. 2014 Jan 24;5(1):79–98. doi: 10.1007/s12975-013-0322-x

Table 3.

Preclinical studies—brain pH and NHE inhibitors in perinatal brain injury (based on [46, 84, 85, 88–90, 93, 101, 102, 107, 110, 111, 138, 145, 158–160])

Paper	Species	Model, study design	NHE inhibitor	Results
In vitro studies
Vornov et al. [46]	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 pH_e (dimethylamiloride and harmaline) slowed pH_i recovery	–Profound protective effects: ↓ pH_e 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 pH_i normalizes fast →acidosis protects: suppressing pH-sensitive mechanisms of injury or blocking Na entry (NHE)
Matsumoto et al. [88]	Ex vivo rodent 1-day rats 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 pH_i	–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. [89]	Ex vivo rodent 14- and 7-day models of rat pups Brain slices	–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 pH_i, ↓ PCr/Pi and ↑ Lac/NAA, and ↓ NTP/PME, at 37°C –Changes delayed with hypothermia (32^o C) or amiloride (pH_i acidified and preserved NTP/PME, at baseline and at 5 h)
Kersh et al. [85]	Ex vivo rodent 3–15-day rat both sex Brain slices	–Hypercapnia (15 % CO₂) –NH₄Cl-induced acidification in brainstem neurons from chemosensitive regions of neonatal rats (brainstem slices from RTNn, NTSn, and LCn)	–Control (DMSO-vehicle) –Amiloride –HOE 642 –S1611 –EIPA	–pH_i 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. [90]	Ex vivo rodent 1–3-day neonatal mice Glial cultures	–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 pH_i regulation in microglia basal conditions –Activation of microglia accelerated pH_i regulation (↑ pH_i, ↑ Na⁺ _i and Ca²⁺i, and production of superoxide anion (SOA) and cytokines (CK)) –HOE 642 abolished pH_i regulation, ↓ production SOA, CK and _iNOS –Hypothesis: NHE1 to maintain microglial pH_i homeostasis (NADPH oxidase and “respiratory” burst)
In vivo studies
Ferimer et al. [158]	Rodent 13 Wistar rats	Cardiac arrest (KCl) in rats followed by resuscitation 7 min later in untreated vs. MIA	MIA Controls (untreated)	–MIA delays normalization of brain pH_i after cardiac arrest in rats –MIA: ↓ cardiac pH in rats postarrest +15 min reperfusion –MIA doesn’t change pH_i from nonischemic value.
Phillis et al. [107]	Rodent 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. [110]	Rodent 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. [93]	Rodent 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. [159]	Rodent 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. [84]	Rodent 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. [101]	Rodent 136 mice –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. [160]	Rodent 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)	Randomised to: –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. [102]	Rodent 9 days 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	Randomised to: –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. [138]	Rodent 6 days 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. [145]	Rodent 6–7 days 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 ↑ pH_e and pH_i 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. [111]	Piglet 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	Randomized to: –Saline placebo –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

Paper

Species

Model, study design

NHE inhibitor

Results

In vitro studies

Vornov et al. [46]

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 pH_e (dimethylamiloride and harmaline) slowed pH_i recovery

–Profound protective effects: ↓ pH_e 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 pH_i normalizes fast

→acidosis protects: suppressing pH-sensitive mechanisms of injury or blocking Na entry (NHE)

Matsumoto et al. [88]

Ex vivo rodent

1-day rats

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 pH_i

–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. [89]

Ex vivo rodent

14- and 7-day models of rat pups

Brain slices

–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 pH_i, ↓ PCr/Pi and ↑ Lac/NAA, and ↓ NTP/PME, at 37°C

–Changes delayed with hypothermia (32^o C) or amiloride (pH_i acidified and preserved NTP/PME, at baseline and at 5 h)

Kersh et al. [85]

Ex vivo rodent

3–15-day rat both sex

Brain slices

–Hypercapnia (15 % CO₂)

–NH₄Cl-induced acidification in brainstem neurons from chemosensitive regions of neonatal rats (brainstem slices from RTNn, NTSn, and LCn)

–Control (DMSO-vehicle)

–Amiloride

–HOE 642

–S1611

–EIPA

–pH_i 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. [90]

Ex vivo rodent

1–3-day neonatal mice

Glial cultures

–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 pH_i regulation in microglia basal conditions

–Activation of microglia accelerated pH_i regulation (↑ pH_i, ↑ Na⁺ _i and Ca²⁺i, and production of superoxide anion (SOA) and cytokines (CK))

–HOE 642 abolished pH_i regulation, ↓ production SOA, CK and _iNOS

–Hypothesis: NHE1 to maintain microglial pH_i homeostasis (NADPH oxidase and “respiratory” burst)

In vivo studies

Ferimer et al. [158]

Rodent

13 Wistar rats

Cardiac arrest (KCl) in rats followed by resuscitation 7 min later in untreated vs. MIA

MIA

Controls (untreated)

–MIA delays normalization of brain pH_i after cardiac arrest in rats

–MIA: ↓ cardiac pH in rats postarrest +15 min reperfusion

–MIA doesn’t change pH_i from nonischemic value.

Phillis et al. [107]

Rodent

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. [110]

Rodent

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. [93]

Rodent

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. [159]

Rodent

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. [84]

Rodent

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. [101]

Rodent

136 mice

–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. [160]

Rodent

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)

Randomised to:

–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. [102]

Rodent

9 days

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

Randomised to:

–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. [138]

Rodent

6 days

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. [145]

Rodent

6–7 days

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 ↑ pH_e and pH_i 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. [111]

Piglet

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

Randomized to:

–Saline placebo

–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

Abbreviations: NHE Na⁺/H⁺ exchanger, NHE1 isoform 1 of NHE, NCX1 Na⁺/Ca²⁺ exchanger-1, NBC Na- and HCO₃-dependent transporter, KCN potassium cyanide, HI hypoxia-ischemia, CA carotid arteries, BBB blood–brain barrier, MIA N-methyl-isobutyl-amiloride (inhibitor of NHE), EIPA N-(N-ethyl-N-isopropyl)-amiloride (highly potent derivative of amiloride for the nonselective inhibition of the NHE system in various cell types), SM-20220 N-(aminoiminomethyl)-1-methyl-1H-indole-2-carboxamide methanesulfonate (a highly selective and specific NHE1 inhibitor, 50 times more potent than EIPA), HOE-642 cariporide mesilate or 4-isopropyl-3-methylsulfonylbenzoyl-guanidine methanesulfonate (a selective NHE1 inhibitor), S1611 (a selective NHE3 inhibitor), Harmaline (a non-amiloride NHE5 inhibitor), NTP/PME nucleotide triphosphate/phosphomonoester, Pi inorganic phosphate, PCr phosphocreatine, Lac/NAA lactate/NAA ratio, RTNn retrotrapezoid nucleus neurons, NTSn nucleus tractus solitarii neurons, LCn locus coeruleus neurons