Isoflurane Anesthesia Initiated at the Onset of Reperfusion Attenuates Oxidative and Hypoxic-Ischemic Brain Injury

This study demonstrates that in mice subjected to hypoxia-ischemia (HI) brain injury isoflurane anesthesia initiated upon reperfusion limits a release of mitochondrial oxidative radicals by inhibiting a recovery of complex-I dependent mitochondrial respiration. This significantly attenuates an oxidative stress and reduces the extent of HI brain injury. Neonatal mice were subjected to HI, and at the initiation of reperfusion were exposed to isoflurane with or without mechanical ventilation. At the end of HI and isoflurane exposure cerebral mitochondrial respiration, H₂O₂ emission rates were measured followed by an assessment of cerebral oxidative damage and infarct volumes. At 8 weeks after HI navigational memory and brain atrophy were assessed. In vitro, direct effect of isoflurane on mitochondrial H₂O₂ emission was compared to that of complex-I inhibitor, rotenone. Compared to controls, 15 minutes of isoflurane anesthesia inhibited recovery of the compex I-dependent mitochondrial respiration and decreased H₂O₂ production in mitochondria supported with succinate. This was associated with reduced oxidative brain injury, superior navigational memory and decreased cerebral atrophy compared to the vehicle-treated HI-mice. Extended isoflurane anesthesia was associated with sluggish recovery of cerebral blood flow (CBF) and the neuroprotection was lost. However, when isoflurane anesthesia was supported with mechanical ventilation the CBF recovery improved, the event associated with further reduction of infarct volume compared to HI-mice exposed to isoflurane without respiratory support. Thus, in neonatal mice brief isoflurane anesthesia initiated at the onset of reperfusion limits mitochondrial release of oxidative radicals and attenuates an oxidative stress. This novel mechanism contributes to neuroprotective action of isoflurane. The use of mechanical ventilation during isoflurane anesthesia counterbalances negative effect of isoflurane anesthesia on recovery of cerebral circulation which potentiates protection against reperfusion injury

Nelfinavir inhibits intra-mitochondrial calcium influx and protects brain against hypoxic-ischemic injury in neonatal mice.

Nelfinavir (NLF), an antiretroviral agent, preserves mitochondrial membranes integrity and protects mature brain against ischemic injury in rodents. Our study demonstrates that in neonatal mice NLF significantly limits mitochondrial calcium influx, the event associated with protection of the brain against hypoxic-ischemic insult (HI). Compared to the vehicle-treated mice, cerebral mitochondria from NLF-treated mice exhibited a significantly greater tolerance to the Ca(2+)-induced membrane permeabilization, greater ADP-phosphorylating activity and reduced cytochrome C release during reperfusion. Pre-treatment with NLF or Ruthenium red (RuR) significantly improved viability of murine hippocampal HT-22 cells, reduced Ca(2+) content and preserved membrane potential (Ψm) in mitochondria following oxygen-glucose deprivation (OGD). Following histamine-stimulated Ca(2+) release from endoplasmic reticulum, in contrast to the vehicle-treated cells, the cells treated with NLF or RuR also demonstrated reduced Ca(2+) content in their mitochondria, the event associated with preserved Ψm. Because RuR inhibits mitochondrial Ca(2+) uniporter, we tested whether the NLF acts via the mechanism similar to the RuR. However, in contrast to the RuR, in the experiment with direct interaction of these agents with mitochondria isolated from naïve mice, the NLF did not alter mitochondrial Ca(2+) influx, and did not prevent Ca(2+) induced collapse of the Ψm. These data strongly argues against interaction of NLF and mitochondrial Ca(2+) uniporter. Although the exact mechanism remains unclear, our study is the first to show that NLF inhibits intramitochondrial Ca(2+) flux and protects developing brain against HI-reperfusion injury. This novel action of NLF has important clinical implication, because it targets a fundamental mechanism of post-ischemic cell death: intramitochondrial Ca(2+) overload → mitochondrial membrane permeabilization → secondary energy failure

Nelfinavir limits post-ischemic mitochondrial cytochrome C release, brain infarct volume and cellular mortality.

<p><b>A, B</b> - Western blot analysis for the presence of Cytochrome C (Cyt C) in cytosolic (Cytosol) and mitochondrial (Mitos) fractions obtained from the ipsilateral hemisphere at five hrs after HI insult. β-actin was used as a loading control and cytochrome C oxidase (COX IV) was used as a purity control of cytosolic fraction and loading control for mitochondrial fraction. Quantitative data presented in (B) are expressed in arbitrary OD units normalized to β-actin. Vehicle –treated mice, n = 3, NLF-treated mice, n = 4. <b>C, D</b> - Cerebral infarct volume and representative TTC-stained brain slices obtained at 24 hrs of reperfusion after HI in the vehicle and NLF-treated mice. <b>E, F</b> – Cell mortality at 6 hours of reperfusion following 12 hours of OGD in HT-22 cells treated with vehicle (n = 7), NLF (n = 7) or RuR (n = 7) compared to naives (n = 4). <b>E</b> - Representative images of HT-22 cells in different experimental conditions stained with Propidium Iodide (red) and Hoechst (blue). Note that amount of red cells predominates in OGD-vehicle group. Confocal microscopy. Scale bar = 50 µm. <b>F</b> - Quantitative evaluation of cell mortality. One-way Anova, only significant difference is shown.</p

Nelfinavir does not inhibit mitochondrial Ca²⁺ uniporter.

<p><b>A</b> – The experiment controlling mitochondrial specificity for TMRE and Rhodamine 123 (R123) fluoroprobes. Note a drastic decrease in Ψm fluorescence following FCCP (0.5 µM) supplementation. Scale bar = 10 µm. <b>B</b> – One of three highly reproducible tracings of mitochondrial Ca²⁺ buffering capacity in organelles pre-treated with RuR (1 µM), NLF (4.4 µM) or vehicle. Note, that only RuR completely inhibited Ca²⁺ up-take by mitochondria, while NLF, virtually, had no effect. <b>C</b> – One of the four highly reproducible tracings of changes in the safranin (Ψm) fluorescence in response to mitochondrial supplementation (indicated) and addition of 10 nmoles of Ca²⁺ pulses to mitochondria pre-incubated with RuR, NLF or vehicle. Note, only the RuR prevented the collapse of Ψm in response to Ca²⁺ challenge. Brain mitochondria were isolated from naïve p10 mice, substrate: succinate-glutamate (see also methods).</p

Nelfinavir improves mitochondrial function.

<p><b>A, B</b> – Intramitochondrial Ca²⁺ content (<b>A</b>) and representative tracings of Ca²⁺ release from mitochondria (<b>B</b>) in naïve (n = 4) and at the end of HI-insult in vehicle (n = 7) or NLF-treated (n = 7) mice. One-way ANOVA. *- p<0.0001 compared to naives. Mito (downward arrow) indicates addition of mitochondria (0.1 mg/ml). Digitonin (downward arrow) shows addition of Digitonin (10 mg/mg of the mitochondrial protein). Digitonin-induced nonspecific fluorescence curve (without mitochondria addition) is indicated as Digitonin. <b>C, D</b> – Mitochondrial Ca²⁺ buffering capacity at five hours of reperfusion (<b>C</b>), with representative tracings (<b>D</b>) in vehicle (n = 11) and NLF-treated (n = 14) HI-mice compared to naïve littermates (n = 7). Mitochondrial Ca²⁺ buffering capacity was defined by the amount of Ca²⁺ needed to open mPTP (spontaneous increase in Ca²⁺ fluorescence). <b>E, F</b> - Mitochondrial ADP-phosphorylating (state 3) and resting (state 4) respiration rates (E), with representative tracing (F) examined in naïve (n = 11) and at five hours of reperfusion in vehicle (n = 16) or NLF-treated mice (n = 15). * p<0.02 compared to Naïve and NLF treated mice.</p

In-vitro and ex-vivo effect of isoflurane on mitochondrial ROS release.

<p>(A)—Mitochondrial H₂O₂ emission rate and representative H₂O₂ fluorescence tracings in HI-mice at the end of HI (n = 6) and at 15 minutes of reperfusion with (n = 6) or without (n = 6) isoflurane anesthesia. (B and C)—H₂O₂ emission rates with representative H₂O₂ fluorescence tracings from mitochondria fueled with succinate (B) or malate-glutamate (C) and exposed to hyperoxic buffer (O₂) in the presence of vehicle (O₂ + Veh, n = 4 and 6), or Isoflurane (O₂ + Iso, n = 4 and 6), or Rotenone (O₂ + Rot, n = 4 and 6) and compared to controls (Normox, n = 4 and 6). P-values and study groups are indicated. * p < 0.01 compared to normoxia.</p

Long-term neurological outcome of the HI-brain injury after isoflurane exposure.

<p>(A)—Navigational memory: time spent in the “platform quadrant” by naïve mice (n = 21) and HI-mice re-oxygenated without (HI, n = 14), or with isoflurane (HI+Iso, n = 14). Representative tracings of swimming path during probe trial in the same groups of mice. (B)—Extent of brain atrophy in the ipsilateral hemisphere and representative Nissl-stained brain images from adult mice treated with isoflurane for initial 15 minutes of reperfusion (HI+Iso, n = 13) or RA (HI, n = 14).</p

Nelfinavir mimics the effect of RuR on mitochondrial response to OGD.

<p><b>A–C,</b> - Confocal microscopy and semi-quantitative analysis of cytosolic cellular Ca²⁺ fluorescence (Fluo4) with simultaneous Ψm fluorescence (TMRE) in cells treated with vehicle (n = 7) or NLF (n = 7), or RuR (n = 7) at 1 hrs following OGD (12 hrs). * p<0.0001 compared to values compared to the naïve cells (100%), and ** p<0.0001 compared to the vehicles. <b>D–F</b> -Confocal microscopy and semi-quantitative analysis for mitochondria-specific Ca²⁺ (Rhod 2) fluorescence alone with Ψm (R123) fluorescence in naïve cells and cells pre-incubated (20 hrs) with vehicle or NLF (4.4 µM), RuR (10 µM). * p<0.0001 compared to naives, ** p<0.0001 compared to the NLF cells. n = 7 in each group. The area outlined with dashed line demonstrates cells with Ca²⁺overloaded mitochondria which lost their Ψm. Scale bar = 40 (merged images) and 20 µm.</p

Nelfinavir mimics the effect of RuR on mitochondrial response to histamine.

<p><b>A–C</b>, - Confocal microscopy and semi-quantitative analysis of cytosolic cellular Ca²⁺ fluorescence (Fluo4) with simultaneous Ψm fluorescence (TMRE) in cells treated with vehicle (n = 6) or NLF (n = 6), before and after histamine (10 mM) challenge. * p<0.0001 compared to values before histamine challenge in the vehicle, and ** p<0.0001 in the NLF treated cells. <b>D–F,</b> - Confocal microscopy and semi-quantitative analysis for mitochondria-specific Ca²⁺ (Rhod 2) fluorescence alone with Ψm (R123) fluorescence in cells pre-incubated (20 hrs) with NLF (4.4 µM), RuR (10 µM) or CsA (1 µM). * p<0.0001 compared to naives, ** p<0.0001 compared to the vehicle-treated cells. n = 6 in each group. Scale bars = 10 µm and 20 µm.</p