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. 2012 Mar 21;32(8):1525–1534. doi: 10.1038/jcbfm.2012.38

Prolonged therapeutic hypothermia does not adversely impact neuroplasticity after global ischemia in rats

Gergely Silasi 1, Ana C Klahr 1, Mark J Hackett 2, Angela M Auriat 3, Helen Nichol 4, Frederick Colbourne 1,5,*
PMCID: PMC3421089  PMID: 22434072

Abstract

Hypothermia improves clinical outcome after cardiac arrest in adults. Animal data show that a day or more of cooling optimally reduces edema and tissue injury after cerebral ischemia, especially after longer intervention delays. Lengthy treatments, however, may inhibit repair processes (e.g., synaptogenesis). Thus, we evaluated whether unilateral brain hypothermia (∼33°C) affects neuroplasticity in the rat 2-vessel occlusion model. In the first experiment, we cooled starting 1 hour after ischemia for 2, 4, or 7 days. Another group was cooled for 2 days starting 48 hours after ischemia. One group remained normothermic throughout. All hypothermia treatments started 1 hour after ischemia equally reduced hippocampal CA1 injury in the cooled hemisphere compared with the normothermic side and the normothermic group. Cooling only on days 3 and 4 was not beneficial. Importantly, no treatment influenced neurogenesis (Ki67/Doublecortin (DCX) staining), synapse formation (synaptophysin), or brain-derived neurotropic factor (BDNF) immunohistochemistry. A second experiment confirmed that BDNF levels (ELISA) were equivalent in normothermic and 7-day cooled rats. Last, we measured zinc (Zn), which is important in plasticity, with X-ray fluorescence imaging in normothermic and 7-day cooled rats. Hypothermia did not alter the postischemic distribution of Zn within the hippocampus. In summary, cooling significantly mitigates injury without compromising neuroplasticity.

Keywords: cardiac arrest, hypothermia, neuroprotection, stroke, synchrotron, temperature

Introduction

Strategies for improving outcome after global (cardiac arrest) and focal ischemia (stroke) are generally divided into those that lessen neurologic impairments by reducing acute brain damage through neuroprotection, and by promoting neuroplasticity. This dichotomy exists partly because neuroprotectants are most effective when administered soon after injury, whereas treatments that promote plasticity and regeneration are generally beneficial somewhat later over days and even weeks (Kleim and Jones, 2008). This is an oversimplification, however, because neurodegeneration sometimes continues for weeks (Colbourne et al, 1999; Dietrich et al, 1993; Valtysson et al, 1994), whereas some forms of plasticity are initiated within minutes of ischemia (Sigler et al, 2009). Accordingly, degenerative and regenerative processes overlap both temporally and spatially (e.g., peri-infarct region) after ischemia (Lo, 2008). This means that neuroprotective treatments applied early after ischemia should work most effectively when they selectively target deleterious processes while allowing or facilitating repair mechanisms. Unfortunately, this ideal situation is unlikely to apply to many neuroprotectants.

Arguably, the best neuroprotective intervention is therapeutic hypothermia, a mild reduction in body and brain temperature. It has been shown to markedly reduce ischemic injury in many models of global and focal ischemia (MacLellan et al, 2009; van der Worp et al, 2010). Notably, studies show superior protection when the treatment is prolonged. For example, 24 hours of mild hypothermia provides permanent and substantial neuroprotection after global ischemia (Colbourne and Corbett, 1995) unlike brief periods that convey little, if any, enduring protection (Dietrich et al, 1993). The use of prolonged hypothermia treatment also improves outcome in out-of-hospital cardiac arrest patients (Bernard et al, 2002; The Hypothermia After Cardiac Arrest Study Group, 2002) and after hypoxia/ischemia in newborns (Shankaran et al, 2005). Focal ischemic and hemorrhagic events may also require prolonged cooling to maximally reduce damage and to treat protracted edema (Clark et al, 2008; Florian et al, 2008; MacLellan et al, 2009). For instance, a recent clinical study with intracerebral hemorrhage patients cooled for 10 days (Kollmar et al, 2010), and others have cooled stroke patients for longer (Gasser et al, 2003). Clearly, such lengthy interventions may interfere with endogenous repair processes that initiate within days and persist for weeks.

For multiple reasons, global ischemia models are a convenient way to study the effects of varying cooling duration on the extent of degenerative and regenerative processes. First, injury is largely restricted to the hippocampal CA1 zone where pyramidal neurons die typically over 2–4 days (Pulsinelli et al, 1982). Second, the mitigating effects of postischemic hypothermia on CA1 injury have been widely studied and, as discussed, these animal findings clearly predicted results in cardiac arrest patients (Bernard et al, 2002; The Hypothermia After Cardiac Arrest Study Group, 2002). Third, plasticity responses within the hippocampus are well documented and are strongly linked to behavioral recovery. For example, mossy fiber sprouting is elevated on the third postischemic day and remains elevated until at least 7 days (Koh et al, 1996; Nishimura et al, 2000; Schmidt-Kastner et al, 1997). Also, ischemia markedly elevates neurogenesis in the subgranular zone of the dentate gyrus (DG) (Liu et al, 1998). A final example is the distribution of the trace element zinc (Zn), which is influenced by ischemia, and involved in neuronal death (Suh et al, 2000) as well as in postischemic plasticity (Nakashima and Dyck, 2009).

Using a broad range of outcome measures, we examined how various cooling durations affect neuroprotection as well as postischemic plasticity. It has been established that ∼48 hours of mild hypothermia results in near complete neuroprotection in rat and gerbil models of global ischemia, especially with early intervention (MacLellan et al, 2009). Accordingly, we evaluated whether maintaining hypothermia therapy beyond this time may impede plasticity while failing to provide added protection (‘diminishing returns'). To examine these possibilities, we used a focal cooling system (Clark and Colbourne, 2007; Silasi and Colbourne, 2011b) that allows the selective cooling of one hemisphere in otherwise normal rats (i.e., no anesthesia confounds). We recently showed that this approach results in considerable CA1 protection (Silasi and Colbourne, 2011b), and it is perhaps the only method that can be used to induce hypothermia for up to 1 week in rodents without risk of serious systemic complications associated with whole body cooling (e.g., hypotension and extensive weight loss; Clark and Colbourne, 2007). After ischemia by the well-established 2-vessel occlusion (2VO) model (Smith et al, 1984), we maintained hypothermia for durations up to 7 days. Then, we quantified the level of neuroprotection in the CA1 zone, the expression of plasticity-related proteins (brain-derived neurotropic factor (BDNF) and synaptophysin), microglial infiltration within the CA1 sector, and neurogenesis in the subgranular zone of the DG. Here, we compared cooling (1 hour after ischemic delay) for 2, 4, and 7 days with a normothermic control group and with animals cooled for 2 days with a 48-hour intervention delay. This last group was to test a treatment that was expected to have no neuroprotective effect, but potentially a harmful effect on plasticity. Comparisons between cooling for 2, 4, and 7 days were to test whether extended cooling, which was not expected to provide additional CA1 protection, would impede postischemic plasticity. A second study measured BDNF with ELISA to evaluate the immunohistochemistry findings observed in the first experiment. Finally, a third experiment used synchrotron-based rapid scanning X-ray fluorescence (RS-XRF) imaging (Popescu and Nichol, 2011) to visualize and quantify the distribution of the trace element Zn in the hippocampus.

Materials and methods

Subjects

All procedures were in accordance with the Canadian Council on Animal Care and were approved by the Biosciences Animal Care and Use Committee at the University of Alberta. Experiments were performed on 83 male Sprague-Dawley rats (Biosciences breeding colony, University of Alberta) weighing ∼300 g and 3 to 4 months old at the time of ischemia. Water and food were available ad libitum, except during fasting before ischemia. Rats were housed singly in standard polycarbonate cages with woodchip bedding on a 12-hour light cycle (on at 0700 h). Group assignment was random and assessment was blinded (e.g., cell counts). However, investigators were not blinded when treatments were administered because it was impossible to be blinded to the cooling method.

Experiment 1

Temperature Device Implantation Surgery

All surgical procedures were performed aseptically under isoflurane anesthesia (4% induction and 2% to 2.5% maintenance in 60% N2O, balance O2). Rats were surgically implanted with a focal cooling plate (1 mm thick, 2 mm wide, 6 mm long; Figure 1) over the right hemisphere according to established methods (Clark and Colbourne, 2007; Silasi and Colbourne, 2011b). The cooling strip was positioned next to the temporal ridge, two anchor screws were threaded into the parietal bone, and dental cement was applied to secure the device. Rats were then tethered via PE50 tubes contained within a flexible metal sheath that extended from the skull to an overhead swivel connected to a cold-water source. A 4-day rest period then followed before ischemia surgery.

Figure 1.

Figure 1

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Diagram illustrating skull placement (A) of the cooling device (B). The device was connected to a cold-water source via tubes connected to an overhead swivel. Thus, rats were awake and mobile during cooling. Only the right hemisphere was cooled in hypothermic rats whereas neither side was cooled in the normothermic group. With this method, the affected hemisphere becomes cooled to ∼33°C as recently illustrated (Silasi and Colbourne, 2011b).

Forebrain Ischemia Surgery

Forebrain ischemia (8 minutes) was produced using the 2VO model (Smith et al, 1984). Rats were fasted for ∼18 hours before surgery to lower blood glucose levels into a consistent range (∼6 to 10 mmol/L). Core temperature was maintained at 37°C through a rectal temperature probe connected to a water blanket (Gaymar TP3E, Orchard Park, NY, USA). To avoid a decrease in brain temperature during ischemia, which significantly contributes to model variability, skull temperature was regulated through a subcutaneous thermocouple probe (model: HYPO-33-1-T-G-60-SMG-M; Omega, Stanford, CT, USA) connected to an overhead infrared lamp (150 W). The tail artery was cannulated for continuous measurement of mean arterial blood pressure (PressureMAT; Pendotech, Princeton, NJ, USA) and to collect blood (100 μL) for regulating pH, PO2, PCO2, and glucose within the normal range (Radiometer ABL 810; Radiometer, Copenhagen, Denmark). The common carotid arteries were isolated bilaterally, and the right jugular vein was cannulated with Silastic tubing connected to a heparinized syringe. Ischemia was induced by withdrawing blood from the jugular vein until mean arterial blood pressure reached ∼35 mm Hg, at which time both common carotid arteries were temporarily occluded using vascular clamps (00400-03; Fine Science Tools, Vancouver, Canada). Blood pressure was maintained at 35 mm Hg during ischemia and then exsanguinated blood was slowly reinfused, the catheters were withdrawn and the neck and tail incisions were sutured.

Treatments

In this experiment, all rats (n=53) received 2VO ischemia followed by either normothermia (NOR; n=19), or focal brain cooling during postischemic days 1 and 2 (HYP1-2; n=8), 3 and 4 (HYP3-4; n=9), 1 through 4 (HYP1-4; n=8), or 1 through 7 (HYP1-7; n=9). Cooling to ∼33°C (Clark and Colbourne, 2007; Silasi and Colbourne, 2011b) was initiated 1 hour after ischemia (for groups that start treatment on day 1) and rewarming occurred over 6 hours. However, the HYP1-7 rats were killed before rewarming.

Euthanasia and Histology

One week after global ischemia, rats were administered an overdose of sodium pentobarbital (100 mg/kg intraperitoneally) and perfused transcardially with phosphate-buffered saline, followed by 10% formalin. The brains were removed and postfixed in formalin for ∼24 hours before being embedded in paraffin and sectioned at 10 μm on a rotary microtome. Hematoxylin and eosin (H&E) was used to stain one series of sections, while adjacent sections were used for immunolabeling (Silasi and Colbourne, 2011a). Before incubation with the primary antibody, antigen retrieval was performed by boiling the sections in 0.1 mol/L citrate buffer (pH 6.3) for 15 minutes in a microwave. Immunolabeling was performed by incubating the sections overnight in antibodies for microglia (Rabbit anti-Iba-1, 1:1,000; Wako product: 019-19741; Wako, Richmond, VA, USA), synaptophysin (Mouse anti-synaptophysin, 1:200; Millipore, clone SY38; Millipore, Billerica, MA, USA), BDNF (Rabbit anti-BDNF, 1:500; Abcam ab72439; Abcam, Cambridge, MA, USA), Doublecortin (DCX; Goat anti-DCX, 1:500, Santa Cruz C-18), and the proliferative marker Ki67 (Rabbit anti-Ki-67, 1:500; Vector VP-K451, Burlington, ON, Canada). The secondary antibodies were from Jackson Laboratories (West Grove, PA, USA), and were applied at a concentration of 1:500 for 2 hours at room temperature. DAPI (1:500; Sigma, Oakville, ON, Canada) was added during the final step of each procedure to visualize cell nuclei.

To quantify the number of remaining intact neurons within the CA1 field, H&E-stained sections were examined under a light microscope ( × 40) by an experimenter unaware of treatment identity. Viable looking neurons with distinct nuclear and cellular membranes (and not eosinophilic) were counted in the medial, middle, and lateral regions (summed) of the left and right CA1 zone cell layers as previously reported (Colbourne and Corbett, 1995; Silasi and Colbourne, 2011a). The number of Iba-1-labeled microglia was also determined in the same regions of interest with an epifluorescent microscope. The total number of DCX and Ki67 double-labeled cells was quantified in one section from each brain to determine if hypothermia altered neurogenesis in the DG. Synaptophysin and BDNF expression was quantified in the mossy fiber bundles in CA3 by performing densitometry analysis (Photoshop CS5, Macintosh, Adobe Systems Canada, Ottawa, ON, Canada) on digital images captured from an epifluorescent microscope. Densitometry measurements were averaged from two nonadjacent sections ∼3.8 mm posterior from Bregma (Paxinos and Watson, 1998).

Experiment 2

Rats were implanted with a cooling device and later subjected to 2VO ischemia as in experiment 1. Rats were then randomized to either the NOR (n=8) or HYP1-7 group (n=8).

Euthanasia and Brain-Derived Neurotropic Factor Enzyme-Linked Immunosorbent Assay

Seven days after ischemia, rats were anesthetized with 4% isoflurane and decapitated. The brains were immediately removed and their hippocampi were dissected free on an ice-cold petri dish. The hippocampal tissue was placed in Eppendorf tubes and immersed in isopentane cooled by dry ice. This process did not take longer than 3 minutes. Samples were then stored at −80°C until used. These samples were homogenized with a Dounce homogenizer (Kontes-7 mL, Vineland, NJ, USA) in ice-cold homogenate buffer solution suggested by the manufacturer of the BDNF sandwich enzyme-linked immunosorbent assay (ELISA, kit CYT306; Chemicon International, Temecula, CA, USA). The homogenate consisted of 100 mL/g of tissue of 100 mmol/L Tris-HCl, 1 mol/L NaCl, 2% bovine serum albumin, 4 mmol/L EDTA-Na+, 0.1% NaN3, 2% Triton X-100, 0.157 μg/mL benzamidine-HCl, 5 μg/mL aprotinin, 0.5 μg/mL antipain, 0.1 μg/mL pepstatin in 10% acetic acid in methanol, 17 μg/mL PMSF in 100% ethanol (reagents from Sigma-Aldrich Canada, Oakville, ON, Canada). Samples were centrifuged (14,000 g for 30 minutes) and supernatants were diluted using the sample diluent solution provided by the ELISA kit. A standard curve (0 to 750 pg/mL) was prepared with standard BDNF and sample diluent solution provided by the manufacturer. Sample dilutions and the standard curve were run in duplicate in each plate. The rest of the procedure was performed by following the protocol provided with the kit. Optical density in each plate was measured at 450 nm using an ELX800 absorbance Microplate reader (Bio-Tek Instruments, Winooski, VT, USA).

Experiment 3

Surgical procedures were similar to experiment 1. Briefly, 2VO ischemia was followed by either HYP1-2 (N=5) or NOR treatment (n=5) and all rats were killed on postischemic day 7. An additional four naive, weight-matched rats were included in the experiment to visualize basal element distribution without ischemia.

Euthanasia and Rapid Scanning X-Ray Fluorescence Imaging

Animals were euthanized by decapitation after isoflurane anesthesia without being perfused. The brains were rapidly removed, frozen in chilled isopentane and stored at −80°C until sectioning (Hackett et al, 2011). Cryostat sections (14 μm) were collected on Ultralene foil (EMS, Hatfield, PA, USA) for RS-XRF imaging.

Elemental analysis was performed at the Stanford Synchrotron Radiation Lightsouce (SSRL) beamline 10-2. The energy of the incident X-ray beam was 13 keV, and a tapered glass capillary was used to focus the beam spot to 35 μm. Beam exposure was 600 ms per 40 μm pixel. Images of total Zn and potassium (K) were acquired and analyzed with Sams' Microanalysis Toolkit (available for download at http://smak.sams-xrays.com/). It is important to note that RS-XRF measures the total amount of an element such as Zn. Therefore, this would include Zn involved in plasticity as well as that not likely to have a direct physiological role in plasticity (i.e., Zn tightly bound in metalloproteins). The distribution of Zn was normalized to the relatively homogenous distribution of K.

Statistical Analyses

Data were analyzed by analysis of variance and when required Tukey post hoc tests (SPSS v18 Mac, IBM Canada Ltd., Markham, ON, Canada). A P value of <0.05 was considered statistically significant, and all data are presented as mean±s.e.m.

Results

Experiment 1

Two rats died prematurely in this experiment (one NOR and one HYP1-4) and their data were excluded from this experiment. The cause of death was not determined.

Focal Brain Cooling Is Neuroprotective and Reduces Microglial Activation in the Cooled Hemisphere only

Hypothermia initiated 1 hour after ischemia was highly neuroprotective in the cooled hemisphere regardless of whether it was maintained for 2, 4, or 7 days (versus NOR, P⩽0.004; among HYP1-2, HYP1-4 and HYP1-7 groups, P⩾0.740). In contrast, hypothermia treatment provided only on postischemic days 3 and 4 did not reduce injury (P∼1, Figure 2A). There were no significant differences among groups in the left (normothermic) hemisphere (P⩾0.154).

Figure 2.

Figure 2

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Hypothermia initiated 1 hour after ischemia was neuroprotective in the cooled (right) hemisphere only (A) to a level approaching that of normal animals. There were significantly more remaining CA1 neurons in the HYP1-2, HYP1-4, and HYP1-7 groups relative to normothermia (NOR) animals. Representative photomicrographs illustrating a comparable amounts of CA1 injury and protection with 2 days of unilateral brain cooling have been published recently (Silasi and Colbourne, 2011b). These neuroprotective treatments, but not HYP3-4, decreased the number of Iba-positive microglia in the CA1 region in the cooled hemisphere (B). An ‘*' denotes P<0.05 versus the NOR group. Data are presented as mean±s.e.m.

Untreated ischemia resulted in extensive microglial infiltration (counts of Iba-1-labeled cells) in the CA1 region, which the three neuroprotective hypothermia protocols all mitigated in the cooled hemisphere (Figure 2B, P⩽0.001). These treatments (HYP1-2, HYP1-4, and HYP1-7), however, were not different (P⩾0.444). The HYP3-4 treatment, which did not reduce CA1 sector damage, was not significantly different in the right hemisphere (versus NOR, P=0.693). For the left side, there were no group differences (P⩾0.103).

Hypothermia Does Not Alter the Rate of Neurogenesis in the Dentate Gyrus

To quantify the effect of hypothermia on neurogenesis in the hippocampus, the number of Ki67-positive cells that also expressed DCX was quantified in the DG of each hemisphere (Figure 3A). There were no significant main effects for either the cooled (right; P=0.253) or normothermic (left; P=0.930) hemispheres in the number of Ki67/DCX colabeled cells (Figure 3B). We did not find any colabeled cells in other regions of the hippocampus, such as the CA fields. We also analyzed just the number of Ki67-positive cells in the DG of each hemisphere (data not shown). There were no significant main effects for either the cooled (right; P=0.058) or normothermic (left; P=0.734) hemispheres. Owing to the trend in the cooled hemisphere, we conducted post hoc comparisons that showed the HYP1-4 having more labeled cells than the NOR group (P=0.029) but not from the other hypothermia groups (P⩾0.156). However, the NOR group had a similar number of cells in each hemisphere (P=0.148) as did the HYP1-4 group (P=0.245). Accordingly, it appears that the HYP1-4 group, like the other hypothermia treatments, did not influence neurogenesis.

Figure 3.

Figure 3

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Neurogenesis was evaluated in the hippocampal dentate gyrus (DG) by counting the number of cells that were double labeled for Doublecortin (DCX) and Ki67 (A). Hypothermia did not significantly alter the rate of neurogenesis in the DG after global ischemia (B). Arrows in panel A denote sample Ki67-positive nuclei that also expressed the immature marker DCX. Data are expressed as mean±s.e.m. NOR, normothermia.

Hippocampal Brain-Derived Neurotropic Factor and Synaptophysin Expression Remains Unaltered by Hypothermia

We quantified the expression of these proteins in the mossy fiber supra-pyramidal bundle in the CA3 region. We found that both BDNF (Figures 4A and 4B) and synaptophysin (Figures 4C and 4D) were highly expressed in mossy fibers relative to other regions. However, hypothermia did not alter BDNF (P=0.631) or synaptophysin (P=0.427) expression.

Figure 4.

Figure 4

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Hypothermia treatment did not alter the expression of brain-derived neurotropic factor (BDNF) (A, B) or synaptophysin (SYN; C, D) in the mossy fiber bundle of the hippocampus. Expression of both proteins was largely restricted to the mossy fiber terminals near CA3 (B, D), and was mostly absent from the pyramidal cell layers. Quantification of optical density was performed in the regions indicated in the insets (B, D). Results are expressed as a percent expression in the cooled (right) hemisphere relative to the normothermic (left) hemisphere. Data are expressed as mean±s.e.m. NOR, normothermia.

Experiment 2

Hypothermia Does Not Affect Hippocampal Brain-Derived Neurotropic Factor Protein Levels

A comparison of the HYP1-7 and NOR groups on the concentration of BDNF protein (ELISA) in the homogenized tissue (Figure 5) revealed no main effects for the left (P=0.887) and right (P=0.980) hippocampi. Only the right side of the HYP1-7 group was cooled. Furthermore, a ratio between the right and left hemisphere was computed, which was not significantly different between the NOR and HYP1-7 groups (P=0.791). This was further supported by the finding that no difference exists between the left and right hippocampi in an analysis that included all subjects (P=0.416). Therefore, BDNF levels were not affected by cooling.

Figure 5.

Figure 5

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Brain-derived neurotropic factor (BDNF) protein levels (ng/mL of homogenized sample) were measured with ELISA. There were neither differences between hemispheres nor was there a treatment effect. Thus, cooling did not influence BDNF levels at this time confirming the immunohistochemistry findings (Figure 4). NOR, normothermia.

Experiment 3

Hippocampal Zinc Distribution Is Maintained After Neuroprotective Hypothermia

Synchrotron RS-XRF imaging was used to visualize the distribution of the total Zn and K within the hippocampus of naïve, NOR, and the HYP1-7-treated rats (Figure 6). We observed that the Zn signal was not a simple reflection of the number of cells as very little Zn was observed within the CA1 region of the hippocampus despite its cell density. Rather, the Zn was predominantly localized to regions rich in mossy fibers, the hilar and CA3 regions (Linkous et al, 2008). The CA1 region contained significantly less Zn relative to the hilar and CA3 regions (P<0.001), whereas K was homogeneously distributed throughout the hippocampus and neocortex. The amount of Zn (expressed relative to K) was similar among groups in the hilar, CA3, and CA1 zones on the left side (P⩾0.065 for main effects). On the right (cooled) side, the CA3 and hilar regions were unaffected by treatment (P⩾0.156) whereas the CA1 zone showed a significant main effect (P=0.049). Further post hoc analysis revealed no significant effects but a trend for the HYP1-7 group to have more Zn than the naïve rats (P=0.052). A comparison of the right and left sides was not significant for the HYP1-7 (P=0.776) and NOR groups (P=0.816). Thus, hypothermia did not affect Zn levels as evidenced by the similar patterns between hemispheres for the three groups.

Figure 6.

Figure 6

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Neither ischemia nor hypothermia significantly altered the distribution of Zinc (Zn) in the hippocampus (expressed relative to K background) at 7 days (A). The CA1 zone contained significantly less Zn than CA3 or dentate gyrus (DG) in both hemispheres, but there were no significant group differences in these regions. The selective distribution of Zn relative to the homogeneous distribution of K is clearly visible in representative rapid scanning X-ray fluorescence (RS-XRF) images of the hippocampus (B; NOR animal). NOR, normothermia.

Discussion

Postischemic hypothermia is a potent neuroprotective treatment that does not appear to impede neuroplasticity. Specifically, we found that prolonged hypothermia did not alter the expression of plasticity-related proteins (BDNF and synaptophysin), the rate of neurogenesis within the DG, or the distribution of the trace element Zn. As expected, cooling did substantially reduce CA1 sector damage, but there was no added benefit from cooling for 4 or 7 days compared with cooling for 2 days, at least in this model and setting (insult severity, treatment delay, etc.). While the additional cooling did not improve neuroprotection, it also did not harm plasticity. Thus, these findings strongly indicate that protracted durations of therapeutic hypothermia will not directly harm recovery after ischemia.

Mossy fiber plasticity, including that measured with synaptophysin labeling, occurs after global ischemia (Nishimura et al, 2000) and other injuries, such as trauma and epilepsy (Li et al, 2002). Although the exact function of synaptophysin is unknown, the protein is expressed within presynaptic membranes and is used as a marker for functional synapses (Hinz et al, 2001; Li et al, 2002). Thus, we evaluated synaptophysin levels after untreated and hypothermia-treated ischemia. Our results show that mossy fiber sprouting as gauged by synaptophysin levels was unaffected by cooling. Additional histological measures, such as the use of Golgi-Cox staining to measure dendritic length and spine density, would strengthen our current findings, as would electrophysiological measures of synaptic integrity. Indeed, patch clamping has been used to evaluate CA1 neurons salvaged by 2 days of postischemic hypothermia, and the effects of cooling in sham-operated animals (Dong et al, 2001). Cooling did not affect resting and regenerative membrane properties (e.g., excitatory postsynaptic currents after Schaffer collateral stimulation) in normal animals when assessed ∼30 days later. Also, CA1 neurons salvaged by hypothermia had normal electrophysiological properties, but this study did not evaluate treatment duration.

One possible mechanism that may mediate plasticity across injury models is the upregulation of neurotrophins, such as BDNF. The mossy fiber terminals have the highest expression of BDNF of any region within the central nervous system (Conner et al, 1997); however, due to a disparity between mRNA and protein expression after ischemia the exact changes that occur have been difficult to determine. Our results show that prolonged hypothermia does not alter BDNF expression at 7 days after ischemia, but we cannot exclude the possibility that cooling had a transient influence at earlier times after ischemia. A distinct advantage of our treatment method is that we restricted cooling to one hemisphere. Thus, we were able to compare the levels of BDNF protein (and other markers of plasticity) between salvaged and unprotected regions within the same animal. We found that neuroprotection did not affect BDNF levels, suggesting that CA1 neuronal death does not independently influence BDNF expression. This finding is somewhat surprising given that hypothermia significantly reduced the number of infiltrating microglial cells within the CA1, and microglia produce BDNF after ischemic injury (Lai and Todd, 2008). Further study with earlier survival times is needed to determine this.

Growth factors in the hippocampus modulate the rate of neurogenesis in the DG. Thus, we measured by the number of Ki67/DCX colabeled cells and found that hypothermia treatment did not affect neurogenesis. Overall, these findings are in line with previous results showing that neuroprotective treatments such as systemic hypothermia (Lasarzik et al, 2009; Silasi and Colbourne, 2011a) or ischemic preconditioning (Liu et al, 1998) do not alter postischemic neurogenesis. Notably, it remains possible that focal brain cooling may enhance the long-term survival of newly generated neurons in the DG, which we found to occur after systemic hypothermia, but this could not be examined with our current survival times.

Synchrotron hard X-ray techniques, such as RS-XRF, are valuable tools to study the distribution of total Zn in unfixed sections of brain tissue (Chwiej et al, 2011; Linkous et al, 2008; Popescu and Nichol, 2011), but the technique has not been applied to models of global ischemia. Thus, we used this precise method of measuring Zn to complement our immunohistochemical quantification of plasticity-related proteins, and to also evaluate potentially toxic effects of Zn. Indeed, several studies document the translocation of Zn from mossy fiber terminals to vulnerable neurons in hilar and CA1 regions during the acute phase of ischemia, which correlates with neurotoxicity in those neurons (Frederickson et al, 2006; Koh et al, 1996). However, synaptic plasticity at mossy fiber terminals is dependent on the presence of Zn (Nakashima and Dyck, 2009), so a protracted depletion of Zn from this region may hinder postischemic plasticity. Our results show that at 7 days after ischemia there are large amounts of Zn remaining in regions of the hippocampus rich in mossy fiber terminals in both hypothermia-treated and normothermic animals. Based on this finding, we conclude that the availability of Zn in mossy fiber terminals is not a limiting factor for plasticity after ischemic injury or neuroprotective hypothermia.

A lack of behavioral testing is an important limitation of the current experimental design. We did not assess behavior for several reasons. First, a causal link between any given postischemic plasticity process (e.g., synaptogenesis), especially subtle changes, and behavioral recovery is often difficult to establish (Whishaw et al, 2008). Second, many cognitive impairments markedly recover spontaneously after ischemia making it difficult to rely on any one simple test (Corbett and Nurse, 1998). Circumventing this problem by using multiple or more demanding tests is indicated, but problematic in the present setting owing to the fact that extensive behavioral testing would undoubtedly influence neuroplasticity by acting as a rehabilitation treatment. Third, we could not test during cooling that lasted for the duration of the experiment in one group. Fourth, behavioral impairments are difficult to detect in rodents after unilateral hippocampal injury because of compensatory pathways including from the uninjured hemisphere. Indeed, we previously encountered this problem when we used the Morris water maze to assess the neuroprotective effects of unilateral brain cooling (Silasi and Colbourne, 2011b). For these reasons, we did not examine behavioral performance after hypothermia treatment in the current study. However, we must concede that extended cooling may harm functional recovery by mechanisms or at times that we did not evaluate. Further study in models of global ischemia is warranted, but experiments in focal ischemia models would perhaps be more easily evaluated (e.g., assessing skilled reaching after motor system damage). Importantly, we have conducted a series of studies in normal rats that show no negative behavioral effects (e.g., skilled reaching, walking, and spontaneous limb usage) of prolonged focal cooling of the motor cortex (unpublished data of Auriat, Klahr, Silasi, MacLellan, Penner, Clark, and Colbourne).

Despite using thin sections to count H&E and immunolabeled cells, a commonly used procedure (e.g., Silasi and Colbourne, 2011a), it is possible that using unbiased stereology methods would have generated slightly different results or picked up very small treatment effects that presently went undetected. Another potential concern is the relatively low number of animals used in the Zn experiment, which was due to having limited RS-XRF scanning time. However, an advantage of our unilateral cooling method is that besides comparing groups we were able to compare hemispheres within animals and this showed no effect of treatment, but without further study we cannot exclude the possibility of small treatment effects. Many previous Zn studies use the Timms histochemical stain to identify chelatable Zn, which is the form likely involved in plasticity. However there are artifacts with this method as well as potential confounds (e.g., group variation in extent of membrane damage affecting staining), which is why we opted to use RS-XRF to determine total Zn levels. Nonetheless, a comparison of methods would be a useful future study and Timms staining may yield different results from RS-XRF. Finally, our unilateral cooling method has advantages, but there are limitations including: (1) the possibility of slight contralateral cooling, especially if deeper cooling treatments are used, (2) the potential to influence the contralateral hemisphere via the extensive commissural connections, and (3) the risk of causing stress. With regard to the latter, we have shown that focal cooling has only minor or no effects on cardiovascular parameters, such as heart rate and blood pressure, and no obvious behavioral or histological effects (Auriat et al, unpublished data), but it is possible that the rats still experience some stress, which we attempt to account for by using tethered control groups. Regardless, none of these potential effects significantly affected CA1 survival in the contralateral-to-cooling hemisphere.

Hypothermia is a potent neuroprotectant in animal models but a lack of adequate phase III clinical data and insufficient knowledge about effective treatment parameters (e.g., duration) and potential harmful effects have hampered its progress for ischemic and hemorrhagic stroke (MacLellan et al, 2009; van der Worp et al, 2010). As reviewed, there is a clear need for prolonged treatment both to rescue vulnerable neurons and to mitigate cerebral edema. Furthermore, animal studies show that when hypothermia is delayed, which is an unfortunate clinical inevitability, longer treatment durations are required to achieve neuroprotection (MacLellan et al, 2009). This presents a potential challenge to endogenous repair mechanisms, such as synaptic reorganization that become active during a similar period after ischemia (Lo, 2008). Our current experiments suggest that in the setting of global ischemia, which is used to predict treatment efficacy in cardiac arrest and ischemic stroke, prolonged hypothermia does not inhibit postischemic plasticity even when treatment is maintained for up to 1 week. While these findings are encouraging, there are important differences in pathophysiology among insults (e.g., hemorrhage versus ischemia). Thus, further study is needed in models of ischemic and hemorrhagic stroke that also take into account important variables such as age, insult severity, and the method of cooling.

Acknowledgments

The authors thank Dr J Stryker for use of a nitrogen chamber. The RS-XRF portion of this research was carried out at the Stanford Synchrotron Radiation Lightsource, a Directorate of SLAC National Accelerator Laboratory and an Office of Science User Facility operated for the US Department of Energy Office of Science by Stanford University.

The authors declare no conflict of interest.

Footnotes

This research was supported primarily by a grant to F Colbourne from the Canadian Institutes of Health Research (CIHR). The RS-XRF data collection was funded by a joint CIHR/Heart and Stroke Foundation of Canada team grant: Synchrotron Medical Imaging (#CIF 99472) awarded to H Nichol, P Paterson, F Colbourne, and others. F Colbourne is a senior medical scholar of Alberta Innovates—Health Solutions. G Silasi has received funding from a focus on stroke doctoral scholarship. A Auriat is a CIHR fellow in Health Research Using Synchrotron Techniques and also supported by a McCormick Fellowship. The SSRL Structural Molecular Biology Program is supported by the DOE Office of Biological and Environmental Research, and by the National Institutes of Health, National Center for Research Resources, Biomedical Technology Program (P41RR001209).

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