Acta Histochemica 116 (2014) 1062–1067
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Acta Histochemica journal homepage: www.elsevier.de/acthis
Delayed remote ischemic postconditioning protects against transient cerebral ischemia/reperfusion as well as kainate-induced injury in rats Rastislav Burda a , Viera Danielisova b , Miroslav Gottlieb b , Miroslava Nemethova b , Petra Bonova b , Milina Matiasova b , Radoslav Morochovic a , Jozef Burda b,∗ a b
University Hospital of L. Pasteur, Kosice, Slovakia Institute of Neurobiology, Slovak Academy of Sciences, Kosice, Slovakia
a r t i c l e
i n f o
Article history: Received 27 March 2014 Received in revised form 29 April 2014 Accepted 30 April 2014 Keywords: Ischemia Reperfusion Delayed remote ischemic postconditioning Hippocampus Rat
a b s t r a c t To test the appropriateness of using delayed remote ischemic postconditioning against damage caused to the hippocampus by ischemia or apoptosis inducing intoxication, we chose 10-min normothermic ischemia induced by four-vessel occlusion or kainate injection (8 mg/kg i.p.) in rats. Ischemia alone caused the number of degenerated CA1 neurons after 7 days lasting reperfusion to be significantly (p < 0.001) increased by 72.77%. Delayed remote ischemic postconditioning lasting 20 min was able to prevent massive increase in the neurodegeneration. The group with 10 min of ischemia and postconditioning after 2 days of reperfusion had only 15.87% increase in the number of apoptotic neurons. Seven days after kainic acid injection the number of surviving neurons was 42.8% (p < 0.001), but the portion of surviving pyramidal cells in the postconditioning group is more than 98%. Our data show that remote postconditioning, performed with 20 min of tourniquet ischemia applied to the hind limb, is a simple method able to effectively stop the onset of neurodegeneration and prevent occurrence of massive muscle cell necrosis, even when used 2 days after the end of the adverse event. Surviving neurons retained a substantial part of their learning and memory ability. © 2014 Elsevier GmbH. All rights reserved.
Introduction The brain is the most sensitive organ to a lack of blood supply. Absence of oxygen lasting more than 4 min results in irreversible damage. The complex architectural, functional as well as cellular structure of the brain allows very marked differences in the sensitivity of individual neuronal populations. However, the most sensitive brain neurons, i.e. hippocampal CA1 (+CA4), dying after the limit of 4 min, represent less than 1% of the total number of neurons in the brain. A lethal dose of ischemia for pyramidal neurons in CA1 in Pulsinelli’s model of transient forebrain ischemia (Pulsinelli and Brierley, 1979) is between 5 and 15 min (LD50 = roughly 8 min). However, the cells do not die immediately. The phenomenon of delayed neuronal death, which is in fact apoptosis triggered by pathological conditions, delays the onset of irreversible changes, depending on the intensity of the initial attack, from a few hours to several days. The delay in the onset of neuronal death opens a therapeutic window enabling, in addition to or in combination with
∗ Corresponding author at: Institute of Neurobiology, Slovak Academy of Sciences, Soltesovej 4, 040 01 Kosice, Slovakia. E-mail address: [email protected] (J. Burda). http://dx.doi.org/10.1016/j.acthis.2014.04.011 0065-1281/© 2014 Elsevier GmbH. All rights reserved.
the classical treatment, the utilization of postconditioning. Zhao et al. (2003) described a technique of “postconditioning”, involving a modified schedule of reperfusion characterized by intermittent restoration of blood flow after a prolonged episode of myocardial ischemia. Our findings also showed how utilization of delayed postconditioning could reverse delayed neuronal death induced by transient global cerebral ischemia (Burda et al., 2005, 2006) and kainate intoxication (Burda et al., 2009). Postconditioning, if it is used before the onset of irreversible changes, is an endogenous defence mechanism with the ability to prevent apoptosis. The problem remains of which postconditioning method to use. Postconditioning indeed means the use of a stressor. In any assessment, suitability and usability in clinical medicine must be a priority. Leaving aside a wide range of inappropriate physical, chemical and biological methods, recently so-called remote ischemic postconditioning has started being promoted. Its tourniquet execution in the form of non-invasive limb ischemia seems to be a safe, easy and cheap method where saving the life or function of the brain greatly outweighs the discomfort of the tourniquet application. The aim of our study was to assess the appropriateness of using clinically applicable delayed remote ischemic postconditioning. We used a non-invasive single 20 min tourniquet ischemia on the hind
R. Burda et al. / Acta Histochemica 116 (2014) 1062–1067
limbs of rats as a postconditioner delayed for 2 days after the end of 10 min transient forebrain ischemia (Pulsinelli and Brierley, 1979) or 2 days after injection of kainic acid (KA). In addition to neurodegeneration and survival, we monitored the functionality of surviving neurons as well. Changes in blood pressure and flow rate were also measured. Materials and methods Fifty eight adult albino Wistar rats of both sexes weighing 250–350 g free of any clinically evident disease were group-housed and maintained on a 12 h light/dark cycle, with free access to water and rodent chow. The animals were bred in the registered animal colony (SK PC 20011) of the Institute of Neurobiology, Kosice, Slovakia. Experiments were performed in accordance with European Community legislation. The Ethics Committee at the Institute of Neurobiology as well as the State Veterinary and Alimentary Administration of the Slovak Republic approved the experiments. The rats were randomized into groups as shown in Table 1. Remote ischemic postconditioning (RIP) was induced using an external elastic band (tourniquet) placed as proximal as possible (Dillon et al., 2006) for 20 min. The rationale for the timing used in this experimental design was based on our experience and the literature (Pignataro et al., 2013). The rats were anesthetized before and throughout the ischemic postconditioning with chloral hydrate (300 mg/kg i.p., Sigma–Aldrich, St. Louis, MO, USA). In accordance with the findings of Tsubota et al. (2010) no occurrence of muscle edema, necrotic changes or functional disorders were detected in rats 24 h after 20 min tourniquet ischemia (RIP only group, n = 8, not shown). The rats were killed 7 days after 10 min of transient forebrain ischemia (Pulsinelli and Brierley, 1979) or kainic acid administration (KA, 8 mg/kg i.p., Sigma–Aldrich, St. Louis, MO, USA, dissolved in saline to 4.0 mg KA/ml immediately before use) by transcardiac perfusion performed under deep anesthesia (chloral hydrate, 400 mg/kg, i.p., 10% solution in saline). Perfusion via the left ventricle started with a washout of blood vessels with 200 ml of 0.9% NaCl. Brains were perfused and fixed with 4% (w/v) paraformaldehyde solution in PBS, removed and postfixed overnight in the same fixative prior to sectioning with a vibratome (Leica VT 1000S, Nussloch, Germany) sectioning. The 33 m coronal sections of brain were prepared at the level of bregma −3.3 ± 0.2 mm for the hippocampus and 1.7 ± 0.2 mm for the striatum. The sections were randomly selected for Fluoro Jade B staining of all degenerating neurons, regardless of the cell death mechanism, and NeuN immunoreaction was used to visualize neurons present in the CA1 region 7 days after intoxication with or without postconditioning. Surviving as well as degenerating neurons were counted in the middle of the linear part of eight different CA1 fields from each animal and expressed per 1 mm of the hippocampal CA1 region. Neuronal cell count was performed by a person who was unaware of the treatment conditions, using ImageJ 1.48i software (National Institutes of Health, Bethesda, MD, USA, http://imagej.nih.gov/ij). Fluoro Jade B staining The sections were mounted on 2% gelatine-coated slides and then dried on a slide warmer at 50 ◦ C for 30 min. The slides were then immersed in a solution containing 1% sodium hydroxide in 80% alcohol for 5 min. This was followed by 2 min in 70% alcohol and 2 min in distilled water. The slides were then transferred to a solution of 0.06% potassium permanganate for 10 min, and subsequently rinsed in distilled water for 2 min. After 20 min in the staining solution containing 0.0004% Fluoro Jade B dye (Histo-Chem Inc., Jefferson, AR, USA), the slides were rinsed three times for 1 min
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in distilled water. Excess water was removed by briefly draining the slides (about 15 s) vertically on a paper towel. The slides were then placed on a slide warmer, set at approximately 50 ◦ C, until they were fully dry (5–10 min). The dry slides were cleared by immersion in xylene for at least a minute before coverslipping with DPX (Sigma–Aldrich). The slides were examined with a Leica DM2500 fluorescence microscope with a Leica camera and LAS V3.6 software (Leica Microsystems, Wetzlar, Germany).
Immunocytochemistry Immunocytochemistry was performed on the prepared coronal free-floating 33 m thick vibratome sections. Sections containing the hippocampus were immunostained for NeuN, a neuronal marker. Briefly, the sections were incubated overnight at 4 ◦ C with monoclonal mouse NeuN antibody (Chemicon Int., Temecula, CA, USA; 1:500) in 0.1 mol/l PBS (pH 7.4) with 0.2% Triton. After washing with 0.1 mol/l PBS (pH 7.4) containing 0.2% Triton, secondary anti-mouse IgG antibody made on horse (Vector Laboratories, Burlingame, CA, USA) was applied for 90 min at room temperature. After further washing, avidin/biotin complex formulation kit (Vectastain ABC Elite, Vector Laboratories, Burlingame, CA, USA) was applied for 90 min, then the slides were rinsed with PBS followed by Tris Buffer (pH 7.6), and reacted with DAB (0.1 mol/l Tris, 0.04% DAB, 0.033% H2 O2 ); the reaction was stopped with phosphate buffer. The slides were dehydrated, cleared, and coverslipped for analysis.
Behavioral analysis Cognitive and memory functions of the rats which underwent experimental procedures were tested with the Morris water maze (Morris, 1984) on the sixth and seventh reperfusion day. A washing tank (150 cm in diameter and 58 cm deep) was filled with 26 ± 1 ◦ C water. Approximately 500 ml of milk was added to the water, making it opaque. A submerged escape platform (20 cm tall and 15 cm diameter) was located in the southeast quadrant of the maze. A variety of extra-maze visual cues were visible from within the maze. The experimenter, who was unfamiliar with the treatment received by the subject, and an assistant, remained at fixed locations approximately 0.5 m away from the outside edge of the tank on each trial. The water maze training procedure lasted 2 days. On the sixth day after kainate intoxication each rat underwent two trials. On each trial, a rat was placed in the water facing the same place at the edge of the pool. The rat was allowed 60 s to locate the platform. If after 60 s it did not find the escape platform, it was guided by the experimenter and allowed to remain on the platform for 10 s. The inter-trial interval for each subject was 5 min, during which the rat was dried and returned to the home cage. On the second day the decisive probe trial was performed: all rats started from the same starting position opposite to the quadrant where the submerged escape platform had been positioned during the tests. The escape latency (the time each subject required to locate the hidden platform after being released) of each subject were measured. The maximum value from each probe taken for statistical analysis was 60 s.
Statistical analysis Data were analyzed with one-way ANOVA followed by Tukey–Kramer’s test using GraphPad InStat 3.1 software (GraphPad Software Inc., La Jolla, CA, USA). Differences were considered significant at p < 0.05.
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Table 1 Scheme of experiment. Group
Sham operated control Ischemia Ischemia with remote isch. postC Kainate intoxication Control Kainate Kainate with remote isch. postC
n
Day 1 Cauterization of vertebral arteries
Day 0 Clasps on carotids for 10 min
Day 2 Tourniquet on hind limb for 20 min
Day 6 Morris water maze trial 1 and 2
Day 7 Morris water maze decisive probe
8 8 8
+ + +
− + +
− − +
+ + +
+ + +
10 10 9
− − −
0.5 ml of saline i.p. 8 mg of kainate i.p. 8 mg of kainate i.p.
− − +
+ + +
+ + +
Fig. 1. Representative photomicrographs of neurodegeneration of CA1 neurons in rats subjected to ischemia visualized by Fluoro Jade B staining (A, B, C), and surviving neurons in the same experimental groups visualized by NeuN immunoreactivity (A , B , C ). The following treatment was used: A, A sham control; B, B 10 min ischemia and 7 days of recovery; C, C 10 min ischemia + 2 days reperfusion + postconditioning (20 min hind limb ischemia) + 5 days reperfusion.
Results No occurrences of muscle edema, necrotic changes or functional disorders were detected 24 h after tourniquet ischemia to the hind limb. Seven days after 10 min of ischemia, when the process of delayed neuronal death is practically terminated, massive neurodegeneration can normally be observed in the selectively vulnerable brain regions, represented mainly by the CA1 region of the hippocampus (Fig. 1B, B ). However, if 2 days after ischemia remote ischemic postconditioning is applied, the occurrence of neurodegeneration is significantly reduced (Fig. 1C), and the CA1 layer of pyramidal neurons remains preserved at a level similar to the control (Fig. 1C , A ). In statistical terms, the number of surviving CA1 neurons (Fig. 2) drops to 25.81% (p < 0.001). In the experimental group in which tourniquet postconditioning was used 2 days after ischemia, more than 80% of the pyramidal cells survived 7 days after the ischemia (p < 0.001 compared to ischemia without postconditioning). The presence of neurodegeneration after 10 min of ischemia without and with subsequent postconditioning is presented in Fig. 3. Ischemia alone caused the number of degenerated CA1 neurons after 7 days lasting reperfusion to be significantly (p < 0.001) increased by 72.77%. Delayed remote postconditioning lasting 20 min was able to prevent massive increase in the neurodegeneration. The group with 10 min of ischemia and RIP after 2 days of reperfusion had only 15.87% increase in the number of apoptotic neurons. The function of CA1 neurons in terms of their ability to participate in learning and memory after ischemia, with or without RIP (Fig. 4), was evaluated using the Morris water maze test. Despite the fact that the chart contains all the data from the test, what is
decisive is the probe from the second day of the test. Here we can see a significant changes in the time needed to find the hidden platform. Looking for the hidden platform took significantly (p < 0.001) longer, from 13.57 ± 2.56 s in sham control to 44.83 ± 4.80 s in the ischemic group without postconditioning. The animals which after ischemia underwent delayed remote postconditioning were able to locate the platform in less than half of the time (22.88 ± 3.72 s, p < 0.05) needed by the ischemic group. These data clearly confirm not only that postconditioning is able to prevent neuronal
Fig. 2. The effects of 10 min ischemia with or without subsequent tourniquet postconditioning (RIP 2 days after ischemia) on the number of surviving neurons visualized by NeuN immunoreaction 7 days after ischemia. Results are expressed as mean ± S.E.M., n = 8 in each group. a Significantly different (p < 0.001) from sham control. b Significantly different (p < 0.001) from remote ischemic postconditioning.
R. Burda et al. / Acta Histochemica 116 (2014) 1062–1067
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Discussion
Fig. 3. The effects of 10 min ischemia with or without subsequent postconditioning (RIP 2 days later) on the number of degenerating neurons visualized by Fluoro Jade B staining 7 days after ischemia. Results are expressed as mean ± S.E.M. n = 8 in each group. a Significantly different (p < 0.001) from sham control. b Significantly different (p < 0.001) from remote ischemic postconditioning.
death but also that surviving neurons retain a substantial part of their function with the ability to learn and remember learned things. Data obtained from the experiment with brain damage induced by intraperitoneal administration of kainic acid (8 mg/kg) are shown in Table 2. The kainate dose did not inducing any epileptogenic activity (tested daily by metal keys ringing). The picture detected after intoxication is very similar to that induced by ischemia. Neurodegeneration in the hippocampal CA1 region caused by kainate amounted to 63.7% seven days after KA injection. This corresponds approximately to the proportion of surviving neurons (42.8%). All these changes are significantly different in comparison to controls, but what is more important is that the proportion of degenerated neurons in the RIP group is less than 1%, while that of surviving pyramidal cells over 98% (both p < 0.001). In the additional group of rats living 28 days after kainate intoxication and subsequent tourniquet postconditioning, 99% of neurons survived (289.68 ± 5.29; n = 5), which is evidence that postconditioning provides long-lasting protection to the brain.
The phenomenon of ischemic tolerance is a surprisingly strong protective tool. The principle “What does not kill you will make you stronger” should be, in the case of ischemic tolerance, formulated “will make you extremely stronger”. Ischemic preconditioning applied 2 days before 30 min of ischemia enabled the survival of 95% of the most sensitive neurons in the brain, the pyramidal neurons in hippocampal CA1 area (Burda et al., 2005). It is necessary to stress that 30-min ischemia represents twice the dose which is able to kill all CA1 neurons, and moreover that 40% of these cells do not survive 5 min of ischemia and subsequent 7 days of reperfusion (Burda et al., 2006). This defence mechanism works, fortunately, also in reverse order. Protective sublethal stress can be used not only prior to (preconditioning) but also after (postconditioning) the lethal stress. Postconditioning opens therapeutic windows whose size depends on the severity of the intervention. In cases of protracted (30–120 min) or persistent focal ischemia (usually MCAO), rapid postconditioning applied some seconds or minutes after ischemia seems to be the most effective. This procedure consists of several periods (usually 10–30 s) of intermittent reperfusion, but a single period of short ischemia (some minutes) after a short period of reperfusion can also be used (Zhao et al., 2006; Pignataro et al., 2008; Xing et al., 2008). Milder attacks, such as global ischemia up to 10 min, provoking so-called delayed neuronal death, which opens a two-day “wide” therapeutic window, can be staved off by delayed postconditioning 48 h after ischemia (Burda et al., 2005, 2006; Danielisova et al., 2006; Zhou et al., 2011; Zhan et al., 2012). Delayed postconditioning not only prevents neuronal death but Nagy et al. (2011) using modest ischemia (2 vessel occlusion) and kainic acid as a postconditioner documented that delayed postconditioning induces reappearance of the normal dendritic spine density in the adult hippocampal CA1 subfield, which results in a marked restoration of experimental LTP induction. Delayed postconditioning can also be used effectively after some apoptosis-inducing intoxications (Burda et al., 2009). In contrast to a combination with antioxidants that block the effect of postconditioning (Puisieux et al., 2004; Burda et al., 2009; Domorakova et al., 2009), a combination of two different protocols of postconditioning (rapid shuttering ischemia and delayed injection of bradykinin) can be used in order to increase the impact of treatment (Danielisova et al., 2012). On the other hand, delayed postconditioning has been
Fig. 4. Results of Morris water maze test performed on day 6 and 7 after ischemia with or without postconditioning. a Significantly different (p < 0.001) versus sham control group. b Significantly different (p < 0.05) versus remote postconditioning group.
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Table 2 Effect of kainic acid intoxication (with or without subsequent remote ischemic postconditioning applied 48 h after injection of kainate) on neurodegeneration, survival and function of CA1 neurons measured 7 days after intoxication.
Fluoro Jade B positivity [neurons/mm] NeuN immunoreactivity [neurons/mm] Morris water maze escape time [s]
Control
Kainic acid 8 mg/kg i.p.
Kainic acid + postconditioning
– 292.43 ± 12.33 13.91 ± 2.72
186.00 ± 17.09a , b 125.00 ± 32.49a , b 36.48 ± 3.45c
0.86 ± 0.13 286.69 ± 5.80 22.25 ± 4.13
Results are expressed as mean ± S.E.M. a Significantly different (p < 0.001) from control. b Significantly different (p < 0.001) from remote ischemic postconditioning. c Significantly different (p < 0.01) from control.
used effectively 3 and 6 h after long-lasting focal ischemia (Ren et al., 2008). The mechanisms of postconditioning are still not clear. An excellent review on this subject was written by Zhao et al. (2012). In our experiments with delayed postconditioning after transient forebrain ischemia, by the effect of Cycloheximide we documented that protein(s) synthesized during the first 5 h after postconditioning are inevitable for the survival of CA1 neurons. Moreover, a strong significant decrease in glutamate concentration seems to be very important (Bonova et al., 2013). This correlates well with the idea of Wieloch et al. (1985). An essential condition for the survival of the pyramidal neurons in CA1 is the restoration of permanently blocked protein synthesis (Thilmann et al., 1986). Although there is no doubt about the efficacy of postconditioning, the current problem is how to apply this in clinical practice. We need to consider the patient in a critical condition after cardiac arrest, stroke or intoxication, when most of the seemingly wide range of stressors is not usable. Repetitive ischemic insult and hypoxia are practically unacceptable. The use of hyperthermia or hypothermia is also debatable. Use of lipopolysaccharide induces fever, and 3-nitropropinic acid is a mitochondrial poison. Application of bradykinin may result in an increase in internal calcium and subsequent excitotoxic glutamate release (Parpura et al., 1994). The use of remote ischemic postconditioning, which is performed by local remote stress, initiates defensive mechanisms in the whole organism, but does not constitute a risk of damage to the limb tissue. Przyklenk et al. (1993) first described that “brief episodes of ischemia in one vascular bed protected the remote, virgin myocardium from subsequent sustained coronary artery occlusion”. Later, remote local ischemia was used as postconditioning through repetitive occlusion and release of selected arteries, but this was changed subsequently into the more palatable non-invasive tourniquet ischemia of skeletal muscles/entire limb (Tsubota et al., 2010; Mansour et al., 2012). Many attempts have been made to determine the optimum protocol for ischemic postconditioning. The development has moved from several periods of ischemia/reperfusion lasting a few seconds through longer-lasting repetitive I/R intervals toward singular periods of ischemia lasting 10–20 min (Saxena et al., 2010; Pignataro et al., 2013). In conclusion, when we used non-invasive singular 20 min ischemia of skeletal muscle 2 days after normothermic transient cerebral ischemia or kainate intoxication, it clearly proved that this stressor is able to effectively prevent delayed neuronal death of the most sensitive neuronal population in the brain, which is the CA1 layer of pyramidal neurons in the hippocampus. We believe that the procedure of tourniquet postconditioning can be used in clinical medicine after cardiac arrest, hypertensive shock and other transient ischemic attacks as well as apoptosis inducing intoxications. The therapeutic window after permanent focal ischemia is substantially shorter, so postconditioning should be used as soon as possible and similarly to other attacks can be used repeatedly. It is important to note that tourniquet application increases blood pressure to about 30% higher values than before
its application, which can be dangerous in the event of possible bleeding. Acknowledgements This study was supported by ERDF grant ITMS 26220220043 and SK VEGA 2/0148/12. The authors thank Dana Jurusova and Andrew Jonathan Billingham for their technical and editorial assistance. References Bonova P, Burda J, Danielisova V, Nemethova M, Gottlieb M. Delayed post-conditioning reduces post-ischemic glutamate level and improves protein synthesis in brain. Neurochem Int 2013;62:854–60. Burda J, Danielisova V, Nemethova M, Gottlieb M, Matiasova M, Domorakova I, et al. Delayed postconditioning initiates additive mechanism necessary for survival of selectively vulnerable neurons after transient ischemia in rat brain. Cell Mol Neurobiol 2006;26:1139–49. Burda J, Danielisova V, Nemethova M, Gottlieb M, Kravcukova P, Domorakova I, et al. Postconditioning and anticonditioning: possibilities to interfere to evoked apoptosis. Cell Mol Neurobiol 2009;29:821–5. Burda J, Matiasova M, Gottlieb M, Danielisova V, Nemethova M, Garcia L, et al. Evidence for a role of second pathophysiological stress in prevention of delayed neuronal death in the hippocampal ca1 region. Neurochem Res 2005;30:1397–405. Danielisova V, Burda J, Nemethova M, Gottlieb M, Burda R. An effective combination of two different methods of postconditioning. Neurochem Res 2012;37:2085–91. Danielisova V, Nemethova M, Gottlieb M, Burda J. The changes in endogenous antioxidant enzyme activity after postconditioning. Cell Mol Neurobiol 2006;26:1181–91. Dillon JP, Laing AJ, Chandler JR, Wang JH, McGuinness A, Redmond HP. Pravastatin attenuates tourniquet-induced skeletal muscle ischemia reperfusion injury. Acta Orthopaedica 2006;77:27–32. Domorakova I, Mechirova E, Dankova M, Danielisova V, Burda J. Effect of antioxidant treatment in global ischemia and ischemic postconditioning in the rat hippocampus. Cell Mol Neurobiol 2009;29:837–44. Mansour Z, Charles AL, Bouitbir J, Pottecher J, Kindo M, Mazzucotelli JP, et al. Remote and local ischemic postconditioning further impaired skeletal muscle mitochondrial function after ischemia-reperfusion. J Vasc Surg 2012;56:774–82, e771. Nagy D, Kocsis K, Fuzik J, Marosi M, Kis Z, Teichberg VI, et al. Kainate postconditioning restores ltp in ischemic hippocampal ca1: Onset-dependent second pathophysiological stress. Neuropharmacology 2011;61:1026–32. Parpura V, Basarsky TA, Liu F, Jeftinija K, Jeftinija S, Haydon PG. Glutamate-mediated astrocyte-neuron signalling. Nature 1994;369:744–7.
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Acta Histochemica journal homepage: www.elsevier.de/acthis
Delayed remote ischemic postconditioning protects against transient cerebral ischemia/reperfusion as well as kainate-induced injury in rats Rastislav Burda a , Viera Danielisova b , Miroslav Gottlieb b , Miroslava Nemethova b , Petra Bonova b , Milina Matiasova b , Radoslav Morochovic a , Jozef Burda b,∗ a b
University Hospital of L. Pasteur, Kosice, Slovakia Institute of Neurobiology, Slovak Academy of Sciences, Kosice, Slovakia
a r t i c l e
i n f o
Article history: Received 27 March 2014 Received in revised form 29 April 2014 Accepted 30 April 2014 Keywords: Ischemia Reperfusion Delayed remote ischemic postconditioning Hippocampus Rat
a b s t r a c t To test the appropriateness of using delayed remote ischemic postconditioning against damage caused to the hippocampus by ischemia or apoptosis inducing intoxication, we chose 10-min normothermic ischemia induced by four-vessel occlusion or kainate injection (8 mg/kg i.p.) in rats. Ischemia alone caused the number of degenerated CA1 neurons after 7 days lasting reperfusion to be significantly (p < 0.001) increased by 72.77%. Delayed remote ischemic postconditioning lasting 20 min was able to prevent massive increase in the neurodegeneration. The group with 10 min of ischemia and postconditioning after 2 days of reperfusion had only 15.87% increase in the number of apoptotic neurons. Seven days after kainic acid injection the number of surviving neurons was 42.8% (p < 0.001), but the portion of surviving pyramidal cells in the postconditioning group is more than 98%. Our data show that remote postconditioning, performed with 20 min of tourniquet ischemia applied to the hind limb, is a simple method able to effectively stop the onset of neurodegeneration and prevent occurrence of massive muscle cell necrosis, even when used 2 days after the end of the adverse event. Surviving neurons retained a substantial part of their learning and memory ability. © 2014 Elsevier GmbH. All rights reserved.
Introduction The brain is the most sensitive organ to a lack of blood supply. Absence of oxygen lasting more than 4 min results in irreversible damage. The complex architectural, functional as well as cellular structure of the brain allows very marked differences in the sensitivity of individual neuronal populations. However, the most sensitive brain neurons, i.e. hippocampal CA1 (+CA4), dying after the limit of 4 min, represent less than 1% of the total number of neurons in the brain. A lethal dose of ischemia for pyramidal neurons in CA1 in Pulsinelli’s model of transient forebrain ischemia (Pulsinelli and Brierley, 1979) is between 5 and 15 min (LD50 = roughly 8 min). However, the cells do not die immediately. The phenomenon of delayed neuronal death, which is in fact apoptosis triggered by pathological conditions, delays the onset of irreversible changes, depending on the intensity of the initial attack, from a few hours to several days. The delay in the onset of neuronal death opens a therapeutic window enabling, in addition to or in combination with
∗ Corresponding author at: Institute of Neurobiology, Slovak Academy of Sciences, Soltesovej 4, 040 01 Kosice, Slovakia. E-mail address: [email protected] (J. Burda). http://dx.doi.org/10.1016/j.acthis.2014.04.011 0065-1281/© 2014 Elsevier GmbH. All rights reserved.
the classical treatment, the utilization of postconditioning. Zhao et al. (2003) described a technique of “postconditioning”, involving a modified schedule of reperfusion characterized by intermittent restoration of blood flow after a prolonged episode of myocardial ischemia. Our findings also showed how utilization of delayed postconditioning could reverse delayed neuronal death induced by transient global cerebral ischemia (Burda et al., 2005, 2006) and kainate intoxication (Burda et al., 2009). Postconditioning, if it is used before the onset of irreversible changes, is an endogenous defence mechanism with the ability to prevent apoptosis. The problem remains of which postconditioning method to use. Postconditioning indeed means the use of a stressor. In any assessment, suitability and usability in clinical medicine must be a priority. Leaving aside a wide range of inappropriate physical, chemical and biological methods, recently so-called remote ischemic postconditioning has started being promoted. Its tourniquet execution in the form of non-invasive limb ischemia seems to be a safe, easy and cheap method where saving the life or function of the brain greatly outweighs the discomfort of the tourniquet application. The aim of our study was to assess the appropriateness of using clinically applicable delayed remote ischemic postconditioning. We used a non-invasive single 20 min tourniquet ischemia on the hind
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limbs of rats as a postconditioner delayed for 2 days after the end of 10 min transient forebrain ischemia (Pulsinelli and Brierley, 1979) or 2 days after injection of kainic acid (KA). In addition to neurodegeneration and survival, we monitored the functionality of surviving neurons as well. Changes in blood pressure and flow rate were also measured. Materials and methods Fifty eight adult albino Wistar rats of both sexes weighing 250–350 g free of any clinically evident disease were group-housed and maintained on a 12 h light/dark cycle, with free access to water and rodent chow. The animals were bred in the registered animal colony (SK PC 20011) of the Institute of Neurobiology, Kosice, Slovakia. Experiments were performed in accordance with European Community legislation. The Ethics Committee at the Institute of Neurobiology as well as the State Veterinary and Alimentary Administration of the Slovak Republic approved the experiments. The rats were randomized into groups as shown in Table 1. Remote ischemic postconditioning (RIP) was induced using an external elastic band (tourniquet) placed as proximal as possible (Dillon et al., 2006) for 20 min. The rationale for the timing used in this experimental design was based on our experience and the literature (Pignataro et al., 2013). The rats were anesthetized before and throughout the ischemic postconditioning with chloral hydrate (300 mg/kg i.p., Sigma–Aldrich, St. Louis, MO, USA). In accordance with the findings of Tsubota et al. (2010) no occurrence of muscle edema, necrotic changes or functional disorders were detected in rats 24 h after 20 min tourniquet ischemia (RIP only group, n = 8, not shown). The rats were killed 7 days after 10 min of transient forebrain ischemia (Pulsinelli and Brierley, 1979) or kainic acid administration (KA, 8 mg/kg i.p., Sigma–Aldrich, St. Louis, MO, USA, dissolved in saline to 4.0 mg KA/ml immediately before use) by transcardiac perfusion performed under deep anesthesia (chloral hydrate, 400 mg/kg, i.p., 10% solution in saline). Perfusion via the left ventricle started with a washout of blood vessels with 200 ml of 0.9% NaCl. Brains were perfused and fixed with 4% (w/v) paraformaldehyde solution in PBS, removed and postfixed overnight in the same fixative prior to sectioning with a vibratome (Leica VT 1000S, Nussloch, Germany) sectioning. The 33 m coronal sections of brain were prepared at the level of bregma −3.3 ± 0.2 mm for the hippocampus and 1.7 ± 0.2 mm for the striatum. The sections were randomly selected for Fluoro Jade B staining of all degenerating neurons, regardless of the cell death mechanism, and NeuN immunoreaction was used to visualize neurons present in the CA1 region 7 days after intoxication with or without postconditioning. Surviving as well as degenerating neurons were counted in the middle of the linear part of eight different CA1 fields from each animal and expressed per 1 mm of the hippocampal CA1 region. Neuronal cell count was performed by a person who was unaware of the treatment conditions, using ImageJ 1.48i software (National Institutes of Health, Bethesda, MD, USA, http://imagej.nih.gov/ij). Fluoro Jade B staining The sections were mounted on 2% gelatine-coated slides and then dried on a slide warmer at 50 ◦ C for 30 min. The slides were then immersed in a solution containing 1% sodium hydroxide in 80% alcohol for 5 min. This was followed by 2 min in 70% alcohol and 2 min in distilled water. The slides were then transferred to a solution of 0.06% potassium permanganate for 10 min, and subsequently rinsed in distilled water for 2 min. After 20 min in the staining solution containing 0.0004% Fluoro Jade B dye (Histo-Chem Inc., Jefferson, AR, USA), the slides were rinsed three times for 1 min
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in distilled water. Excess water was removed by briefly draining the slides (about 15 s) vertically on a paper towel. The slides were then placed on a slide warmer, set at approximately 50 ◦ C, until they were fully dry (5–10 min). The dry slides were cleared by immersion in xylene for at least a minute before coverslipping with DPX (Sigma–Aldrich). The slides were examined with a Leica DM2500 fluorescence microscope with a Leica camera and LAS V3.6 software (Leica Microsystems, Wetzlar, Germany).
Immunocytochemistry Immunocytochemistry was performed on the prepared coronal free-floating 33 m thick vibratome sections. Sections containing the hippocampus were immunostained for NeuN, a neuronal marker. Briefly, the sections were incubated overnight at 4 ◦ C with monoclonal mouse NeuN antibody (Chemicon Int., Temecula, CA, USA; 1:500) in 0.1 mol/l PBS (pH 7.4) with 0.2% Triton. After washing with 0.1 mol/l PBS (pH 7.4) containing 0.2% Triton, secondary anti-mouse IgG antibody made on horse (Vector Laboratories, Burlingame, CA, USA) was applied for 90 min at room temperature. After further washing, avidin/biotin complex formulation kit (Vectastain ABC Elite, Vector Laboratories, Burlingame, CA, USA) was applied for 90 min, then the slides were rinsed with PBS followed by Tris Buffer (pH 7.6), and reacted with DAB (0.1 mol/l Tris, 0.04% DAB, 0.033% H2 O2 ); the reaction was stopped with phosphate buffer. The slides were dehydrated, cleared, and coverslipped for analysis.
Behavioral analysis Cognitive and memory functions of the rats which underwent experimental procedures were tested with the Morris water maze (Morris, 1984) on the sixth and seventh reperfusion day. A washing tank (150 cm in diameter and 58 cm deep) was filled with 26 ± 1 ◦ C water. Approximately 500 ml of milk was added to the water, making it opaque. A submerged escape platform (20 cm tall and 15 cm diameter) was located in the southeast quadrant of the maze. A variety of extra-maze visual cues were visible from within the maze. The experimenter, who was unfamiliar with the treatment received by the subject, and an assistant, remained at fixed locations approximately 0.5 m away from the outside edge of the tank on each trial. The water maze training procedure lasted 2 days. On the sixth day after kainate intoxication each rat underwent two trials. On each trial, a rat was placed in the water facing the same place at the edge of the pool. The rat was allowed 60 s to locate the platform. If after 60 s it did not find the escape platform, it was guided by the experimenter and allowed to remain on the platform for 10 s. The inter-trial interval for each subject was 5 min, during which the rat was dried and returned to the home cage. On the second day the decisive probe trial was performed: all rats started from the same starting position opposite to the quadrant where the submerged escape platform had been positioned during the tests. The escape latency (the time each subject required to locate the hidden platform after being released) of each subject were measured. The maximum value from each probe taken for statistical analysis was 60 s.
Statistical analysis Data were analyzed with one-way ANOVA followed by Tukey–Kramer’s test using GraphPad InStat 3.1 software (GraphPad Software Inc., La Jolla, CA, USA). Differences were considered significant at p < 0.05.
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Table 1 Scheme of experiment. Group
Sham operated control Ischemia Ischemia with remote isch. postC Kainate intoxication Control Kainate Kainate with remote isch. postC
n
Day 1 Cauterization of vertebral arteries
Day 0 Clasps on carotids for 10 min
Day 2 Tourniquet on hind limb for 20 min
Day 6 Morris water maze trial 1 and 2
Day 7 Morris water maze decisive probe
8 8 8
+ + +
− + +
− − +
+ + +
+ + +
10 10 9
− − −
0.5 ml of saline i.p. 8 mg of kainate i.p. 8 mg of kainate i.p.
− − +
+ + +
+ + +
Fig. 1. Representative photomicrographs of neurodegeneration of CA1 neurons in rats subjected to ischemia visualized by Fluoro Jade B staining (A, B, C), and surviving neurons in the same experimental groups visualized by NeuN immunoreactivity (A , B , C ). The following treatment was used: A, A sham control; B, B 10 min ischemia and 7 days of recovery; C, C 10 min ischemia + 2 days reperfusion + postconditioning (20 min hind limb ischemia) + 5 days reperfusion.
Results No occurrences of muscle edema, necrotic changes or functional disorders were detected 24 h after tourniquet ischemia to the hind limb. Seven days after 10 min of ischemia, when the process of delayed neuronal death is practically terminated, massive neurodegeneration can normally be observed in the selectively vulnerable brain regions, represented mainly by the CA1 region of the hippocampus (Fig. 1B, B ). However, if 2 days after ischemia remote ischemic postconditioning is applied, the occurrence of neurodegeneration is significantly reduced (Fig. 1C), and the CA1 layer of pyramidal neurons remains preserved at a level similar to the control (Fig. 1C , A ). In statistical terms, the number of surviving CA1 neurons (Fig. 2) drops to 25.81% (p < 0.001). In the experimental group in which tourniquet postconditioning was used 2 days after ischemia, more than 80% of the pyramidal cells survived 7 days after the ischemia (p < 0.001 compared to ischemia without postconditioning). The presence of neurodegeneration after 10 min of ischemia without and with subsequent postconditioning is presented in Fig. 3. Ischemia alone caused the number of degenerated CA1 neurons after 7 days lasting reperfusion to be significantly (p < 0.001) increased by 72.77%. Delayed remote postconditioning lasting 20 min was able to prevent massive increase in the neurodegeneration. The group with 10 min of ischemia and RIP after 2 days of reperfusion had only 15.87% increase in the number of apoptotic neurons. The function of CA1 neurons in terms of their ability to participate in learning and memory after ischemia, with or without RIP (Fig. 4), was evaluated using the Morris water maze test. Despite the fact that the chart contains all the data from the test, what is
decisive is the probe from the second day of the test. Here we can see a significant changes in the time needed to find the hidden platform. Looking for the hidden platform took significantly (p < 0.001) longer, from 13.57 ± 2.56 s in sham control to 44.83 ± 4.80 s in the ischemic group without postconditioning. The animals which after ischemia underwent delayed remote postconditioning were able to locate the platform in less than half of the time (22.88 ± 3.72 s, p < 0.05) needed by the ischemic group. These data clearly confirm not only that postconditioning is able to prevent neuronal
Fig. 2. The effects of 10 min ischemia with or without subsequent tourniquet postconditioning (RIP 2 days after ischemia) on the number of surviving neurons visualized by NeuN immunoreaction 7 days after ischemia. Results are expressed as mean ± S.E.M., n = 8 in each group. a Significantly different (p < 0.001) from sham control. b Significantly different (p < 0.001) from remote ischemic postconditioning.
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Discussion
Fig. 3. The effects of 10 min ischemia with or without subsequent postconditioning (RIP 2 days later) on the number of degenerating neurons visualized by Fluoro Jade B staining 7 days after ischemia. Results are expressed as mean ± S.E.M. n = 8 in each group. a Significantly different (p < 0.001) from sham control. b Significantly different (p < 0.001) from remote ischemic postconditioning.
death but also that surviving neurons retain a substantial part of their function with the ability to learn and remember learned things. Data obtained from the experiment with brain damage induced by intraperitoneal administration of kainic acid (8 mg/kg) are shown in Table 2. The kainate dose did not inducing any epileptogenic activity (tested daily by metal keys ringing). The picture detected after intoxication is very similar to that induced by ischemia. Neurodegeneration in the hippocampal CA1 region caused by kainate amounted to 63.7% seven days after KA injection. This corresponds approximately to the proportion of surviving neurons (42.8%). All these changes are significantly different in comparison to controls, but what is more important is that the proportion of degenerated neurons in the RIP group is less than 1%, while that of surviving pyramidal cells over 98% (both p < 0.001). In the additional group of rats living 28 days after kainate intoxication and subsequent tourniquet postconditioning, 99% of neurons survived (289.68 ± 5.29; n = 5), which is evidence that postconditioning provides long-lasting protection to the brain.
The phenomenon of ischemic tolerance is a surprisingly strong protective tool. The principle “What does not kill you will make you stronger” should be, in the case of ischemic tolerance, formulated “will make you extremely stronger”. Ischemic preconditioning applied 2 days before 30 min of ischemia enabled the survival of 95% of the most sensitive neurons in the brain, the pyramidal neurons in hippocampal CA1 area (Burda et al., 2005). It is necessary to stress that 30-min ischemia represents twice the dose which is able to kill all CA1 neurons, and moreover that 40% of these cells do not survive 5 min of ischemia and subsequent 7 days of reperfusion (Burda et al., 2006). This defence mechanism works, fortunately, also in reverse order. Protective sublethal stress can be used not only prior to (preconditioning) but also after (postconditioning) the lethal stress. Postconditioning opens therapeutic windows whose size depends on the severity of the intervention. In cases of protracted (30–120 min) or persistent focal ischemia (usually MCAO), rapid postconditioning applied some seconds or minutes after ischemia seems to be the most effective. This procedure consists of several periods (usually 10–30 s) of intermittent reperfusion, but a single period of short ischemia (some minutes) after a short period of reperfusion can also be used (Zhao et al., 2006; Pignataro et al., 2008; Xing et al., 2008). Milder attacks, such as global ischemia up to 10 min, provoking so-called delayed neuronal death, which opens a two-day “wide” therapeutic window, can be staved off by delayed postconditioning 48 h after ischemia (Burda et al., 2005, 2006; Danielisova et al., 2006; Zhou et al., 2011; Zhan et al., 2012). Delayed postconditioning not only prevents neuronal death but Nagy et al. (2011) using modest ischemia (2 vessel occlusion) and kainic acid as a postconditioner documented that delayed postconditioning induces reappearance of the normal dendritic spine density in the adult hippocampal CA1 subfield, which results in a marked restoration of experimental LTP induction. Delayed postconditioning can also be used effectively after some apoptosis-inducing intoxications (Burda et al., 2009). In contrast to a combination with antioxidants that block the effect of postconditioning (Puisieux et al., 2004; Burda et al., 2009; Domorakova et al., 2009), a combination of two different protocols of postconditioning (rapid shuttering ischemia and delayed injection of bradykinin) can be used in order to increase the impact of treatment (Danielisova et al., 2012). On the other hand, delayed postconditioning has been
Fig. 4. Results of Morris water maze test performed on day 6 and 7 after ischemia with or without postconditioning. a Significantly different (p < 0.001) versus sham control group. b Significantly different (p < 0.05) versus remote postconditioning group.
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Table 2 Effect of kainic acid intoxication (with or without subsequent remote ischemic postconditioning applied 48 h after injection of kainate) on neurodegeneration, survival and function of CA1 neurons measured 7 days after intoxication.
Fluoro Jade B positivity [neurons/mm] NeuN immunoreactivity [neurons/mm] Morris water maze escape time [s]
Control
Kainic acid 8 mg/kg i.p.
Kainic acid + postconditioning
– 292.43 ± 12.33 13.91 ± 2.72
186.00 ± 17.09a , b 125.00 ± 32.49a , b 36.48 ± 3.45c
0.86 ± 0.13 286.69 ± 5.80 22.25 ± 4.13
Results are expressed as mean ± S.E.M. a Significantly different (p < 0.001) from control. b Significantly different (p < 0.001) from remote ischemic postconditioning. c Significantly different (p < 0.01) from control.
used effectively 3 and 6 h after long-lasting focal ischemia (Ren et al., 2008). The mechanisms of postconditioning are still not clear. An excellent review on this subject was written by Zhao et al. (2012). In our experiments with delayed postconditioning after transient forebrain ischemia, by the effect of Cycloheximide we documented that protein(s) synthesized during the first 5 h after postconditioning are inevitable for the survival of CA1 neurons. Moreover, a strong significant decrease in glutamate concentration seems to be very important (Bonova et al., 2013). This correlates well with the idea of Wieloch et al. (1985). An essential condition for the survival of the pyramidal neurons in CA1 is the restoration of permanently blocked protein synthesis (Thilmann et al., 1986). Although there is no doubt about the efficacy of postconditioning, the current problem is how to apply this in clinical practice. We need to consider the patient in a critical condition after cardiac arrest, stroke or intoxication, when most of the seemingly wide range of stressors is not usable. Repetitive ischemic insult and hypoxia are practically unacceptable. The use of hyperthermia or hypothermia is also debatable. Use of lipopolysaccharide induces fever, and 3-nitropropinic acid is a mitochondrial poison. Application of bradykinin may result in an increase in internal calcium and subsequent excitotoxic glutamate release (Parpura et al., 1994). The use of remote ischemic postconditioning, which is performed by local remote stress, initiates defensive mechanisms in the whole organism, but does not constitute a risk of damage to the limb tissue. Przyklenk et al. (1993) first described that “brief episodes of ischemia in one vascular bed protected the remote, virgin myocardium from subsequent sustained coronary artery occlusion”. Later, remote local ischemia was used as postconditioning through repetitive occlusion and release of selected arteries, but this was changed subsequently into the more palatable non-invasive tourniquet ischemia of skeletal muscles/entire limb (Tsubota et al., 2010; Mansour et al., 2012). Many attempts have been made to determine the optimum protocol for ischemic postconditioning. The development has moved from several periods of ischemia/reperfusion lasting a few seconds through longer-lasting repetitive I/R intervals toward singular periods of ischemia lasting 10–20 min (Saxena et al., 2010; Pignataro et al., 2013). In conclusion, when we used non-invasive singular 20 min ischemia of skeletal muscle 2 days after normothermic transient cerebral ischemia or kainate intoxication, it clearly proved that this stressor is able to effectively prevent delayed neuronal death of the most sensitive neuronal population in the brain, which is the CA1 layer of pyramidal neurons in the hippocampus. We believe that the procedure of tourniquet postconditioning can be used in clinical medicine after cardiac arrest, hypertensive shock and other transient ischemic attacks as well as apoptosis inducing intoxications. The therapeutic window after permanent focal ischemia is substantially shorter, so postconditioning should be used as soon as possible and similarly to other attacks can be used repeatedly. It is important to note that tourniquet application increases blood pressure to about 30% higher values than before
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