| IntroductionHypoxic damage to brain is very common phenomenon and is still the leading cause of death in clinical. The main mechanisms of ischemic injury to brain include the release of excitatory amino acid (EAA) and the receptor activation of EAA, intracellular calcium overload, depletion of ATP and so on. However, until now most of the therapeutic methods and drugs based on the above mechanisms have little effects on human ischemic brain injuries in clinical. Some scholars suggested that the N - methyl - D - aspartate (NMDA) receptor over -activation undoubtedly contributes to the post - ischemic neuronal death, but it was not the predominant mechanism, proteolysis activated by intracellular calcium overload might be main cause for the ischemic brain damage.Hypoxia or ischemia could cause neuronal membrane potential changes, showed that slow depolarization, followed by rapid depolarization and membrane potential increase to "0"mv and maintain at this level for the rest of ischemia, called persistent depolarization. The rapid depolarization was commonly used as the marker of irreversible ischemic or hypoxic damage to neurons, but the exact mechanisms of rapid depolarization is unclear. Some research found the mass release of EAA from neurons and the extra - cellular K~+ concentration increase after ischemia or hypoxia. But there were much less researches about the effects of EAA and K~+ on the neuronal membrane potential.Recent studies have confirmed that the ischemia or hypoxia can activate the calpains and cathepsins, both of them belong to the cysteine protease superfami-ly. The activation of calpains and cathepsins could cause a series of cascade e-vents and eventually cause neuronal death. Some researchers using biochemistry and morphorological indexes found that the inhibitors of calpains and cathepsins could effectively protect the neurons from ischemic or hypoxic damages. But up till now, there is no research using isolated hippocampal slices to explore the direct effects of inhibitors of both calpains and cathepsins on hypoxia injury with electrophysiological techniques.In this experiment, we would use intracellular recording techniques to stud-y: 1. The effects of hyperkaemia artificial cerebral - spinal fluid (aCSF) and blocker of NMD A receptor MK801 on the neuronal electrophysiological changes of CAl region of isolated hippocampal slices in rats;2. The inhibitor of calpains PD150606 on the neuronal electrophysiological changes and the activation of caspase-3 in CAl region. 3. The inhibitor of cathepsin B CA074Me on the neuronal electrophysiological changes and the activation of caspase - 3 of CAl region.Part 1. The effects of hyperkaemia artificial cerebral - spinal fluid and the blocker of NMD A receptor MK801 on the neuronal electrophysiological changes of CAl region in isolated hippocampal slices of ratsMaterialsExperiment animals: Adult male Sprague - Dawley ( SD) rats (90 - 120 days) were provided by Health Science Center at Brooklyn, State University of New York.Experiment instrumentsChemical and reagents: NaCl ( Fisherchemicals, USA );KC1 ( Fisherchemi-cals, USA );KH2 PO4 ( Sigma Cor. USA );NaHC03 ( Fisherchemicals, USA);Glucose ( Fisherchemicals, USA );MgSO4 ( Sigma, USA );CaCI2 ( Sigma, USA) ,NMDA(Sigma, USA)MK801(Sigma, USA)0MethodsThe preparation of hippocampal slice: 26 SD rats were anesthesized with isoflurane and then decapitated and the brain was removed from the animal rapidly and placed it in the cold oxygenated aCSF. 400 jim thickness of hippocampal slices were prepared in the cold aCSF which was saturated with mixed gas of 95% O2 and 5% C02, then the slices were incubated at room temperature for 2 hours before experiment.The hippocampal slices were randomly divided into 6 groups: Con ( n = 10), MK801 (n = 10), NMDA (n=8), K15 (n=8), K30 (n=8) and K0 (n = 8) groups respectively. The slices in Con, K0 and MK801 groups were subjected to lOmin of hypoxia, or given aCSF that had no K+ or 100|xM MK801 during hypoxia, followed by 60min of re -oxygenation;the slices in K15, K30 and NMDA groups were treated with aCSF which contained 15mM>30mM KCI or 25uM NMDA for lOmin, followed by normal aCSF for 60min. The rate of neuro-nal slow depolarization, the time to rapid depolarization and the amplitude of rapid depolarization were recorded using intracellular recording technique, the neuronal response to the intracellular current injection and Schaffer collateral pathway stimuli was observed at the end of experiment.Results1. The hippocampal slices perfused with aCSF that contained 15mM or 30mM KCI had the same electrophysiological changes as that of slices in Con group. The rate of slow depolarization in K15 and K30 groups were 0. 41 ± 0. 17mv/s and 0. 45 ±0. 16mv/s respectively, which were significant higher than that in Con and K0 groups (0.22 ±0.05mv/s, 0.20 ±0.05mv/s respectively, P<0.05).The time to rapid depolarization of K15 and K30 groups were 154 ± 17 seconds and 153 ±22 seconds respectively, which were significant smaller than that in Con and K0 groups.At the end of experiment, all neurons in all groups had no response to in-tracellular current injection and Schaffer collateral pathway stimulation.2. The time to rapid depolarization was delayed in MK801 group. It was significant longer than that in Con and NMDA groups. Meanwhile, MK801 could inhibit the amplitude of neuronal rapid depolarization.MK801 could decrease the rate of slow neuronal membrane potential depolarization during hypoxia. The rate in MK801 group was 0.08 ±0.03mv/s,significant lower than that in Con and NMDA groups.MK801 could enhance the neuronal functional recovery from hypoxia injury. Among the 9 out of 10 neurons in MK801 group, the response to intracellu-lar current injection and Schaffer collateral stimuli recovered after 60min of re -oxygenation. But there was no recovery in NMDA and Con groups.DiscussionHie release of excitatory amino acid ( EAA) from neurons was one of the main pathological - physiological changes in brain ischemic damages. The research using micro - dialysis technique had confirmed that the EAA concentration in normal brain is 1 ~ 5 jxM, but it begin to increase after 1 ~ 2min of ischemia or hypoxia, and could increase to 16 ~30|xM after 10 ~ 15min of ischemia. The calcium influx was triggered by the NMDA receptors activation and caused intracellular calcium overload. In this experiment, we found that the neuronal electrophysiological changes induced by NMDA were the same as that during ischemia, and MK801 could significantly inhibit such changes during ischemia, and enhance the neuronal functional recovery from hypoxia injury.The stability of potassium in the extracellular fluids and certain grade between intra - and extra - cell are the basis for the neuronal resting membrane potential. Some researches have confirmed that hypoxia or ischemia can cause the potassium increase of the extracellular fluid in hippocampal. Hie potassium could increase to the level of 16mM even before the neuronal rapid depolarization and could increase to the level of 45mM when the neuronal rapid depolarization occurred. In this experiment, we found that the slices perfused with aCSFthat contained 15mM or 30mM KC1 had electrophysiological changes, which was the same as that in Con group. But the aCSF with no K + could not block the slow and rapid neuronal depolarization induced by hypoxia. The possible reason might be that the extra - cellular potassium concentration might be determined by the potassium efflux induced by the inhibition of Na+ - K+ - ATP.Part 2. The effects of calpain inhibitor PD150606 on the neuronal hy-poxic injury and the electrophysiological mechanismMaterialsExperiment animal and experiment instruments are the same as that in part 1;PD 150606 was'obtained from Calbiochem;DMSO was obtained from Sigma;PI and the kit for the Caspase - 3 measurement was offered from Promega.MethodsThe methods of hippocampal slice preparation were the same as that in part 1. The slices from 12 SD rats were randomly divided into Con ( n =6), PD150606(PD, n = 10) and DMSO groups(n =8). The slices in Con group were treated with aCSF at 37*0 for 60min before intracellular recording, and the slices in PD and DMSO groups were incubated with 50|xM PD150606 or 0.3% DMSO for 60min before intracellular recording. All slices in each group were subjected to 5min hypoxia and followed by 60min of re - oxygenation. The electrophysiological indexes were the same as that in part 1. After the electrophysiological experiment, all slices were incubated with normal aCSF for another 3 hours, then the activated caspase - 3 positive neurons and the death neurons were measured.Results1. Compared with Con and DMSO group, PD150606 significantly delayed the time to rapid depolarization and inhibited amplitude of rapid depolarization(P <0.05). After 60min of re - oxygenation, 8 out of 10 neurons in the PD group had its functional recovery.2. The activated caspase - 3 positive neurons in PD group was significant less than that in Con and DMSO groups.3. The death neurons in PD group was significant less than that in Con and DMSO groups.DiscussionCalpains belong to the cysteine protease, which are activated dependant on the increase of calcium concentration in cytosol. Many studies have confirmed that the increase of calpains activity is related to the hypoxia or ischemic damage to neurons. PD 150606 is an inhibitor of calpains, which can easily across the neuronal membrane. The results in our experiment showed that PD 150606 could delay the time to rapid depolarization and inhibit the amplitude of rapid depolarization, and it could enhance the neuronal functional recovery after re - oxygenation. Meanwhile, we also found that PD150606 could inhibit the caspase - 3 activated , which mean that PD 150606 could inhibit the neuronal apoptosis induced by hypoxia.Part 3. The effects of cathepsin B inhibitor CA074Me on the neuronal hypoxic injury and the electrophysiological mechanismMaterialsExperiment animal and experiment instruments are the same as that in part 1;CA074Me was obtained from Calbiochem;DMSO was obtained from Sigma;PI and the kit for the Caspase - 3 measurement was offered from Promega.MethodsThe methods of hippocampal slice preparation were the same as that in part 1. The slices from 12 SD rats were randomly divided into Con, CA074Me andDMSO groups. The slices in Con group were treated with aCSF at 37 X. for 60min before intracellular recording, and the slices in CA074Me and DMSO groups were incubated with 75|iM CA074Me or 0. 3% DMSO for 60min before intracellular recording. All slices in each group were subjected to 5min hypoxia and followed by 60min of re - oxygenation. The electrophysiological indexes were the same as that in part 1. After the electrophysiological experiment, all slices were incubated with normal aCSF for another 3 hours, then the activated caspase - 3 positive neurons and the death neurons were measured.Results1. The cathepsin B inhibitor CA074Me had little effects on the rate of slow depolarization. But it could delay the time to rapid depolarization and inhibit the amplitude of rapid depolarization, and could enhance the neuronal functional recovery.2. It could inhibit the caspase 3 activation in pyramidal neurons, the caspase 3 positive neurons number in CA1 region in CA074Me group was 19 ±7 units per millimeter, which was significantly less than that in Control and DMSO groups (81 ± 11 units/mm and 97 ± 13units/mm respectively, P <0. 05).3. The number of death neurons in CA1 region was 89 ± 11 units/mm in CA074Me group, which was significant less than that in DMSO and Con groups.DiscussionCathepsin B is most abundant lysosomal protease of papain family. It could digest cell proteins, nucleic acids, complex carbohydrates and lipids when it leaked out from lysosomal and was activated by the decrease of pH. CA074Me is a selective inhibitor of cathepsin B, some studies with morphorolgical index have confirmed that CA074 can protect the brain from the ischemic damages. In our experiment, we found that CA074Me could delay the time to neuronal rapid depolarization and inhibit the amplitude of rapid depolarization, and enhance thefunctional recovery. Meanwhils, it could also inhibit the activation of caspase -3, which mean that it might inhibit the neuronal apoptpsis after hypoxia. The mechanisms for its neuroprotection might be related to the inhibition of proteoly-sis by CA074Me. |
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