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

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

Blood gases during isoflurane anesthesia with or without MV.

<p>* p < 0.002 compared to Iso. MV group. Data are Mean ± SEM</p><p>Blood gases during isoflurane anesthesia with or without MV.</p

Post-HI isoflurane anesthesia attenuates oxidative brain damage and extent of brain injury.

<p>(A and B)—Mitochondrial aconitase activity and expression of 3-Nitrotyrosine in brains obtained from naïve mice (n = 11 and 7) and in HI-mice reperfused for initial 15 minutes with isoflurane (n = 12 and 9) or RA (n = 10 and 6). (C)—representative 3-Nitrotyrosine western blot. (D)—Infarct volume and representative TTC-stained cerebral images of HI-mice reperfused without (RA, n = 50), or with isoflurane anesthesia: for initial 15 min (n = 35), or initial 30 min (n = 35), or delayed (30–45 minutes, n = 16).</p

Mechanical ventilation enhances neuroprotection of isoflurane.

<p>(A)—CBF during HI and reperfusion in mice re-oxygenated with RA (n = 4) or Isoflurane for initial 15 minutes (Iso—RA, n = 4), or 30 minutes (Iso, n = 4), or mice re-oxygenated with isoflurane combined with mechanical ventilation (Iso+Vent, n = 4). * p < 0.02 between groups, Dashed square indicates analyzed area. (B)—Infarct volume in mice re-oxygenated under isoflurane anesthesia for 15 or 30 minutes with (n = 22) or without (n = 35) mechanical ventilation. * p = 0.017 compared to the mice ventilated for 30 minutes.</p

Experimental design.

<p><b>Mitochondrial phosphorylating respiration rates after HI</b>. (A)—HI-mice, upon reperfusion, were exposed to either room air or isoflurane with or without mechanical ventilation (MV) for different time of reperfusion. (B)—Changes in SaO₂ in naive mice (n = 6), and mice exposed to 2 Vol% isoflurane with (n = 6) or without (n = 4) 30% oxygen supplementation, * p < 0.01. (C)—Mitochondrial phosphorylating and uncoupled respiration rates in naïve mice (n = 10), HI-mice at the end of HI-insult (n = 5), and at 15 minutes of reperfusion under isoflurane (HI+Iso, n = 14) or without (HI+RA, n = 14) isoflurane anesthesia. (D)—Representative cerebral mitochondrial respiration tracings from HI-mice tested at the end of HI (End of HI) and 15 minutes of reperfusion with isoflurane exposure (15’ Rep+Iso) or room air (15’ Rep+RA).</p