The Institutional Animal Care and Use Committee of the University of Alaska Fairbanks (UAF) approved all procedures. Arctic ground squirrels were trapped on the northern slope of the Brooks Range, Alaska, approximately 20 miles south of the Toolik Field Station of UAF (68°38′ N, 149°38′ W; elevation 809 m) in July 2004 and 2005 under permit from Alaska Department of Fish and Game. On arrival at UAF, AGS were screened for salmonella and quarantined for at least 14 days. All AGS were housed individually in cages at approximately 18°C, fed approximately 40 g of Mazuri Rodent Chow per day, and kept on natural lighting for 64° latitude where the light:dark cycle changes over days from 20:4 to 16:8 h. While housed in environmental chambers, AGS were fed rodent chow ad libitum although animals do not eat when hibernating.
In early fall, AGS were fed 10 to 15 sunflower seeds each day for 2 weeks before being moved to environmental chambers where they were housed at approximately 2°C with a 4:20 h light:dark cycle. All AGS used were trapped between 2 and 3 months of age. Female ibeAGS (total of 8 ibeAGS) and female hAGS (total of 11 hAGS) were seasonally matched, cold adapted, experienced prolonged torpor bouts of regular frequency and comparable duration, and were of similar age and weight. Summer euthermic female AGS (a total of three) had experienced one hibernation season in captivity with regular hibernation bouts. These AGS were moved to 18°C with a 12:12 h light:dark cycle in mid-May and tissue was sampled in July and early August.
Animals were habituated to handling to avoid arousal from hibernation in response to movement from the chambers to the laboratory before euthanasia. Briefly, habituation trials were begun after AGS had been through at least three regular hibernation bouts lasting a minimum of 3 days each and three periods of interbout euthermy lasting approximately 24 h each. Arctic ground squirrels were habituated to handling by being handled in a 2°C chamber and progressing through trials A through C as described below. As respiration rate increases before heart rate or body temperature during arousal (Toien et al, 2001
), it was used as a sensitive measure of responsiveness and recorded before and 5 mins and 1 h after handling. The trials were performed as follows: trial A, pick up briefly and return to cage; trial B, move AGS with their cotton nest to sanitized cooler for 5 mins; trial C, move AGS with nest to sanitized cooler and push cooler on a cart for 5 mins. Arctic ground squirrels were considered habituated once in trial C and if the change in respiration rate did not exceed 1 respiration per minute at 5 mins and 1 h after handling for 3 consecutive days.
The ‘shavings added’ method was used to monitor the state of AGS. Shavings were placed on the back of the AGS and checked every 24 h. An AGS was considered ‘torpid’ (hibernating) if the shavings remained on its back or considered to have been through an arousal if the shavings were disturbed or missing. This ‘shavings added’ or ‘sawdust method’ is a reliable indicator of prolonged torpor (Pengelley and Fisher, 1966
). Animals in the hAGS group were euthanized after a minimum of 2 days in torpor. Animals in the ibeAGS group were aroused by transfer to 18°C to 21°C 14 to 16 h before euthanasia, after having spent a minimum of 24 h in the current torpor bout.
Acute Slice Preparation
Hippocampal slices were prepared from juvenile (5- to 11- month-old) ibeAGS and hAGS and from 13- to 14-month-old summer euthermic AGS.
Euthermic animals were anesthetized using 5% halothane (maintained at 1% to 3%) with oxygen at a constant flow rate of 1.5 L/min while rectal body temperature and weight were recorded, and then animals were euthanized by decapitation. Hibernating AGS were euthanized without anesthetization as it is difficult to anesthetize hibernating animals to the same degree as euthermic animals because of the profound differences in respiration rate, pharmacokinetics, and response to anesthetics. Using a sterile technique, the whole brain was removed and placed in ice-cold oxygenated aCSF (120 mmol/L NaCl, 25 mmol/L NaHCO3, 10 mmol/L glucose, 3.3 mmol/L KCl, 1.2 mmol/L NaH2PO4, 2.4 mmol/L MgSO4, 1.8 mmol/L CaCl2 (pH 7.33 ± 0.01)) for 40 secs. Hippocampi were dissected and placed immediately into ice-cold oxygenated aCSF for 20 secs. Coronal hippocampal slices were simultaneously cut at a thickness of 400 μm using an MX-TS tissue slicer (SD Instruments, Grants Pass, OR, USA) and gently transferred to 20mL glass vials using wide-bore pipettes containing fresh aCSF to recover for 1 h at room temperature with constant bubbling with 95% O2/5% CO2.
OGD and Inhibitor Treatment
Hippocampal slices were exposed to OGD by transferring the slices to deoxygenated aCSF (pH 7.32 ± 0.01) lacking glucose that had been previously bubbled with 95% N2/5% CO2 for the time indicated at 37°C. Control slices were transferred to another vial containing oxygenated aCSF and subjected to all subsequent transfers in parallel. The partial pressure of oxygen was monitored continually throughout the OGD insult using a miniature Clark-style electrode (Instech Laboratories, Plymouth Meeting, PA, USA) inserted in the vial. pO2 during OGD was 7.7 ± 2.1mmHg for ibeAGS over 119 ± 1.5 mins and 13.1 ± 2.4mmHg for hAGS over 116 ± 1.8 mins (P>0.05). Alternatively, slices were incubated in 300 μmol/L sodium cyanide (NaCN) and 2 mmol/L iodoacetate (IAA) in deoxygenated aCSF for 2 h at 37°C. For activation of ERK1/2, 200 nmol/L of phorbol dibutyrate (PdBU; BIOMOL, Plymouth Meeting, PA, USA) was added to oxygenated aCSF at 37°C. For inhibition of MAPK activation, 10 μmol/L of the MEK1 inhibitor U0126 (BIOMOL) or 20 μmol/L of the JNK inhibitor SP600125 (Calbiochem, San Diego, CA, USA) was added to the slices during the 1 h slice recovery period after slice preparation and throughout OGD or normoxia. Slices were then incubated for 1 h at 37°C in aCSF constantly bubbled with 95% O2/5% CO2 (pO2 was at least 760mmHg) to mimic reperfusion for cell death analysis or as indicated. Slices were randomly distributed for cell death analysis, homogenized for MAPK analysis, or sonicated for ATP analysis as indicated. As a positive control for detection of cell death by propidium iodide (PI) staining, slices were treated with 300 μmol/L NaCN, 2 mmol/L IAA, and 0.1% Triton X-100 in phosphate-buffered saline (pH 7.6) for 20 mins immediately after slice preparation.
Quantification of Cell Death by PI and Hoescht 33341 Double-Staining
For cell death analysis, triplicate slices were incubated immediately after 1 h recovery in aCSF at 37°C for 20 mins with 2.5 μg/mL Hoechst 33342 (Sigma, St Louis, MO, USA) to identify all cells and with 5 μg/mL PI (Molecular Probes, Eugene, OR, USA) to identify dead and dying cells with permeabilized membranes. Slices were then transferred to fresh aCSF to wash out excess dye. Images were acquired on a Nikon Eclipse TE2000-U inverted microscope (Nikon, Melville, NY, USA) equipped with MetaMorph software (Universal Imaging Corporation, Downingtown, PA, USA). Slices were illuminated with a halogen bulb to obtain bright-field images for identification of CA1 region and with a mercury lamp and a Texas Red filter to obtain PI fluorescent images and a UV filter for Hoescht epifluorescence. Images were acquired for 20 ms. Metamorph software was used to set the threshold intensity to identify PI- and Hoescht-positive cells. The number of PI-positive cells and the number of Hoescht-positive cells were quantified by automated cell counting using Metamorph. Percent cell death was calculated by dividing the number of cells that were positive for Hoescht and PI by the number of cells positive for Hoescht. This method eliminates from the calculation any artifactual staining of cells that were PI-positive but not Hoescht-positive.
Determination of ATP
Triplicate slices were combined with 200 μL of ice-cold 5% trichloroacetic acid. A probe sonicator was used to disrupt the tissue using 3 to 4 bursts of 3 secs each. The solution was then centrifuged for 1 min at 16,000 g and the supernatant was assayed for ATP activity using the ENLITEN ATP assay system (Promega, Madison, WI, USA) by comparison with a concurrently generated standard curve. The pellet was resuspended in 200 μL of 1 mol/L NaOH and protein concentration was determined using the Bio-Rad Rc protein assay kit (Bio-Rad, Hercules, CA, USA).
Blood Ketone and Glucose Determination
Blood ketone and glucose levels were assessed from core blood from the carotid artery immediately after decapitation. Blood was sampled directly onto ketone or glucose test strips and levels were determined using the Precision Xtra Blood Glucose and Ketone Monitoring System (Abbott Laboratories, Abbott Park, IL, USA) according to the manufacturer’s instructions (n = 3 ibeAGS, 4 hAGS).
Immunoblot Analysis of MAPK Activation
Three hippocampal slices were homogenized in ice-cold RIPA lysis buffer (50 mmol/L Tris-HCl (pH 7.6), 0.02% sodium azide, 0.5% sodium deoxycholate, 0.1% SDS, 1% NP-40, 150 mmol/L NaCl, 1 mmol/L phenylmethylsulfonyl fluoride, 1 μ
g/mL aprotinin, 1 μ
g/mL antipain, 10 μ
g/mL leupeptin, 1 mmol/L orthovanadate) using a hand-held homogenizer and left on ice for 20 mins. Insoluble material was removed by centrifugation and protein concentrations were determined using the Bio-Rad Rc protein assay kit. Proteins (10 μ
g for ERK or 20 μ
g for JNK and p38) were separated on 10% SDS–polyacrylamide gel electrophoresis gels and transferred to nitrocellulose membranes. The membranes were blocked for 1 to 2 h with 5% (w/v) skim milk powder in TBST (10 mmol/L Tris-HCl, 150 mmol/L NaCl, 0.1% Tween 20) and incubated overnight at 4°C with the appropriate primary antibody. Phosphorylated proteins were detected using antibodies recognizing ERK phosphorylated on Thr202/Tyr204, JNK phosphorylated on Thr183/Tyr185, and p38 phosphorylated on Thr180/Tyr182 (Cell Signaling Technology Inc., Beverly, MA, USA), which have been used previously on AGS protein extracts (Zhu et al, 2005
). All antibodies were diluted in TBST. The membranes were washed with TBST and incubated with the appropriate horseradish peroxidase-conjugated secondary antibody (Bio-Rad) for 1 h at room temperature. Immunoreactive bands were visualized using enhanced chemiluminescence (Pierce, Rockford, IL, USA). To reprobe the membranes, bound antibodies were eluted by incubation with 10 mmol/L Tris-HCl (pH 2) and 150 mmol/L NaCl for 30 mins. Equal loading was then confirmed by reprobing the membranes with antibodies recognizing total ERK, JNK, or p38 (Cell Signaling Technology Inc.). To quantitate results, scans of ECL exposures were saved as TIFF files and analyzed using ImageQuant 5.2 software (Molecular Dynamics, Sunnyvale, CA, USA).
Antibody reactivity and specificity to AGS proteins is shown by the observation that protein bands from AGS were of equivalent (and expected) molecular weight as protein bands detected simultaneously from rat protein. In addition, the detected phospho-proteins were of the same molecular weight as the corresponding total protein band.
Data were analyzed with one- or two-way analysis of variance (ANOVA). Significant main effects or interactions were subjected to post hoc analysis (Tukey or t-test where indicated) (SAS software, SAS Institute Inc., Cary, NC, USA). P < 0.05 was considered statistically significant. Data are expressed as mean ± s.e.m.