1. RESUSCITATIVE HYPOTHERMIA ACADEMIC INDUSTRY ROUNDTABLE Hypothermia: future directions Midori A. Yenari, MD Depts. of Neurosurgery & Neurology Stanford Stroke Center & Stanford University Stanford, CA

2. Introduction Hypothermia is a robust neuroprotectant. Compelling pre-clinical data to justify clinical trials Questions at preclinical level: duration/delay, permanent occlusion, combination with thrombolysis, combination with other treatments?

3. Summary: Hypothermia in Experimental Stroke Depth: 34.5C or lower results in comparable neuroprotection, but lower temperatures are associated with compromised hemodynamics; most consistent results with 30-33C **Duration: At least one hour, provided cooling begins soon after ischemia onset. Several hours (6-12 h) if cooling is delayed by more than several hours **Delay: consistent protection with 2-3 h delay with at least 2 h cooling; can delay up to 6 h provided cooling is maintained for 1-2d Long term: protection observed out to 2 mos with 2 h intraischemic cooling, or 1 h delay with prolonged cooling (2d); 70% protection in global model out to 6 mos, but extent of protection decreases over time **Permanent vs. Temporary: mixed results

4. Protect against permanent ischemia? Less consistent data compared to temporary ischemia Protects: Intraischemic hypothermia for 6 h ↓infarct size @ 6 h (Baker et al, Exp Neurol 1991) Intraischemic hypothermia for 24 h ↓infarct size @ 48 h (Yamamoto et al, Stroke 2001) ↓ infarct size @ 24 h when cooling (30-34.5C) delayed up to 1 h & maintained for 1 h (Kader et al, Neurosurgery 1992) Doesn’t protect: No difference in infarct size when cooling (33C) was instituted during ischemia, and maintained for 1 h (Ridenour et al, Stroke 1992) No protection when cooling to 30-36C was instituted shortly after occlusion and maintained for 2 h (Morikawa et al, JCBFM 1992)

5. Optimize duration/delay Brief intraischemic hypothermia (1-2 h) protects, but also protects when cooling begins 2-3 h after ischemia onset Prolonging the duration of cooling to several hours seems to lengthen the temporal therapeutic window What are the limits in rodents? In humans?

6. Combination hypothermia & rt-PA Embolic model in rats Hypothermia (32C), normothermia (37C) or hyperthermia (39C) for 2 h pre/post embolization rt-PA 2 h after embolization Angiographic recanalization best @ 39C rt-PA Rx itself ↓ inf. Size ~50% @ 48h: largest infarcts (39C), smallest (32C), hypothermia ↓inf. size by ~70% No further improvement with rt-PA/hypothermia—is lack of difference due to robust protection by hypothermia? Interaction of rt-PA and temperature? Can hypothermia prolong the window for rt-PA Rx? Reduce hemorrhage? (Meden et al, Br Res, 1994)

7. Effect of temperature on clot lysis rt-PA is the only approved treatment for acute stroke combination rt-PA and mild hypothermia? how does temperature influence clot lysis?

8. Effect of temperature on clot lysis Thrombin stabilized, 24 h aged clots from whole blood of donor rabbits. Incubate in sterile PBS at 24, 30, 33, 35, 37 & 40C. Incubate with rt-PA (concentrations corresponding to serum levels in rabbits given doses of 1, 3 and 6 mg/kg) Measure pre- and post-incubation weights

9. Clot lysis is dose dependent (Yenari et al, Thrombosis research, 1995) clot lysis is dose dependent each 1 ug/ml increase in rt-PA increases clot lysis by 4%

10. Clot lysis & temperature rt-PA: ~0.5% decreased clot lysis/1C drop control: 0.5% increased in clot lysis/1C drop (Yenari et al, Thrombosis research, 1995)

11. Combination RX with hypothermia & gene therapy (Zhao et al.) HSV viral vectors Overexpress potentially neuroprotective genes Bcl-2 as a prototypical anti-apoptosis, anti- necrosis gene

13. Bcl-2, an anti-apoptotic protein, to treat stroke -6-15 h 0 1h 1.5h 5h 48h Inject vectorocclude reperfuse end experiment* Delay vector delivery

14. Striatal Bcl-2 overexpression protects neurons from tMCAO (Lawrence et al, JCBFM, 1995)

15. 0 1h 2h 5h 48h Occlude reperfuse Combination Bcl-2 gene therapy & hypothermia Cool to 33C end experiment Inject vectors* Groups: 33C-control 33C-Bcl-2 37C-control 37C-Bcl-2

16. * vs 37ºC-Bcl-2, p<0.01 # vs. 33C-control, p<0.01 Hypothermia prolongs the temporal therapeutic for Bcl-2 gene therapy

17. Conclusions Permanent occlusion? Optimal duration/delay? Combination Rx with thrombolytics? Combination Rx with neuroprotectants?