes [46, 89, 90, 91, 92], inhibition of microglia , and less intracellular calcium accumulation [87, 88]. This anoxia-induced alkalization was also ameliorated in NHE1−/− CA1 neurons. In parallel, cell death was reduced in wild-type neurons treated with HOE 642 or in NHE1−/− neurons in cultured mouse cortical neurons undergoing 3 h oxygen–glucose deprivation (OGD) and 21 h reoxygenation (from ∼70 % to 40–50 %) .
Vornov et al. , described that pHi decreased by 0.2 pH units, but then recovered when inhibition was removed in rat cortical neuronal cultures undergoing metabolic inhibition for 20 min. NHE1 inhibitors, dimethylamiloride or harmaline, significantly reduced the postinhibition pHi recovery, showing less brain injury in these compared to those with a fast pHi normalization.
As mentioned above, NHE1 activity also affects cellular Na+ levels. A small but significant (∼2-fold) increase in neuronal intracellular concentration of Na+ ([Na+]i) results after 2 h OGD, reaching to ∼7-fold increase during 1 h reoxygenation. HOE 642 attenuated the rise in [Na+]i following OGD. NHE1−/− neurons did not exhibit significant increase in [Na+]i. This supports that NHE1 activity is elevated upon reoxygenation [83, 86]. Three hours of OGD and 21 h reoxygenation lead to cell death in ∼70 % of the NHE1+/+ neurons. However, cell death is significantly reduced in wild-type neurons treated with HOE 642 or in NHE1−/− neurons .
In vivo studies mainly in rodents have found that NHE1 inhibitors reduce brain injury in hypoxia–ischemia models  and decrease infarct size in a focal ischemia model . In NHE1 heterozygous mice, there was a significant decrease in infarct size of around 30 % compared with normal NHE+/+ mice following middle cerebral artery occlusion (MCAO) , similar to those NHE+/+ mice treated with HOE 642. It implies that the disruption of Na+ and Ca2+ homeostasis contributes to ischemic neuronal damage. These studies firmly demonstrate the dominant role of NHE1 among other NHE isoforms in cerebral ischemic brain damage.
Other studies show neuroprotection in ischemic models. SM-20220 (N-(aminoiminomethyl)-1-methyl-1H-indole-2-carboxamide methanesulfonate), a highly selective NHE1 inhibitor, given intravenously 1 h after MCAO significantly reduced the extent of cerebral edema and Na+ content after 2 h ischemia and 4 h reperfusion and infarct volume after 22 h reperfusion . SM-20220 decreased infarct size in both transient and permanent MCAO models. The reduction of infarct size improved when the treatment is delayed for 5, 30, or 60 min after the onset of ischemia . SM-20220 led to ∼50 % decrease in infarct size at 72 h reperfusion. Other groups have shown similar reductions in infarct volume and edema with other NHE blockers [95, 96, 97].
Some NHE blockers, specifically SM-20220  and MIA , have been found to reduce the number of neutrophils in the ischemic hemisphere. SM-20220 attenuates leukocyte adhesion and migration in the mesenteric artery  and improves endothelial dysfunction . NHE1 blockers (EIPA and HOE 642) [84, 90, 101, 102] reduced microglial activation posthypoxia ischemia and hence the microglial phagocytosis and astrocytosis in CA1 region. Cengiz et al.  found that HOE 642 given either prehypoxia–ischemia or starting 10 min after the insult reduced neurodegeneration in the acute stage in hippocampus, striatum, and ipsitaleral thalamus, and improved the striatum-dependent motor and spatial learning skills at 8 weeks.
NHE1 inhibitors are neuroprotective in astrocytes in a similar way to neurons. The intracellular Na+ overload is substantially mediated through activation of the ERK1/2 pathways . In a more severe model of in vitro ischemia, cultured astrocytes were superfused with a solution, which mimics the ionic composition of the ischemic extracellular space (a hypoxic, acidic, ion shifted ringers (HAIR) solutions at 37 °C) [103, 104]. Exposure to HAIR caused a rapid decline in astrocyte pHi. This is consistent with the finding that NHE1 activity is inhibited by low extracellular pH (pHe) . When exposed to normal buffer again, astrocytes rapidly alkalized and a significant rebound of pHi over the baseline took place . Bondarenko et al.  have reported that HAIR and reoxygenation leads to ∼40 % cell death in astrocytes, which is significantly reduced in the presence of NHE blockers such as EIPA or HOE 694 during HAIR and reoxygenation, or during reoxygenation alone.
NHE inhibitors have also been shown to preserve neurological function after ischemic injury. In a model of ischemia and hypothermia in piglets, Castella et al.  observe a rapid neurological recovery in subjects receiving HOE 642 just at the onset of cooling. Several studies [84, 107] demonstrate that EIPA not only protects gerbil hippocampal neurons from ischemic injury but also reduces the magnitude of the ischemia-induced locomotor hyperactivity at both 24 h and 6 days after reperfusion. Ischemic injury to the gerbil forebrain produces an increase in locomotor activity, which is related to the degree of pyramidal neuronal damage in the CA1 region of the hippocampus [84, 108]. In another Mongolian gerbil model, the recovery time of consciousness significantly improved (lower neurological scores at 2 h postreperfusion) following a 30 min transient global cerebral ischemia in those treated with SM-20220, compared to the vehicle group . This improvement in neurological deficit persisted until 24 h postreperfusion.
In summary, NHE inhibitors (or genetically modified NHE1−/−) are neuroprotective in animal models. They reduce the alkalinization of the cell (inhibition of phospholipases and kinases [107, 110] and delayed secondary energy failure ), and decrease the calcium and sodium intracellular overload, reducing cell edema and cell damage. They suppress microglial activation, especially in hippocampal regions (pyramidal neurons of the CA1 area in rodents)  and in striatum and ipsilateral thalamus in a focal ischemia model . Improved functional outcome also follows NHE inhibition .
NHE Inhibition Following Transient Hypoxia–Ischemia in the Piglet Model of Perinatal Asphyxia
Robertson et al.  used the piglet model of perinatal asphyxia to study the effect of NHE1 inhibition with MIA given 10 min after the end of hypoxia–ischemia and eight hourly thereafter. MIA improved cerebral energy metabolism in the thalamic area in the 48 h after hypoxia–ischemia on MRS biomarkers and reduced cell death and microglial activation in some brain areas. The use of clinically relevant MRS biomarkers and reduced cell death in a large animal model of perinatal asphyxia is an important step towards the clinical translation of NHE inhibitors. It is important to assess whether NHE blockade would augment hypothermic neuroprotection in this devastating disease.
Brain pHi and Seizures
Relation Between Brain pHi and Seizures
Neonatal seizures are the most common manifestation of a neurological disorder in the newborn period. Neonatal encephalopathy is the most frequent cause of seizures at term. In some studies, the duration of electrographic seizures in newborn babies is associated with worse MRI appearances  and poor neurodevelopmental outcome . Newborn babies with neonatal encephalopathy as a cause of seizures tend to have a higher seizure burden than those with a stroke . Increased morbidity and mortality, brain injury, and poor neurodevelopmental outcome have been associated with a higher seizure burden [113, 114, 115, 116, 117, 118, 119, 120]. Therefore, addressing neonatal seizures is a priority, but current drugs are largely ineffective and are not free of serious side effects on the neonatal brain [121, 122, 123].
Therapeutic hypothermia not only improves outcomes of babies with neonatal encephalopathy by reducing the rate of death and disability at 18 months of age, but it also appears to reduce seizure burden . Cooling appears either to act directly to reduce seizure burden or to augment the action of conventional anticonvulsants. Xenon gas, which is currently being used in clinical phase II trials to augment hypothermic neuroprotection in babies, appears to reduce seizure burden when administered with cooling . The data are consistent with those from preclinical models .
There is however still an unresolved controversy whether seizures worsen outcome by themselves or whether they are associated to those with a more severe degree of encephalopathy, hence the higher mortality and morbidity [126, 127]. The molecular and cellular mechanisms underlying birth-asphyxia seizures are unknown, but understanding the seizure-triggering mechanisms plays a key role in the design of novel therapeutic strategies.
A profound acidosis (blood pH 7.00 or lower) at the time of birth is an essential criterion in the diagnosis of perinatal asphyxia [128, 129]. However, postasphyxia seizures do not coincide with the maximal blood acidosis but are typically first observed during the recovery period after a delay that usually ranges in human babies from 2 to 16 h . A wide range of observations have shown that changes in extra- and intracellular pH exert a strong modulatory effect on brain excitability under normal and pathophysiological conditions, whereby an alkalosis enhances excitability while an acidosis has an opposite effect [131, 132, 133, 134, 135, 136, 137, 138]. The immature brain appears to be particularly sensitive to changes in pHi. Recent experiments have shown that changes of 0.05 pH units on neonatal hippocampal slices have a profound effect on endogenous network activity .
In a rat pup model of neonatal asphyxia, brain alkalosis after recovery from asphyxia played a key role in the triggering of seizures . Brain pH was measured with intracortical ion sensitive microelectrodes, and EEG was recorded from parietal cortex in freely moving rat pups with simultaneous video recording . There was a robust correlation between postasphyxic brain pH changes and seizure burden; that is, the more alkaline the brain pH after asphyxia, the higher the seizure burden. The alkaline brain pH overshoot after asphyxia reached values of up to 0.40 pH units above normal brain pH; these are similar finding to previous studies in rats , piglet [3, 111], and human newborn [4, 5]. Some pups received MIA (the NHE inhibitor) intraperitoneally 30 min before asphyxia; in those rat pups pretreated with MIA, seizure burden was reduced. These data add weight to the evidence that acidosis suppresses neuronal excitability, whereas alkalosis does the opposite [133, 139, 140, 141]. Taking this further, there is accumulating evidence that brain pHi plays a key role in modulating neuronal survival after injury [131, 142, 143].
In another study in neonatal rat pups by the same group, a rise in brain pH was accompanied by seizure activity following injection of sodium bicarbonate . The authors suggested that a “graded restoration of normacapnia” should be recommended for resuscitation of neonatal encephalopathy as this may reduce seizures and improve outcome .
The same group explored mechanisms of postasphyxia brain alkalosis  in the rat pup exposed to one hour of asphyxia (simultaneous hypoxia and hypercapnia). They observed that the postasphyxia brain alkalosis was generated in the absence of a rise of blood pH. The increase in both extracellular and intracellular pH recorded in the brain implied an enhanced net loss of acid equivalents from brain tissue across the BBB. BBB disruption itself was excluded by the absence of increased sodium fluorescein extravasation into the brain and by electrophysiological characteristics of the BBB . The net efflux of acid equivalents from the brain across the BBB was abolished by MIA, suppressing the postasphyxia rebound alkalosis of the brain extracellular pH without having any significant effect on the weight of the pups. Remarkably, MIA also abolished postasphyxia seizures (reduced seizure burden by 88 %) when applied preinsult in rat pups [138, 145] and ameliorated brain injury when applied before hypoxia–ischemia in neonatal mice . These findings support the conclusion that activation of NHE in the BBB leads to brain alkalosis and consequent seizures.
Therapeutic hypothermia is now standard care for infants in the developed world with moderate to severe neonatal encephalopathy . It is unknown if NHE inhibition combined with therapeutic hypothermia might augment hypothermic neuroprotection after perinatal asphyxia. Some in vitro studies suggest that moderate hypothermia (reducing temperature from 37 to 20 °C) itself increases NHE activity [147, 148], while others suggest that hypothermia produces a partial inhibition of NHE activity . The combined effects of an NHE inhibitor with hypothermia could significantly augment hypothermic neuroprotection especially as they act on separate pathways. Importantly, studies suggest that the potency of NHE inhibition under hypothermic conditions is not influenced by a change in temperature  and NHE inhibition was beneficial in a perfused heart model even after hypothermic ischemia . Preclinical large animal studies of combined therapy with cooling are high priority.
Perinatal hypoxic–ischemic brain injury in term babies is still a significant problem throughout the world. Therapeutic hypothermia has improved outcome, especially of those with a moderate insult. Unfortunately, around 40 % of infants treated with therapeutic hypothermia still have adverse outcomes. Experimental data suggest that the addition of another agent to cooling may enhance overall protection either additively or synergistically. Experimental models have demonstrated the central role of NHE in maintaining brain pHi and ion homeostasis, and in microglial activation in some areas of the brain. NHE inhibitors are a promising neuroprotective agent in animal models, improving brain energy metabolism in in vivo MRS, reducing cell death, seizure burden, and brain injury with improvement of functional outcome. Preclinical studies are urgently needed to assess possible augmentation of hypothermic neuroprotection with NHE inhibition.
This work was undertaken at UCH/UCL who received a proportion of funding from the UK Department of Health's NIHR Biomedical Research Centres funding scheme.
Conflict of Interest
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