Kym Campbell

Rodent Stroke Model Guidelines for Preclinical Stroke Trials (1st Edition)

Translational stroke research is a challenging task that needs long term team work of the stroke research community. Highly reproducible stroke models with excellent outcome consistence are essential for obtaining useful data from preclinical stroke trials as well as for improving inter-lab comparability. However, our review of literature shows that the infarct variation coefficient of commonly performed stroke models ranges from 5% to 200%. An overall improvement of the commonly used stroke models will further improve the quality for experimental stroke research as well as inter-lab comparability. Many factors play a significant role in causing outcome variation; however, they have not yet been adequately addressed in the Stroke Therapy Academic Industry Roundtable (STAIR) recommendations and the Good Laboratory Practice (GLP). These critical factors include selection of anesthetics, maintenance of animal physiological environment, stroke outcome observation, and model specific factors that affect success rate and variation. The authors have reviewed these major factors that have been reported to influence stroke model outcome, herewith, provide the first edition of stroke model guidelines so to initiate active discussion on this topic. We hope to reach a general agreement among stroke researchers in the near future with its successive updated versions.

In Search of Clinical Neuroprotection After Brain Ischemia. The Case for Mild Hypothermia (35°C) and Magnesium

Background and Purpose— Brain injury after stroke and other cerebral ischemic events is a leading cause of death and disability worldwide. Our purpose here is to argue in favor of combined mild hypothermia (35°C) and magnesium as an acute neuroprotective treatment to minimize ischemic brain injury. Methods and Results— Drawing on our own experimental findings with mild hypothermia and magnesium, and in light of the moderate hypothermia trials in cardiac arrest/resuscitation and magnesium trials in ischemic stroke (IMAGES, FAST-Mag), we bring attention to the advantages of mild hypothermia compared with deeper levels of hypothermia, and highlight the existing evidence for its combination with magnesium to provide an effective, safe, economical, and widely applicable neuroprotective treatment after brain ischemia. With respect to effectiveness, our own laboratory has shown that combined mild hypothermia and magnesium treatment has synergistic neuroprotective effects and reduces brain injury when administered several hours after global and focal cerebral ischemia. Conclusions— Even when delayed, combined treatment with mild hypothermia and magnesium has broad therapeutic potential as a practical neuroprotective strategy. It warrants further experimental investigation and presents a good case for assessment in clinical trials in treating human patients after brain ischemia.

Combined magnesium and mild hypothermia (35 °C) treatment reduces infarct volumes after permanent middle cerebral artery occlusion in the rat at 2 and 4, but not 6 h

BACKGROUND AND PURPOSE Using transient focal and global cerebral ischemia models in the rat, we have previously shown that MgSO4 is not neuroprotective unless it is combined with mild hypothermia. This study establishes a therapeutic time window for combined MgSO4 and mild hypothermia treatment after permanent middle cerebral artery occlusion (MCAO). METHODS Rats were subjected to permanent intraluminal thread MCAO and animals were treated 2, 4 or 6 h after ischemia with a MgSO4 infusion (360 micromol/kg, then 120 micromol/kg/h) and mild hypothermia (35 degrees C) or with vehicle for 24 h. At the 2 h time point, treatment with hypothermia alone and MgSO4 alone were also assessed. Infarct volumes were measured 48 h after MCAO induction. RESULTS After permanent MCAO, combined MgSO4 and hypothermia treatment reduced infarct volumes by 54% at 2 h (P = 0.048) and by 39% at 4 h (P = 0.012), but there was no treatment effect detected at 6 h or in the hypothermia alone or MgSO4 alone groups. CONCLUSIONS These findings support our earlier work highlighting the neuroprotective effect of MgSO4 when combined with mild hypothermia, even when treatment is delayed by several hours.

Magnesium treatment and spontaneous mild hypothermia after transient focal cerebral ischemia in the rat

There is evidence from global cerebral ischemia experiments in the rat that the neuroprotection attributable to magnesium treatment depends on the concurrent presence of at least mild hypothermia. We set out to determine to what extent spontaneous hypothermia occurred after transient middle cerebral artery occlusion in the rat, and whether this hypothermia influenced the outcome of magnesium treatment. We found that rectal temperatures from 30 min to 3h after recovery from anaesthesia/surgery were 1 °C lower than in the period from 4 to 6h. Striatal infarcts were significantly reduced by 32% in animals treated with 360 μmol/kg MgSO(4) intravenously immediately prior to ischemia. A higher magnesium dose of 720 μmol/kg had not effect on infarct volume. Having previously established that these two doses of magnesium are ineffective in normothermic animals using this model, we conclude that the mild spontaneous hypothermia contributed to the observed neuroprotective effect of magnesium in this study, and that previous studies of magnesium in cerebral ischemia have likely been confounded in this way.

The effect of blood pressure (37 vs 45 mmHg) and carotid occlusion duration (8 vs 10 min) on CA1-4 neuronal damage when using isoflurane in a global cerebral ischemia rat model.

This study presents our findings on the extent of neuronal damage in the hippocampal CA1-4 subfields following global (forebrain) cerebral ischemia in rats when using different blood pressure levels (37 vs 45 mmHg) and bilateral carotid occlusion durations (8 vs 10 min) under isoflurane anesthesia. We observed that global ischemia induced at a blood pressure of 37 mmHg resulted in high-grade CA1 neuron injury (>90%) at either duration of carotid occlusion. In contrast, global ischemia induced at a blood pressure of 45 mmHg resulted in either high-grade CA1 neuronal loss or a neuronal loss of ≈50% or less. We also noted that a post-reperfusion EEG recovery time (return of burst suppression spikes) of >12 min was associated with an 85% rate of high-grade CA1 neuronal injury. Neuronal loss in the other hippocampal subfields did not differ significantly between any of the 4 different model parameters tested. In these subfields ≈55% neuronal loss occurred in the CA2 subfield, and ≈30% in the CA3 and CA4 subfields. These findings highlight the need to assess different model parameters in order to achieve consistent high-grade CA1 neuronal damage, which, among other experimental outcomes, will improve the ability to uncover therapeutic effects using the least possible animals when assessing a neuroprotective treatment.

Lack of neuroprotection of inhibitory peptides targeting Jun/JNK after transient focal cerebral ischemia in Spontaneously Hypertensive rats

In this study, we have assessed the ability of two TAT-fused peptides PYC36d-TAT and JNKI-1d-TAT (JNKI-1 or XG-102), which respectively inhibit jun proto-oncogene (c-Jun) and c-Jun N-terminal kinase (JNK) activation, to reduce infarct volume and improve functional outcome (adhesive tape removal) after transient focal cerebral ischemia in Spontaneously Hypertensive (SH) rats. PYC36d-TAT and JNKI-1d-TAT peptide batches used for experiments were tested in vitro and protected cortical neurons against glutamate excitotoxicity. Rats were treated intravenously with three different doses of PYC36d-TAT (7.7, 76, or 255 nmol/kg), JNKI-1d-TAT (255 nmol/kg), d-TAT peptide (255 nmol/kg), or saline (vehicle control), 10 minutes after reperfusion after 90 minutes of middle cerebral artery occlusion (MCAO). Contrary to other stroke models, no treatment significantly reduced infarct volume or improved functional score measurements compared with vehicle-treated animals when assessed 48 hours after MCAO. Additionally, assessment of the JNKI-1d-TAT peptide, when administered 1 or 2 hours after reperfusion after 90 minutes of MCAO, also did not improve histological or functional outcomes at 48 hours after occlusion. This study is the first to evaluate the efficacy of PYC36d-TAT and JNKI-1d-TAT using the SH rat, which has recently been shown to be more sensitive to AMPA receptor activation rather than to NMDA receptor activation after cerebral ischemia, and which may have contributed to the negative findings.

Does remote ischemic preconditioning prevent delayed hippocampal neuronal death following transient global cerebral ischemia in rats

Objective: To determine if remote ischemic preconditioning (RIPC) induced by transient limb ischemia is protective against delayed hippocampal neuronal death in rats undergoing transient global cerebral ischemia (GCI). Method: Animals were randomized into 3 groups: Group I (Control, n = 5) underwent sham procedure, namely, general anesthesia x 2, without cerebral ischemia; Group II (RIPC + GCI, n = 5) was subjected to RIPC, induced by transient left hind limb ischemia under general anesthesia prior to GCI; Group III (GCI only, n = 5) underwent sham procedure under general anesthesia prior to GCI. Twenty-four hours after the RIPC or sham procedure, a transient GCI was induced for 8 minutes in Groups II and III by means of bilateral common carotid artery occlusion and hypotension. Hippocampal CA1 neurons were histologically examined at 7 days after ischemia. Results: There was no significant difference between the RIPC group and the ischemia only group. The number of neurons in the RIPC group were 0.90 (95% CI 0.20, 4.08) times the number in the ischemia group (p=0.89). The number of neurons in the RIPC group were 0.03 (95% CI 0.01, 0.10) times the number in the Control group (p=0.0001). Conclusion: Second window of the RIPC does not prevent hippocampal CA1 neuronal death at 7 days after transient global cerebral ischemia.

Efficacy of mild hypothermia (35°C) and moderate hypothermia (33°C) with and without magnesium when administered 30min post-reperfusion after 90min of middle cerebral artery occlusion in Spontaneously Hypertensive rats.

In this study we compared the efficacy of mild (35°C) and moderate (33°C) hypothermia alone and when combined with magnesium in a transient focal cerebral ischaemia rat model. Spontaneously Hypertensive rats were subjected to 90min of transient intraluminal thread middle cerebral artery occlusion (MCAO). Thirty minutes after reperfusion animals were treated with mild (35°C/24h) or moderate (33°C/24h) hypothermia combined with either magnesium (intravenous MgSO4 infusion: 360μmol/kg, then 120μmol/kg/h for 24h) or a similar volume of saline. Control animals were maintained normothermic (37°C/24h) and received vehicle infusion (saline for 24h). Infarct volumes and functional assessment (bi-symmetrical adhesive tape removal) were measured 48h after MCAO induction. After transient MCAO, only moderate hypothermia and mild hypothermia combined with magnesium treatment significantly reduced infarct volumes by 32.9% (P=0.01) and by 24.8% (P=0.046), respectively. Mild hypothermia alone reduced infarct volume by 23.8%, but did not reach statistical significance (P=0.054), while moderate hypothermia combined with magnesium reduced infarct volume by 17.3% (P=0.17). No treatment improved adhesive tape removal time. In summary, moderate hypothermia and mild hypothermia with or without magnesium can reduce infarct volume, however magnesium may reduce the efficacy of moderate hypothermia. Given the potential advantages of mild hypothermia over moderate hypothermia in terms of side-effects and induction, and the potential beneficial effects of magnesium, these findings have important implications for the use of hypothermia for stroke.

FAST-Mag protocol with or without mild hypothermia (35°C) does not improve outcome after permanent MCAO in rats

The current study assessed the neuroprotective efficacy of magnesium using a FAST-Mag trial treatment protocol alone, and in combination with mild hypothermia, in Sprague Dawley rats subjected to permanent, middle cerebral artery occlusion (MCAO). Treatment with magnesium (MgSO4.7H2O) consisted of an intravenous loading dose (LD: 360 μmol/kg) and a 24 hour infusion (120 μmol/kg/h), while mild hypothermia at 35°C was maintained for 24 hours. Treatment groups consisted of animals receiving: i) saline; ii) magnesium LD/infusion at 1.5 h/2.5 h post-MCAO; iii) magnesium LD/infusion at 1.5 h/2.5 h post-MCAO and hypothermia commencing at 2.5 h post-MCAO; iv) magnesium LD and hypothermia at 1.5 h and magnesium infusion at 2.5 h post-MCAO; v) hypothermia commencing at 1.5 h post-MCAO and magnesium LD/infusion at 2.5 h post-MCAO; and vi/vii) hypothermia commencing at 1.5 h or 2.5 h post-MCAO. No treatment significantly reduced infarct volumes or improved adhesive tape removal time when measured 48 hours after MCAO. These findings indicate that FAST-Mag treatment alone or with mild hypothermia may not provide benefit after ischemic stroke, associated with permanent cerebral artery occlusion.

Comparison of the Efficacy of Mild Hypothermia (35°C) and Moderate Hypothermia (33°C), Alone or Combined with Magnesium Treatment, When Commenced 2 or 4 Hours After Global Cerebral Ischemia in Rats

We first assessed the neuroprotective efficacy of mild hypothermia (35°C/24 hours) alone and in combination with magnesium (intravenous loading dose: 360 μmol/kg; 48 hours infusion: 120 μmol/kg/h) commencing 4 hours after global ischemia. Treatment with mild hypothermia alone (CA1 survival rate: 8.7%±0.9%) or with magnesium (9.0%±2.9%) did not significantly increase hippocampal CA1 neuronal survival compared with saline-treated controls (7.1%±0.7%). Next, we assessed mild hypothermia (35°C/24 hours) and moderate hypothermia (33°C/24 hours), alone or in combination with magnesium, when commenced 2 hours after global ischemia. At this time point, all treatments significantly increased CA1 neuronal survival compared with saline controls (CA1 survival rates: mild hypothermia: 13.6%±1.8%; mild hypothermia + magnesium: 19.4%±8.7%; moderate hypothermia: 15.9%±4.1%; moderate hypothermia + magnesium: 21.1%±11.2%; saline control: 6.7%±1.6%; p<0.001). Although a trend for increased neuroprotection was observed when hypothermia was combined with magnesium, it did not reach statistical significance. It also appears that when a 24-hour hypothermia treatment is commenced earlier (≤2 hours postischemia) there is no difference in efficacy between mild and moderate hypothermia.