Clinical trials investigating the effects of peri-ischemic hypothermia on neurological outcome show inconsistent results. Although hypothermia preserves neurologic function in patients successfully resuscitated from cardiac arrest after ventricular fibrillation1,2 and reduces the risk of death or disability in neonates with hypoxic-ischemic encephalopathy,3,4 hypothermia failed to improve clinical outcome in patients with traumatic brain injury5 or during intracranial aneurysm surgery.6 Because neurologic outcome after cerebral ischemia may improve over time after therapeutic hypothermia, it is feasible that this is in part related to cerebral regeneration by postischemic endogenous neurogenesis. Most studies have focused on the neuroprotective aspects of hypothermia, and potential impact on neuroregenerative repair mechanisms in the brain have rarely been suggested.7
Ischemic neurodegeneration was previously considered to result in permanent loss of neurons with no capacity for cellular regeneration. This view has been challenged by the evidence that certain brain areas retain the capability to generate new neurons in adulthood.8 Newly generated neurons in the hippocampus make functional synaptic connections and possess neurophysiological characteristics of mature neurons.9,10 Furthermore, they extend axonal projections to the hippocampal CA3 region,11,12 and the time course of cognitive recovery after brain lesions is coincident with the integration of newly generated granule cells into the hippocampal circuitry.13 The nature and potential extent of hypothermic effects on neurogenesis after cerebral lesions are unknown. Therefore, we investigated whether mild therapeutic hypothermia leads to a long-term increase in postischemic neurogenesis 28 days after forebrain ischemia in the rat, with special attention to the timepoint of application.
All experiments were approved by the local governmental authorities (Landesuntersuchungsamt Rheinland-Pfalz, Koblenz, Germany, approval number 1.5 177-07/051-13) and performed in accordance with the German animal protection law. Eighty-five fasted male Sprague Dawley rats (weight 359 ± 42 g, age 9-12 wk; Charles River Laboratories, Sulzfeld, Germany) were included in the study (Table 1). The effect of intraischemic and postischemic hypothermia on the extent of neurogenesis was analyzed 28 days after cerebral ischemia. Nonanesthetized and nonischemic naïve animals were studied as reference for natural neurogenesis.
Rats were anesthetized in a bell jar saturated with sevoflurane (Abbott GmbH, Wiesbaden, Germany), intubated, and mechanically ventilated (683 Small Animal Ventilator, Harvard Apparatus, Holliston, MA) with 3.5% sevoflurane in oxygen and air (fraction of inspired oxygen = 0.33). A temperature probe was placed into the right temporal muscle. With exception of animals rendered hypothermic during or after cerebral ischemia, the pericranial temperature was maintained constant at 37.5°C with a servo-controlled heating lamp and pad (TCAT-2DF Controller, Physitemp Instruments, Clifton, NJ). Catheters were inserted into the right femoral artery and vein and the right jugular vein for arterial blood pressure monitoring, blood withdrawal, and drug administration. Loose ligatures were placed around both common carotid arteries for later clamping. Incisions were infiltrated with 0.5% bupivacaine (0.2 mL, AstraZeneca GmbH, Wedel, Germany).
Sevoflurane was adjusted to 0.9 volume% (vol%) end-tidal and combined with a sufentanil infusion (Janssen-Cilag GmbH, Neuss, Germany) at the rate of 2.5 μg · kg−1·h−1 (Syringe Pump, Model 11 Plus, Harvard Apparatus, Holliston, MA) to ensure minimal effects of sevoflurane on cerebral blood flow. Animals were randomly assigned to treatment groups (Table 1). After an equilibration period of 30 min, forebrain ischemia was induced by blood withdrawal to a mean arterial blood pressure of 40 mm Hg (8-10 mL) and bilateral common carotid artery clip-occlusion. After 14 min of ischemia, clips were removed and the withdrawn blood, which was kept at body temperature on a heating pad in heparinized syringes, was reinfused slowly over 15 min to avoid cerebral hyperemia and systemic hypertension.
Animals were kept in anesthesia (sevoflurane 0.9 vol% end-tidal supplemented with sufentanil 2.5 μg · kg−1 · h−1) for a recovery period of 90 min to enable hypothermia and the rewarming period. Physiological variables were recorded before cerebral ischemia (baseline), at the end of ischemia (ischemia), after reperfusion (reperfusion), and at the end of the recovery period (recovery). For sham-operated animals, variables were recorded at corresponding times. After completion of the recovery period, catheters were removed, wounds were closed, and the animals were tracheally extubated. Pericranial temperature was continuously monitored until the animals showed adequate motor activity. Rats were then returned to their cages.
Pericranial temperature was decreased within 15 min from 37.5°C to 33°C by switching off the heating lamp/pad and cooling the body surface with ice bags. For animals with intraischemic hypothermia, the cool-down period was initiated 15 min before blood withdrawal and 33°C was attained with the beginning of hemorrhagic hypotension, whereas for animals with postischemic hypothermia, the cool-down period was initiated with the onset of reperfusion and 33°C was attained 15 min after the onset of reperfusion. The pericranial temperature of 33.0°C was maintained over 45 min by partly removing and replacing the ice bags, and the rewarming period was initiated 23 min after the onset of reperfusion for the intraischemic hypothermia group and 60 min after the onset of reperfusion for the postischemic hypothermia group. Rats were rewarmed over 45 min to a pericranial temperature of 37.5°C (0.1°C/min). Sham-operated animals were cooled and rewarmed at corresponding times. The designated temperature for each study section was continuously monitored and adjusted according to requirements (Table 2). During the hypothermic period, α-stat strategy was used to control arterial carbon dioxide tension.
5-Bromo-2-Deoxyuridine (BrdU) Labeling and Tissue Preparation
For labeling of proliferating cells, 100 mg/kg BrdU (Sigma-Aldrich Chemie GmbH, Munich, Germany) was injected intraperitoneally every day during the first 7 days of survival. As a thymidine analog, BrdU is incorporated into deoxyribonucleic acid on mitotic division. After an observation period of 28 days, animals were deeply anesthetized with sevoflurane and transcardially perfused with 100 mL saline 0.9% and 100 mL paraformaldehyde 4% in phosphate buffer (0.2 mol/L). Brains were removed, postfixed in paraformaldehyde-phosphate buffer for 24 h, and placed in 30% sucrose. Forty-micrometer sagittal brain sections were prepared and stored in a cryoprotection solution (glycerol, ethylene glycerol, and 0.1 mol/L phosphate buffer) at −20°C.
Evaluation of Neurogenesis After 28 Days
Immunohistochemistry and immunofluorescence stainings were performed as previously described.14 In brief, sections for immunohistochemistry staining were incubated with the primary mouse anti-BrdU antibody (monoclonal mouse immunoglobulin G [IgG], 1:500; Roche Diagnostics Corp., Indianapolis, IN) followed by an incubation with the biotinylated secondary donkey anti-mouse antibody (biotin-SP-conjugated donkey anti-mouse IgG, 1:500; Jackson ImmunoResearch Laboratories, West Grove, PA). Sections for immunofluorescence staining were incubated with the primary rat anti-BrdU antibody (monoclonal rat IgG, 1:500; Oxford Biotechnology, Oxford, UK) and the primary mouse anti-neuron-specific nuclear protein antibody (monoclonal mouse IgG, 1:250; Chemicon International, Temecula, CA) followed by an incubation with the secondary antibodies (fluorescein isothiocyanate-conjugated donkey anti-rat IgG, 1:500, rhodamine red-X-conjugated donkey anti-mouse IgG, 1:500; Jackson ImmunoResearch Laboratories).
Blinded stereologic analysis of immunohistochemically stained BrdU-positive cells was performed manually using light microscopy assuming equal distribution of BrdU-positive cells throughout the dentate gyrus. BrdU-positive cells were counted in a 1-in-10 series of sections (400 μm apart) spanning the entire dentate gyrus, and results were multiplied by 10. To determine the percentage of newly generated neurons, BrdU-positive cells were analyzed for colabeling of BrdU and neuron-specific nuclear protein with an immunofluorescence microscope (Axiovert 200, Zeiss GmbH, Göttingen, Germany) combined with an ApoTome and Axiovision software (Zeiss GmbH). The resulting percentages were multiplied with the stereologically estimated amount of BrdU-positive cells assessed by immunohistochemical staining to estimate the amount of newborn neurons in the dentate gyrus.15 In addition, the CA1 and CA3 regions were analyzed for the existence of cells colabeled for BrdU and neuron-specific nuclear protein.
Evaluation of Histopathological Damage and Volume of Dentate Gyrus After 28 Days
Histopathological damage was assessed in the hippocampal CA1 and CA3 regions in sections stained with hematoxylin and eosin in a blinded fashion. The amount of injured neurons (eosinophilic cytoplasm and pyknotic nuclei) was graded according to the following score (hematoxylin-eosin index): 0 = no pathologic change, 1 = 1%-10% pathologic changes, 2 = 11%-50% pathologic changes, and 3 = >50% pathologic changes.
The area of the dentate gyrus was measured in every tenth section of the brain using Optimas 6.51 software (Media Cybernetics, Silver Spring, MD). The volume of the dentate gyrus was calculated by multiplying the results with the thickness of the slice (40 μm) times 10 (as every 10th slice was analyzed).
The numbers of newly generated neurons are presented as median and quartiles. Confirmatory Mann-Whitney U-tests were performed for the comparison of each ischemic group with the corresponding sham-operated group and with the naïve group as well as for the pairwise comparisons between the ischemic groups. The nonparametric Mann-Whitney U-tests were applied because some of the data did not show a normal distribution. The global significance level was defined at 5% and, applying a Bonferroni adjustment to these 9 comparisons, a P value was considered statistically significant if P ≤ 0.05/9 = 0.0056. Pairwise comparisons of the histopathological damage were analyzed on an explorative level by Mann-Whitney U-tests. Statistical calculations were performed with SPSS 12.0 (Chicago, IL). Physiological variables and animal weight are presented as mean ± sd. No statistical tests were performed for physiological variables to avoid type I error inflation because of their large number. However, comparability of physiological variables across experimental groups was assessed on a descriptive level.
Physiological variables are listed in Table 3. Mean arterial blood pressure decreased in the ischemic groups to 40 mm Hg according to blood withdrawal. Mean arterial Pao2 ranged between 135 and 197 mm Hg. Mean arterial Paco2 varied between 35 and 40 mm Hg. Because of blood withdrawal, the hemoglobin concentration decreased by an average of 3.0 g/dL at the end of ischemia. This effect was reversed upon reperfusion. Average plasma glucose concentrations of the pooled ischemic groups increased during ischemia (from 79 ± 20 to 163 ± 70 mg/dL) but recovered after reperfusion (121 ± 38 mg/dL) and at recovery (75 ± 15 mg/dL). With exception of higher plasma glucose levels for groups IHypoIsch and PHypoIsch at the end of ischemia (182 ± 70 and 183 ± 94 mg/dL), values remained within the physiological range of rats.
Forty-five minutes of hypothermia did not affect neurogenesis in sham-operated rats (IHypoSham median 5500 [Q1: 4600 and Q3: 7500] new neurons and PHypoSham median 5400 [Q1: 4300 and Q3: 6500] new neurons) compared with normothermic rats (median 4600 [Q1: 3600 and Q3: 6100] new neurons) and naïve rats (median 6000 [Q1: 4000 and Q3: 7300] new neurons; Fig. 1). Twenty-eight days after cerebral ischemia, BrdU-positive neurons increased to median 18,800 [Q1: 13,500 and Q3: 22,500] new neurons with normothermic conditions (P < 0.0056 versus NormoSham and naïve). Likewise, newly generated neurons increased to median 15,200 [Q1: 12,800 and Q3: 21,300] new neurons with 45 min of hypothermia beginning before the start of ischemia (P < 0.0056 versus IHypoSham and naïve) and to median 14,300 [Q1: 6300 and Q3: 18,700] new neurons with 45 min of postischemic hypothermia (P < 0.0056 versus naïve). Compared with neurogenesis after normothermic ischemia, intraischemic and postischemic hypothermia did not affect the ischemia-induced increase in new neurons (P = 0.637 NormoIsch versus IHypoIsch; P = 0.043 NormoIsch versus PHypoIsch; and P = 0.212 IHypoIsch versus PHypoIsch). No newborn neurons colabeled for BrdU and NeuN were detected in the CA1 and CA3 regions (data not shown).
Sham-operated and naïve animals showed no histopathological damage. In ischemic rats, neuronal injury of the CA1 region was more than 50% with normothermia and postischemic hypothermia, whereas intraischemic hypothermia reduced neuronal damage to <10% (Fig. 2A). The CA3 region showed minor damage of up to 50% with normothermic ischemia and postischemic hypothermia, whereas intraischemic hypothermia also reduced neuronal damage to <10% (Fig. 2B).
Volume of the Dentate Gyrus
The average volume of the dentate gyrus was similar for all groups (0.8 ± 0.1 mm3), regardless of cerebral ischemia or pericranial temperature (data not shown).
The present data show that cerebral ischemia resulted in an increased number of new neurons in the dentate gyrus after 28 days. Peri-ischemic hypothermia did not affect postischemic endogenous neurogenesis regardless of application time compared with normothermia. The postischemic increase in newly generated neurons was not altered by the severity of histopathological damage, because a comparable amount of newly generated neurons was present despite differences in histopathological damage. In summary, these data suggest that 28 days after cerebral ischemia, peri-ischemic hypothermia as well as the severity of postischemic histopathological damage seem to have no significant impact on postischemic endogenous neurogenesis.
Despite the common application of therapeutic hypothermia in clinical practice, no study has investigated hypothermia-induced effects on postischemic neurogenesis. Two studies have assessed the impact of hypothermia on neurogenesis in nonischemic mammals, demonstrating a suppression of neurogenesis, which is in contrast to the present data. However, one study was performed in 7-day-old rats,16 when the developing brain is most vulnerable because it is on the peak of synaptogenesis.17 For this reason, synaptic integration of newly generated neurons in newborn rats is not comparable with adult rats and even slight disturbances in natural homeostasis, such as a decrease in brain temperature, may lead to neuronal death. In the other study, nonanesthetized adolescent rats were immerged into cold water and subsequently kept at 20°C, which resulted in a suppression of neurogenesis, whereas rewarming to 30°C protected against the reduction in new cell formation.18 Because neurogenesis is a multifactorial process and diminished by stressful experiences,19 the stress response of nonanesthetized animals immerged in cold water without a rewarming period may explain the results.
Transient cell proliferation in the dentate gyrus occurs after various ischemic challenges, such as traumatic brain injury,20 focal cerebral ischemia,21 and global cerebral ischemia.14 There is controversy about whether the formation of histopathological damage leads to the increase in neurogenesis. Although some studies showed a positive correlation between histopathological damage and endogenous neurogenesis, other studies had contradictory results.14,22–24 In this study, a comparable increase in newly generated neurons was shown after 28 days in all ischemic groups despite differences in histopathological damage. Similarly, sublethal ischemic stimuli, such as ischemic preconditioning, are capable of inducing neurogenesis without formation of histopathological damage.25 The present data support the view that postischemic neurogenesis is triggered by mechanisms other than the histopathological damage, and they direct future research toward intraischemic events that are not affected by mild hypothermia. For example, neurogenesis- and growth factor-associated genes were similarly expressed at 32°C and 37°C after rat forebrain ischemia in the hippocampus.26 Although these aspects have not been investigated here, they are potentially important in the modulation of postischemic neurogenesis.
Forty-five minutes of hypothermia beginning before ischemia provided long-term neuroprotection, whereas 45 min of hypothermia beginning after reperfusion did not reduce neuronal damage. However, 30-60 min of postischemic hypothermia could not achieve relevant neuroprotection after global ischemia in gerbils.27 The opposite effects of intraischemic and postischemic hypothermia on the severity of histopathological damage may be explained by differences in excitotoxic glutamate release. Glutamate release triggered by cerebral ischemia is a key mechanism in the formation of neuronal damage after brain injury.28 Intraischemic hypothermia completely inhibits the ischemia-induced increase in synaptic levels of glutamate,29 whereas postischemic hypothermia fails to do so.30,31 For neuroprotection by postischemic hypothermia, the duration may need extension, because the therapeutic benefit of postischemic hypothermia positively correlates with duration. For example, hypothermic periods of 0.5-6 h initiated immediately after reperfusion after global ischemia resulted in progressive protection from ischemic damage. However, relevant protection was not observed before hypothermic periods of 2 h, indicating that hypothermia, even when initiated immediately after reperfusion requires a duration of 2 h or more to be effective.27 Forty-five minutes of postischemic hypothermia as applied in this study was, therefore, most likely inadequate for long-term neuroprotection. Furthermore, the mildly increased intraischemic plasma glucose level in rats with postischemic hypothermia may also have contributed to the damage seen in this group.
The present findings may be limited by the fact that sevoflurane and sufentanil were used as background anesthesia. Peri-ischemic sevoflurane administration has been shown to enhance neurogenesis with increasing concentrations.14 Concerning the application of sufentanil, μ-opioid receptors take part in the regulation of progenitor cell survival.32 However, because the number of newly generated neurons in sham-operated rats was not different from naïve animals, an overall effect of the anesthetics on neurogenesis is most unlikely. It also cannot be excluded that sevoflurane affected the degree of histopathological damage because it has preconditioning effects. However, even if there was an overall preconditioning effect of sevoflurane, it should not have influenced comparability because the same dose of sevoflurane was given in each animal.
The BrdU labeling paradigm may present a further limitation of the study. Previous studies, describing a short-term reduction of nonischemic neurogenesis by hypothermia, used a single injection of BrdU immediately before or after hypothermia and assessed the effects within 1-24 h.16,18 In contrast, the BrdU labeling protocol of this study was designed for the assessment of changes induced during the first postischemic week and their long-term sustainability. Therefore, short-term effects of hypothermia on neurogenesis may not have become evident. However, the recommendations for standards regarding preclinical neuroprotective and restorative drug development Stroke Therapy Academic Industrie Roundtable (STAIR) have emphasized the necessity to follow animals for longer time periods, because initial effects may be lost over time.33
The significance of the present findings may also be questioned because no new neurons migrated to the lesion site, as observed for neuroblasts in the striatum34 and in the cortex.35 However, axonal projections to the hippocampal CA3 region11,12 and the coincidence of cognitive recovery with the integration of newly generated granule cells in the hippocampal circuitry after brain lesions13 suggest that newly generated dentate granule neurons contribute to hippocampal regeneration without further migration.
In conclusion, all ischemic groups showed a comparable increase in the number of newly generated neurons after 28 days despite differences in the peri-ischemic pericranial temperature and in the postischemic histopathological damage. These findings suggest that postischemic neurogenesis is induced by intraischemic events that are most likely not affected by hypothermia and the resulting histopathological damage. Therefore, this study demonstrated that mild peri-ischemic hypothermia did not increase postischemic neurogenesis and that differences in histopathological injury did not affect the amount of newly generated neurons 28 days after cerebral ischemia.
The authors gratefully acknowledge Frida Kornes (Technician, Department of Anesthesiology, Medical Center of the Johannes Gutenberg-University, Mainz, Germany) for excellent technical assistance. Data in this study form part of doctoral theses presented by Natascha Benz (Resident, Department of Anesthesiology, Medical Center of the Johannes Gutenberg-University, Mainz, Germany) and Matthias Lörscher (Resident, Department of Anesthesiology, Medical Center of the Johannes Gutenberg-University, Mainz, Germany) to the Medical Faculty, Medical Center of the Johannes Gutenberg-University, Mainz, Germany.
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